MXPA99011954A - Retentate chromatography and protein chip arrays with applications in biology and medicine - Google Patents

Abstract

This invention provides methods of retentate chromatography for resolving analytes in a sample. The methods involve adsorbing the analytes to a substrate under a plurality of different selectively conditions, and detecting the analytes retained on the substrate by desorption spectrometry. The methods are useful in biology and medicine, including clinical diagnostics and drug discovery.

Description

CHROMATOGRAPHY OF RETENATO AND ARRANGEMENTS OF PROTEIN FRAGMENTS WITH APPLICATIONS IN BIOLOGY AND MEDICINE RECIPROCAL REFERENCE TO RELATED APPLICATION This application claims the benefit of the priority dates of the patent application, pending, 60 / 054,333, filed on June 20, 1997, and the patent application, also pending, 60 / 067,484, filed on 1 December 1997, whose contents are incorporated herein by reference in their entirety.

RESEARCH OR SPONSORED DEVELOPMENT FEDERAL ENTE Not applicable.

BACKGROUND OF THE INVENTION This invention relates to the field of separation science and analytical biochemistry. The methods of this invention have application in biology and medicine, which include the analysis of genetic function, differential gene expression, protein discovery, cellular and clinical diagnostics, and drug classification. The cellular function, both normal and pathological, depends, in part, on the genes expressed by the cell (ie, the function of the gene). The expression of the gene has both qualitative and quantitative aspects. That is, the cells may differ both in terms of particular gene expressed, and in terms of the relativ level of expression of the same gene. The differential expression of the gene can be manifested, for example, by differences in the expression of the proteins encoded by the gene, or modifications after the translation of the expressed proteins. For example, proteins can be decorated with carbohydrate or phosphate groups, or they can be processed through the splitting of peptides. Thus, at the biochemical level, a cell represents a complex mixture of organic biomolecules. A goal of functional genomes ("proteomes is the identification and characterization of organic biomolecules that are differentially expressed between cell types." Comparing expression one can identify molecules that may be responsible for a particular pathological activity of a cell. Identification of a protein that is expressed in cancer cells, but not in normal cells, is useful in diagnosis and, finally, for drug discovery treatment of the pathology.At the end of the Human Genome Project, all human genes are They have cloned in sequence and organized into databases, and this world of the "post-genome", the ability to differentially identify the expressed proteins, will lead, in turn, to the identification of the genes that encode them. The genetic analysis can be applied to problems of cellular function. Genetics require tools that can solve the complex mixture of molecules in a cell, the amount of it and its identification, even when they are present in trace quantities. However, the current tools of analytical chemistry for this fi are limited in each of these areas. A widely used biomolecular separation method is gel electrophoresis. Frequently, a first separation of the proteins by the isoelectric focus on a gel is coupled with a second separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The result is a map that solves the proteins, according to the dimensions of the isoelectric point (net charge) and size (that is, the mass). However, this method is limited in several ways. First, the method provides information only about the characteristics of a biomolecule - mass and isoelectric point ("pl"). Second, the resolution power in each of the dimensions is limited by the resolving power of the gel. For example, molecules whose mass differs by less than 5% or less than 0.5 pl, are often difficult to solve. Third, gels that have a charge capacity, and thus sensitivity, one may not be able to detect biomolecules that are expressed in small quantities. Quarter, proteins and small peptides with a more molecular below 10-20 kDa, are not observed. Other analytical methods can overcome one or more of these limitations, but they are difficult to combine efficiently. For example, analytical chromatography can separate biomolecules based on a variety of analyte / adsorbent interactions, but it is difficult to multidimensional and time-consuming analysis. Also, the methods are limited in sensitivity. Clinical diagnoses require the ability to specifically detect known markers of diseases. However, the development of such diagnoses is hampered by the time necessary to prepare reagents that specifically bind to the markers, or that can discriminate the marker in a complex mixture. Drug discovery requires the ability to quickly classify agents that modulate the ligand / receptor compound interactions. Often, the step limiting the regimen in such classifications is the ability to detect the interaction of ligand / receptor compound. Thus, fast and specific methods to identify binding events would be an advance in the technique. Until now, the process of identifying a potential marker or member of a ligand / receptor compound pair, to produce an agent that specifically binds to the marker or member, has been difficult. In one method, normal and diseased tissue are compared to identify the mRNA species or expressed sequence tags ("EST") that rise or fall in the diseased tissue. These species are isolated and the polypeptides that they encode are produced through routine recombinant DNA methods. Then, the polypeptides are isolated and used as immunogens to raise specific antibodies to the marker. The antibodies can be used in, for example, ELISA assays, to demonstrate the amount of labeling in a patient sample. This process is long and tedious. It may take nine months to a year to produce such antibodies, with much time being spent on developing the protocols to isolate a sufficient amount of the polypeptide for immunization. Also, the method relies on the hope that differences in RNA expression are expressed as differences in protein expression. However, this assumption is not always reliable. Therefore, methods in which differentially expressed proteins are directly detected and where specific ligations can be generated in a significantly shorter time would be of great benefit in this field. Thus, the tools for solving complex mixtures of organic biomolecules, which identify individual biomolecules in the mixture and identification of specific molecular recognition events, which involve one or more target analytes, are suitable for analytical biochemistry, biology and medicine.

COMPENDIUM OF THE INVENTION This invention provides devices and method for the retentate chromatography. Retene chromatography is a combinatorial method to provide high resolution of analyte information in complex mixtures through the use of multi-dimensional separation methods. It provides a unified analyte detection and functional analysis capability for medical biology and is characterized by an integral, simple operating system for the direct detection of analyte expression patterns associated with the function of genes, protein function, function cellular and function d the complete organisms. In one aspect, this invention provides a unified operating system for the discovery or diagnosis of gene function, function of proteins or the function of complete macromolecule sets. More particularly, analytes can be resolved in a variety of two-dimensional formats, thus providing multidimensional information. The analytes are first separated into at least two different first dimensions, based on their ability to adsorb to a stationary phase under at least different selectivity conditions, such as the anionic / cationic potential, hydrophobicity / hydrophilicity or specific biomolecular recognition. Then the analytes are separated into a second dimension, based on mass, or desorption spectrometry (eg, laser desorption mass spectrometry), which also provides for the detection of separated analytes. The nature of the adsorbent to which the analytes adsorb, provides the physico-chemical information about the analyte. Thus, this invention provides a molecular discovery and diagnostic device, which is characterized by the inclusion of analyte processing capabilities, both multiple and parallel. Because the analytes are directly detected, the invention makes possible the simultaneous transmission of two or more independent objective analyte signals, from the same "circuit" (ie, the location of the steerable fragment) during a single operation of the unit. Retenate chromatography is different from conventional chromatography in several ways. First, in the chromatography of the retenate, the analytes that are retained on the adsorbent are detected. In conventional chromatographic methods, the analytes are eluted separated from the adsorbent before detection. There is no convenient routine or resource for detecting the analyte, which is not eluted and separated from the adsorbent in conventional chromatography. Thus, the chromatography of the retenate provides direct information about the structural chemical characteristics of the retained analytes. Second, the coupling of adsorption chromatography with detection of desorption spectrometry, provides extraordinary sensitivity, in the femtomolar interval, and unusual resolution. Third, in part because it allows the direct detection of analytes, retentate chromatography provides the ability to rapidly analyze retentions with a variety of different conditions of selectivity, thus providing rapid, multi-dimensional characterization of the analytes in a sample. Fourth, the adsorbents can be attached to a substrate an array of adressable, predetermined locations. This allows parallel processing of analytes exposed to different adsorbent sites (ie, "affinity sites" "zones") in the array under different conditions of elution. The chromatography of the retenate has many uses in biology and medicine. These uses include combinatorial biochemical separation and purification of analytes, and study of differential gene expression and molecular recognition, diagnostic and drug discovery events. A basic use of retenat chromatography as an analytical tool involves exposing a sample to a combinatorial classification of different combinations of adsorbents / eluents and detecting analyte behavior under different conditions. This both purifies the analyte and identifies the useful conditions for detecting the analyte in a sample. The substrates that have the adsorbents identified in this way can use analyte-specific analyte detectors. In a progressive extraction method, a sample is exposed to a first combination of adsorbent / eluent washing, reduced of analytes, which are adsorbed by the first adsorbent, is exposed to a second adsorbent, for the reduction of the other analytes. The selectivity conditions identified for retaining the analytes can also be used in preparative purification procedures in which an impure sample, containing an analyte, is exposed, in sequence, to adsorbents which it retains, the impurities are removed and the analyte The retentate is collected from the adsorbent for a subsequent routine. One aspect of the invention is that each kind of molecular recognition event type (eg, the interaction of the target adsorbent and the target analyte), characterized by a particulate selectivity condition of a steerable location within the array, is directly detected, while the associated molecules are located (ie, "retained") in the steerable location. That is, selection and detection by direct means does not require the elution, recovery, amplification or labeling of the target analyte. Another aspect of the present invention is that the detection of one or more convenient molecular recognition events, at one or more locations, within the steerable array, does not require the removal or consumption of more than a small fraction of the total adsorbent-analyte. Thus, the unused portion can be further investigated after one or more "secondary process" events, conducted directly in your (ie, within the boundary of the steerable location) for the purpose of structure function elucidation, which include the assembly disassembled, modified, or amplified (directly or indirectly). Adsorbents with improved specificity for an analyte can be developed by an interactive process, referred to as "progressive resolution", where the adsorbents or eluents tested to retain an analyte s test with additional variables to identify combinations with better binding characteristics. Another method allows the rapid creation of substrates with specific antibody adsorbents for an analyte. The method involves attaching the analyte to an adsorbent, classifying the phagocyte display collections for those phagocytes that bind to the analyte. The chromatography of the retenate has use in molecular and cellular biology, as well. Analytes that are differentially present in two samples (eg proteins differentially expressed in cell extracts) can be identified by exposing the samples to a variety of adsorbent / eluent combinations for analysis by desorption spectrometry, thereby making use of The high power d resolution of system information that other systems d separation and detection can not be matched. The unknown target proteins can be identified by determining the physicochemical characteristics, which include the molecular mass, based on the chemical characteristics of the adsorbent / eluent combination, and this information can be used to classify data bases for proteins that have profiles Similar. The methods in the separation biochemistry and the adsorbents produced from these methods are useful in diagnosis. More particularly, adsorbents, chemical or biospecific, can be developed to detect important diagnostic markers. In certain embodiments, a substrate may have an array of selected adsorbent zones for a combination of diagnostic markers for a disease or syndrome. The chromatography of the retenate is also useful in the discovery of drugs. A member of a pair of receptor / ligated compound is attached to an adsorbent, and its ability to bind to its binding partner is tested for the presence of the agent. Due to the rapidity with which l adsorption can be tested, the combinatorial collections of agents can be easily tested in their ability to modulate the interaction. In one aspect, this invention provides a method for high resolution of information of at least one analyte in a sample. The method is a combinatorial separation method that includes the separation and detection of multiple analytes in parallel. This method comprises the steps of a) exposing the analyte to at least different conditions of selectivity, each condition of selectivity defined by the combination of an adsorbent and an eluent, to allow retention of the analyte by the adsorbent.; and b) detecting the analyte retained under the different selectivity conditions, by the desorption spectrometry. The detection of the analyte retained under the different conditions of selectivida provides a high resolution of analyte information. In one embodiment, each different condition of selectivity is defined at a different predetermined location for the parallel process. In another embodiment, the method comprises the steps of i) exposing and analyte to a first selectivity condition at a defined location, to allow retention of the analyte by the adsorbent; ii) detecting the analyte retained under the first selectivity condition by the desorption spectrometry; iii) washing the adsorbent under a different condition of selectivity at the defined location, to allow retention of the analyte to adsorbent; and iv) detecting the analyte retained under the second selectivity condition, by desorption spectrometry. In another embodiment, the analyte is an organic biomolecule, a multimeric molecular complex or a macromolecular conjunct. In another embodiment, the organic biomolecule is an enzyme, an immunoglobulin, a cell surface receptor or an intracellular receptor. In another embodiment, the adsorbent comprises an anion, a cation, a hydrophobic interaction adsorbent, a polypeptide, a nucleic acid, a carbohydrate, a lectin, a dye, a reducing agent, a hydrocarbon or combinations thereof. In another embodiment, the adsorbent is attached to a substrate comprising: glass, ceramic, a magnetic material, an organic polymer, a conductive polymer, or a native biopolymer, a metal or a metal coated with an organic polymer. In another embodiment, the adsorbent is in the form of a microemulsion, a latex, a layer or a strip. In another embodiment, the locations on the substrate are arranged in a line or an orthogonal array. In another modality, the adsorbents are located on a substrate at different locations before the analytes are exposed to selective conditions. In another embodiment, the adsorbents are located on a substrate in different locations, after the analytes are exposed to the conditions of selectivity. In another embodiment, the different selectivity conditions comprise different ligation conditions or different elution conditions.

In another embodiment, the detection step comprises detecting the mass of the analyte by the laser desorption spectrometry. In another embodiment, the selectivide conditions are selected to optimize the retention of the analyte by an adsorbent. In another embodiment, this at least one analyte and more than one analyte. In another embodiment, the plurality d selectivity conditions are defined by different adsorbents and the same eluent. Another embodiment further comprises the step d supplying a substrate comprising adsorbents and steerable locations, each absorbent being an adsorbent d a selectivity condition, identified to retain and analyte. In another embodiment, the elution conditions differ according to pH, regulation capacity, ionic strength, a characteristic water structure, detergent type, detergent strength, dielectric constant hydrophobicity. In another embodiment, the plurality d selectivity conditions are defined by the same eluent. In another embodiment, this invention provides a method for sequential extraction of analytes from a sample. It is a separation in series, combinatorial and a method of development --- d-e purification for multiple analytes and parallel. The method comprises the steps of: a) exposing a sample comprising the analytes to a first selectivity condition, to allow the retention of analytes by a first adsorbent and to create a non-retained sample; b) collecting the non-retained sample comprising the analytes, exposing the non-retained sample to a second selectivity condition, to allow the retention of the analytes by a second adsorbent and to create a non-retained sample; and c) detecting the analyte retained under the different selectivity conditions by the desorption spectrometry. In another aspect, this invention provides a substrate for desorption spectrometry, comprising an adsorbent, whose ligation characteristics vary in a gradient along one or more linear axes. In another aspect, this invention provides a method for progressively identifying a selectivity condition with improved resolution, for an analyte in a sample. The method comprises the steps of: (a) identifying a selectivity condition that retains an analyte in a sample by: (i) exposing a sample to a set of selectivity conditions, each condition of selectivity defined by at least one binding characteristic and at least one elution characteristic, - (ii) detecting and retained analyte under each condition of selectivity by the desorption spectrometry; e (iii) identifying a selectivity condition that retains the analyte; and (b) identifying a selectivity condition with improved resolution for the analyte by: (i) selecting at least one binding characteristic or elution characteristic of the identified selectivity condition jle and adding it to a constant set characteristic of the selectivity; (ii) exposing the sample to a modified set of conditions of selectivity, in which each condition of selectivity in the modified game comprises: (1) the characteristics of selectivity in the constant game and (2) the characteristic of ligature or elution characteristic. , which is not in constant play; and (iii) identifying a selectivity condition- from the set modified by the desorption spectrometry that retains the analyte with improved resolution compared to the previously identified selectivity condition. One embodiment comprises the step of repeating step (b) at least once. Another embodiment comprises repeating step (b) until a selectivity condition is identified that retains only the target analyte of the sample. In another aspect, this invention provides a substrate for desorption spectrometry, comprising an adsorbent of a selectivity condition identified to solve an analyte by the method of progressive resolution. In one embodiment, the substrate comes in the form of a set that also comprises an eluent of the selectivity condition or instructions in the use of the eluent in combination with the adsorbent. In another aspect, this invention provides a method for the preparative purification of an analyte from an impure sample. The method comprises the steps of: a) exposing the sample to a substrate, under a plurality of different selectivity conditions; detect the analyte retained under the different selectivity conditions by the desorption spectrometry; and identify the selectivity conditions under which the analyte is retained; b) purifying the analyte by repeating, for a plurality of different selectivity conditions, a sequence of steps comprising: i) exposing the sample to an adsorbent, under the identified selectivity condition, to allow retention of the analyte by the adsorbent; ii) separating the analyte from impurities, which are not retained by the substrate; and iii) collecting the adsorbent analyte. In another aspect, this invention provides a method for preparing a substrate for detecting at least one analyte in a sample. This method is a combinatorial method for the design and identification of specific analyte adsorbents. It is useful in detecting target analyte. The method comprises the steps of: a) exposing the sample to at least two different conditions of selectivity, each condition of selectivity defined by the combination of an adsorbent and an eluent, to allow retention of the analyte by the adsorbent; b) identifying by desorption spectrometry at least one selectivity condition under which the analyte is retained; and c) preparing a substrate comprising at least one adsorbent of an identified selectivity condition. In one embodiment, the identification step comprises the identification of at least one selectivity condition under which a plurality of analytes are retained. In another embodiment, the preparation step comprises preparing a substrate that includes a plurality of adsorbents that retain the analyte under an elution condition, such as a multiple adsorbent. In another aspect of this invention, a method is provided for diagnosing in a subject a disease characterized by at least one diagnostic marker. This is a combinatorial method for the simultaneous detection of multiple diagnostic markers. The method comprises the steps of a) supplying a substrate for use in the desorption spectrometry, comprising at least one steerable location, each steerable location including an adsorbent that resolves at least one of the diagnostic markers, under an elution condition.; b) exposing the substrate to a biological sample from the subject, under the elution condition, to allow retention of the diagnostic marker, and c) detecting the diagnostic marker retained by the desorption spectrometry. The detection of the retained diagnostic marker provides a diagnosis of the disease. In another aspect, this invention provides a set for detecting an analyte in a sample, which comprises: (1) a substrate, for use in the desorption spectrometry, including at least one steerable location, each steerable location comprising an adsorbent , which resolves an analyte under a selectivity condition comprising the adsorbent and an eluent, and (2) the eluent or instructions for exposing the sample to the selectivity condition. In one embodiment, the set is characterized by a plurality of diagnostic markers and the substrate comprises a plurality of steerable locations, each steerable address comprising an adsorbent that resolves at least one of the diagnostic markers. In another aspect, this invention provides a substrate for desorption spectrometry, comprising at least one adsorbent in at least one steerable location, wherein this at least one adsorbent resolves a plurality of diagnostic markers for a pathological condition of a sample of a patient.

In another aspect, this invention provides a method for selecting identity candidates for an analyte protein. This method is a combinatori method for the identification of proteins, based on at least do physico-chemical properties. The method comprises the steps of a) determining a set of values that specify the matching parameters for at least a first second physical-chemical characteristic of a protein analyte in a sample by: i = exposing the analyte to a plurality of different conditions e selectivity, in which the adsorption of the protein analyte to the substrate is mediated by a base of attraction, which identifies the physical-chemical properties of the protein analyte; ii) detect the analyte retained under the different conditions of selectivity by desorption spectrometry; and b) executing, in a programmable digitable computer, the steps of i) accessing a database, comprising, for each member of a set of reference polypeptides, a set of values that specify at least a first and second characteristic. physicochemical parameters of the reference polypeptides; ii) enter the set of values that specify the physical-chemical characteristics of the protein analyte; iii) classifying the reference polypeptides that have sets of values within the correspondence parameters from the database. The classified reference polypeptides provide identifiable candidates for the protein analyte. The unclassified reference polypeptides are those excluded as identity candidates. In another aspect, this invention provides a method for analyte sequence retention. This method is a method of inspection of the macromolecular or supramolecular multimeric conjunct. It is useful as a method for the discovery of drugs, by the interference of molecular recognition. The method comprises the steps of a) exposing a first sample to a primary adsorbent and to an eluent, to allow the retention of a first analyte by the adsorbent, and detect the analyte adsorbed by the desorption spectrometry, so that the first analyte retained arrives. to be a secondary adsorbent; b) exposing a second sample to the secondary adsorbent and to an eluent, to allow the retention of a second analyte by the secondary adsorbent, and detect the second analyte adsorbed by desorption spectrometry, whereby the second analyte retained becomes a tertiary adsorbent . In another aspect, this invention provides a method for detecting an enzyme in a sample. The method comprises the steps of: a) supplying a solid phase which includes an adsorbent and an enzyme substrate bound to the adsorbent, in which the activity of the enzyme on the enzyme substrate produces a product having a more molecular characteristic; b) exposing the substrate to the sample; and c) detecting the product by desorption spectrometry. The detection of the product provides detection of the enzyme. In another aspect, this invention provides a method for determining whether an analyte is differentially present (eg, differentially expressed) in a first and second biological sample. The method is useful for the combinatorial method of inspection of the expression of the difference gene, by the display of differential protein. The method comprises the steps of: a) determining a first retention map for the analyte in the first sample for at least one selectivity condition; b) determining a second retention map for the analyte in the second sample for the same condition d selectivity; and c) detect a difference between the first and second retention maps. A difference in the retention maps provides a determination that the analyte is differentially present in the first and second samples. In one embodiment, the method to determine if a protein is differentially expressed between two different cells, and the first and second samples comprise the cells or the material of the cells. In another embodiment, the method for determining whether an agent alters the expression of a protein in a biological sample further comprises the step of administering the agent to a first biological sample, but not to the second biological sample. In another modality, the first biological sample is derived from a healthy subject and the second biological sample is from a subject that suffers from a pathological condition. The sample can be selected from, for example, blood, urine, serum tissues. The analytes that are found will be increased and the samples of the pathological subjects are diagnostic markers d candidates. Generally, the confirmation of a diagnostic marker involves the detection of the marker in many subjects. In another aspect, this invention provides a method for identifying a ligand compound to a receptor. The method comprises the steps of: a) supplying a substrate that includes an adsorbent, in which the receptor s binds the adsorbent; b) exposing the receptor bound to a sample containing a ligand compound under conditions to allow ligation between the receptor and the ligand compound; and c) detecting the bound ligand compound by the desorption spectrometry. In another aspect, this invention provides a classification method for determining whether an agent modulates the binding between an objective analyte and an adsorbent. This is a combinatorial method for drug discovery. The method comprises the steps of: a) supplying a substrate comprising an adsorbent to which the target analyte is attached under an elution condition; b) exposing the substrate to the target analyte and the agent, under the condition of elution, to allow the ligation between the target analyte and the adsorbent; c) detecting the amount of ligation between the target analyte and the adsorbent by the desorption spectrometry; and d) determining whether the measured quantity is erent from a binding control amount, when the substrate is exposed to the target analyte under the elution condition without the agent. A erence between the measured amount and the amount of control indicates that the agent modulates the ligation. In one aspect, this invention provides a method for detecting a genetic package, which contains a polynucleotide encoding a polypeptide agent which specifically binds to a target adsorbent. That is, in one aspect, a combinatorial method for selecting the specific phagocytes of the analyte from a collection of exposure, which includes the use of objective proteins isolated by the mapping of the retentate or the target proteins generated in itself by transcription and translation. vi tro The method comprises the steps of: a) providing a substrate comprising a target adsorbent; b) supplying an exhibit collection comprising a plurality of erent genetic packages, each erent genetic package comprises a polynucleotide that includes a nucleotide sequence encoding a polypeptide agent, and each erent genetic package has a surface on which s exhibits the encoded polypeptide agent; c) exposing the substrate to the display collection under elution conditions, to allow specific ligation between a polypeptide agent and the target adsorbent, whereby a genetic package, which includes the polypeptide agent, is obtained on the substrate, and d) detecting a genetic package retained on the substrate by desorption spectrometry. In one embodiment of this method, the exhibition collection is a phagocyte display collection. And another modality, the phagocyte is M13. In another embodiment, the polypeptide is a single chain antibody. In another embodiment, the target analyte is a polypeptide analyte that is erentially expressed between cells of erent phenotypes. In another embodiment, the substrate comprises a cell or a cell membrane. In one embodiment, the step of supplying the substrate comprises the target adsorbent which includes the steps of: i) providing a substrate comprising or adsorbent, wherein the adsorbent retains an objective analyte, under an elution condition; and ii) exposing the adsorbent to the target analyte, under the elution condition, to allow retention of the target analyte by the adsorbent, whereby the target analyte becomes the target adsorbent. In one embodiment, the target analyte is an objective polypeptide and the step of ii) exposing the adsorbent comprises the step of producing the target polypeptide in itself over the adsorbent, by translating it in vi tro of a polynucleotide encoding the target polypeptide, and may further comprise amplifying the polynucleotide sequence itself on the substrate.

In another embodiment, the substrate comprises (1) an adsorbent that binds to an anchored polypeptide and (2) at least one target genetic package, which has a surface that exhibits the anchored polypeptide and an objective adsorbent polypeptide, this genetic package objectiv comprises a polynucleotide that includes a d-nucleotide sequence encoding the target adsorbent, in which the target genetic package is bound to the adsorbent via the anchorage peptide. In another embodiment, the method further comprises any of the following steps: forming a sequence of nucleotides encoding the polypeptide agent; isolates the retained genetic package or produces the polypeptide agent d. In another aspect, this invention provides a substrate for desorption spectrometry, comprising an adsorbent that binds to an anchorage polypeptide, displayed on a surface of a genetic package, in which the surface of the genetic package also exhibits a target polypeptide and wherein this genetic package comprises a polynucleotide, which includes a nucleotide sequence encoding the target polypeptide. In another aspect, this invention provides a method for detecting the translation of a polynucleotide. The method comprises the steps of: a) supplying a substrat comprising an adsorbent, for use in the desorption spectrometry; b) contacting the substrate with the polynucleotide, which encodes a polypeptide and with agents for translation within the polynucleotide, whereby the polypeptide is produced; c) exposing the substrate to an eluent, to allow retention of the polypeptide by the adsorbent; and d) detects the polypeptide retained by desorption spectroscopy. The detection of the polypeptide provides detection of the translation of the polynucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a substrate containing a plurality of absorbent zones in the form of a belt. This belt contains six different adsorbent assemblies, classified according to their base of attraction. (hydrophobic, ionic, coordinated covalent and mixed function).

The strip contains several zones for each type of adsorbent, which allows the interrogation of the zones at different times, with different eluents, or to archive and par the subsequent analyzes. Figure 2 illustrates an orthogonal array of adsorbents (surface interaction potentials) and predetermined "steerable locations." The arrangement also takes the form of a plate.This arrangement includes various adsorbents.In the exposure to the analyte, each strip can be washed by a variety of eluents (selectively threshold modifiers) The retention analysis under different selectivity conditions results in a retention map or recognition profile Figure 3 is a representation of the quantitative analysis of analytes by desorption of the analyte from given locations in the arrangement and quantitative detection of the analyte desorbed by mass spectrometry d laser desorption.

Figure 4A illustrates an example of a computer system used to run the software (program), which can be used to analyze the data generated by the present invention. Figure 4A shows a computer system 1 d that includes: a monitor 3, a screen 5, u cabinet 7, a keyboard 9 and a mouse (mouse) 11. This mouse 11 can have one or more buttons, such as buttons 13 d mouse. The cabinet 7 houses a CD-ROM drive and a hard drive (not shown) that can be used to store and retrieve computer programs, which includes the code embodying the present invention. Although CD-ROM 17 is shown as a storage medium that can be read by computer, other storage media that can be read by computer, such as floppy disks, DRAM memory, hard drives, volatile memory, tape, etc. can use. Cabinet 7 also accommodates familiar computer components (not shown) as a processor, memory and similar. Figure 4B shows a block diagram of computer system 1, used to run the software, which can be used to analyze the data generated by the present invention. As in Figure 4A, computer system 1 d includes monitor 3 and keyboard 9. Computer system 1 further includes subsystems, such as central processor 102, system memory 104, or input / output controller 106, an exhibit adapter 108, a removable disk 112, a fixed disk 116, network interface 118, and horn 120. The removable disk 112 is representative of removable media that can be read by computer, such as diskettes, tapes, CDs -ROM, removable hard disk, volatile memory, and similar. The fixed disc 116 is representative of an internal hard disk, DRAM memory, or the like. Other computer systems suitable for use with the present invention include additional or minor subsystems. For example, another computer system may include more than one processor 102 (i.e., a multi-processor system) or a cache memory. Figures 5A-5F show retention maps for the lysozyme under selectivity conditions, which include six different adsorbents and several different eluents. Figures 6A-6B show the resolution at a low and high molecular mass of the analytes in the human serum, by an immobilized metal adsorbent. Figures 7A-7B show the resolution at a low and high molecular mass of analytes in human serum by a variety of adsorbents, which use the same eluent. Figures 8A-8B show the resolution at a low and high molecular mass of analytes in the urine of a young child by a variety of adsorbents that use water as the eluent. Figure 9 shows the resolution of analytes in the urine of a young child, using a hydrophobic phenyl adsorbent and three different eluents, resulting in the discovery of selective retention of one of the analytes (*) by the condition of Tween washing. Figures 10A-10D show the resolution of analytes in a cell culture medium of two different breast cancer cell lines. * Figure 11 shows a retention map composed of the urine of a small child exposed to selectivity conditions, defined by six different adsorbents and three different eluents. Figure 12 shows a two-dimensional polyacylamid gel (pl and apparent molecular mass) of a small child's urine. Figure 13 shows a panning method with phagocyte display collections for those phagocytes that has a surface protein that specifically binds to the target analyte. -The substrate illustrated in the upper part shows that even a few specifically bound phagocytes can be detected by desorption spectrometry through the detection of the many coating proteins that phagocyte contains.

In the background, the substrate with several adsorbent zones develops so that the target analyte binds specifically. The phagocyte is exposed to the zones. The bound phagocytes are detected by desorption spectrometry. Phagocytes attached to another area can grow to be isolated. Figure 14 shows how a ligand compound agent, in this case a single chain antibody, identified by a panning method, can be used as an adsorbent to attach the target protein for use in protein interaction studies -protein. An objective is purified in your (zone 2) and used for the panoramic taking of a phagocyte display collection (zone 4). A single chain antibody is isolated and bound to a substrate (zone 6) as an adsorbent. The target is then adsorbed to the single-chain antibody. This objective is now attached for the study of protein-protein interactions (zone 8). Figure 15 shows a method for classifying drug candidates for their ability to interfere with the binding of proteins to a ligand compound, in this case a single chain antibody. A single-chain antibody specific for a target protein is attached to an area on a substrate through, for example, an anti-phagocyte antibody, which, by itself, can be attached through protein A or the protein G. E single chain antibody is exposed to target protein and drug candidates. The ability of the drug to bind to the analyte protein and to interfere with the ligand compound, which binds to the analyte, is monitored by desorption spectrometry. Figure 16 shows a method for classifying drug candidates by the ability to interfere with protein binding to a ligand compound. The method is similar to that illustrated in the previous figure, except that it monitors the ability of the drug to interfere with the analyte bound by the binding, by itself, to the compound ligated by the desorption spectrometry. Figure 17 shows a method for classifying drug candidates for the ability to interfere with the target protein (Target Protein I) that binds to a secondary ligand compound (Target II Protein). As in the two previous figures, the objective is attached to the substrate, which becomes, by itself, an adsorbent for the league compound. In this case, the analyte is attached through a single chain antibody. The goal is then exposed to the ligand compound and the drug candidates. The ability of the drug to interfere with the ligature between the analyte and the ligand compound (for example, by ligation to the target analyte) is monitored by the desorption spectrometry. Figure 18 shows a flow chart starting with the identification of differentially expressed mRNA or polypeptides and ending with the creation of a diagnostic platform to specifically bind the polypeptide for detection by the desorption spectrometry. Figures 19A-19D show a retention map of the Hemophilus lysate on an adsorbent array. Figur 19A: anionic adsorbent; Figure 19B: normal fas absorbent; Figure 19C: Ni (II) adsorbent; Figure 19D: hydrophobic adsorbent. Figures 20A-20C show the progressive resolution of an analyte in the Hemophilus lysate. The adsorbent in each case was an anionic adsorbent. Fig. 20A: In a first stage, after exposure to the sample, the zone was washed with 150 μl of 20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0. In a second stage, the adsorbent and sodium phosphate characteristic of eluent were added to a constant set of characteristics. A new feature of elution s added. Figure 20B: In addition to 20 mM sodium phosphate, p of 7.0, the zone was washed with 0.05% Triton XlOO and 0.15 M d NaCl (150 μl, in total). Figure 20C: In addition to 20 mM sodium phosphate, pH 7.0, the zone was washed with 100 mM imidazole, 0.15 M NaCl (150 μl in total). Figures 21A-21D show the results of a comparison between the components in normal human serum and diseased serum. Figure 21A: Map of the normal serum retentate over a Cu (II) site of the adsorbent array. Figure 2IB: Retentive map of the diseased serum on Cu (II) site of adsorbent array. Figure 21C: Retained analytes from both serum samples were combined in an overlapping manner. To simplify the presentation, each crest of the retained analyte was converted to a bar, the bars in dashes represent the analytes retained from a normal serum and the solid bars represent the analytes retained from a diseased serum. Figure 2ID: To differentiate more clearly the differences between the two samples, a comparison projection was generated, where the ratio of the analytes retained from the samples was calculated and exhibited. The two analytes marked with "*" show significant increases in the sick serum (5 to 10 times of increase). Figures 22A-22D show a comparison of the retenate maps for the control, urine of sick and drug-treated mice, on a Cu (II) adsorbent, and quantification of the amount of a marker in the diseased urine and treated with drug.KER Figures 23A-23D show retinal maps of analytes in the urine of four human cancer patients, shown in the "gel visa" format. The difference maps between patients 1, 2 and 3 showed two common analytes that occur in increasing amounts in these patients. Figures 24A-24E show the detection of the M13 phagocyte by mass spectrometry of laser desorption, through the detection of the protein coat of the gene VIII. Dilutions of 1012 original phagocytes per milliliter vary from 1: 10 to 1: 100, 000,000. Figures 25A-25B show the capture of M13 phagocytes by desorption spectrometry, using the antibody -anti-M13 as an adsorbent. Figure 22 shows the M13 phagocyte captured with crests representing the proteins of gene VIII and gene III. L Figure 22B is a control showing the crests representing the antibody adsorbent (loaded in single and double form). Figures 26A-26D show the adsorption of the M13 phagocyte carrying a sencill anti-tat chain antibody, by the tat protein adsorbent. Simple resistance is shown under phagocyte dilutions of 1: 10 to 1: 10,000.

Figures 27A-27B shows retaining maps of the TGF-β binding to the adjunct TGF-β receptor fusion protein, at 1 μg / ml (Figure 27A) and at 100 ng / ml (Figur 27B). The solid line shows the ligation without the presence of the receptor free of TGF-β. The dashed line shows the ligation in the presence of the TGF-β receptor. Figures 28 to 31 show the resolving power of the retentate chromatography. Figures 28A-28C show the resolution of proteins of the Hemophilus lysate using hydrophobic, cationic and hydrophobic adsorbents.

Cu (II), at molecular masses from 0 kD to 30 kD. Each analyte retained is represented by a bar, the height of the bar represents the intensity of the analyte retained. Figures 29A-29C show the resolution of proteins of the Hemophilus lysate using hydrophobic, cationic and Cu (II) adsorbents, at molecular masses of about 30 kD to 100 kD. Figure 30 shows the combined resolution of 0 kD to 30 kD of the Hemophilus proteins from each of the three adsorbents. Figur 31 shows the combined resolution of 20 kD to 100 kD of the Hemophilus proteins from each of the three adsorbents. Figure 32 shows the ligation of the GST fusion protein to a normal adsorbent.

Figures 33A-33B show the ligation of a specific ligand compound to the GS fusion receptor attached to an array of adsorbent (Figure 33A) and that lacks binding to the ligand compound to a control array that does not include the receptor. GST fusion (Figure 33B).

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS Unless defined otherwise, all technical and scientific terms, used herein, have the meanings commonly understood by a person skilled in the art to which this invention pertains. The following references provide an expert in the subject with a general definition of many of the terms used in this invention. Singleton et al., DICTIONARY OR MlCROBIOLOGY AND MOLECULAR BIOLOGY (2 a Ed., 1994); THE CAMBRIDGE DICTIONARY QF SCIENCE AND TECHNOLOGY (WalEd., 1988); THE GLOSSAR OF GENETICS, 5th ED., R. RIEGER ET AL. (EDS.), SPRINGER VERLA (1991); AND HALE & MARHAM, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, the following terms have the meanings ascribed to them, unless otherwise specified. "Analyte" refers to "a component of a sample, which is conveniently retained and detected." The term "can refer to a single component or a set of components in the sample." Adsorbent "refers to any material capable of adsorbing. An "analyte." The term "adsorbent" is used herein to refer to both a single material ("monoplex adsorbent") (e.g.pic , a compound or functional group) to which the analyte is exposed, and a plurality of different materials ("multiplex adsorbent") to which the sample is exposed. The adsorbent materials in a multiplex adsorbent are referred to as' adsorbent species. "For example, a steerable location on a substrate may comprise a multiplex adsorbent, characterized by many different adsorbent species (e.g., anion exchange materials, chelators or antibodies). , which have different ligation characteristics. "Adsorb" refers to the detectable ligation between an absorbent and an analyte, or before or after washing, with an eluent (selective threshold modifier). "Substrate" refers to a solid phase to which an adsorbent is attached or deposited. "Ligature characteristic" refers to a chemical or physical characteristic that dictates the attraction of an adsorbent by an analyte Two adsorbents have different ligation characteristics if, under same elution conditions, the adsorbents bind the same analyte with different degrees of affinity, ligation characteristics include , for example, the degree of interaction promoted by salt, degree of hydrophobic interaction, degree of hydrophilic interaction, degree of electrostatic interaction and others described here. "Ligation conditions" refers to the binding characteristics to which an analyte is exposed. "Eluent" refers to an agent, tylly a solution, that is used to mediate the adsorption of an analyte to an adsorbent. Eluents are also referred to as "selectivity threshold modifiers". "Elution characteristic", refers to the characteristic that dictates the ability of a particular eluent (selectivity threshold modifier) to mediate the adsorption between an analyte and an adsorbent. Two eluents have different elution characteristics if, when placed in contact with an analyte and adsorbent, the degree of affinity of the analyte for the adsorbent differs. The characteristics of the elution include, for example, the pH, ionic strength, modification of the water structure, detergent strength, modification of the hydrophobic interactions, and others described herein.

"Elution conditions" refers to the elution characteristics to which an analyte is exposed. "Selectivity characteristic" refers to a peculiarity of the combination of an adsorbent having particular ligation characteristics, and an eluent having particular elution characteristics, which dictate the specificity with which the analyte is retained to the adsorbent, after the washing with the eluent. "Selectivity conditions", refers to the characteristics of the selectivity to which an analyte is exposed. "Base for attraction," refers to the chemical and / or physico-chemical properties that cause one molecule to be attracted to another. "Force of attraction" refers to the intensity of the attraction of one molecule to another (also known as affinity). "Solve", "resolution" or "analyte resolution" refers to the detection of at least one analyte in a sample. The resolution includes the detection of a plurality of analytes in a sample by subsequent differential detection separation. The resolution does not require the complete separation of an analyte from all other analytes in a mixture. Rather, any separation that allows the distinction between at least two analytes is sufficient. "High resolution information", refers to the resolution of an analyte in a way that allows not only the detection of the analyte, but also at least a physical-chemical property of the analyte to be evaluated, for example, the molecular mass . "Desorption Spectrometry" refers to the method for detecting an analyte in which this analyte is exposed to energy that desorbs the analyte from a stationary phase in a gas phase, and the analyte desorbed or a distinguishable portion thereof, is detected directly by a detector, without an intermediate step of capturing the analyte in a second stationary phase. "Detect" refers to identifying the present, absence or quantity of the object to be detected. "Retention" refers to an adsorption of an analyte by an adsorbent, after washing with an eluent. "Retention data" refers to the data indicating the detection (which optionally includes the detection mass) of a retained analyte, under a particular selectivity condition. "Retention map" refers to a retention datum specifying a set of values for an analyte retained under a plurality of selectivity conditions. "Recognition profile" refers to a relative retention that specifies a set of values of an analyte under a plurality of selectivity conditions. "Complex" refers to analytes formed by the union of 2 or more analytes. "Fragment" refers to the products of the chemical, enzymatic or physical decomposition of an analyte. The fragments can be in a neutral or ionic state. "Differential expression" refers to a detectable difference in the quantitative qualitative presence of an analyte. "Biological sample", refers to a sample derived from a virus, cell, tissue, organ or organism, including, without limitation, lysates or homogenates of cells, tissues or organs, or samples of bodily fluids, such as blood, urine or cerebro-spinal fluid. "Organic biomolecules" refers to an organic molecule of biological origin, for example, steroids, amino acids, nucleotides, sugars, polypeptides, polynucleotides, complex carbonates or lipids.

"Small organic molecule" refers to organic molecules of a size comparable to those organic molecules generally used in pharmaceutical products. The term excludes organic biopolymers (eg proteins, nucleic acids, etc.). Preferred small organic molecules vary in size from about 5000 Da, up to about 2000 Da or up to about 1000 Da. "Biopolymer" refers to a polymer, of biological origin, for example polypeptides, polynucleotides, polysaccharides or polyglycerides (such as di- or triglycerides). "Polypeptide", refers to a polymer compound of amino acid residues, related to naturally occurring structural variants, and their synthetic analogues that occur not naturally, linked by means of peptide ligations, related to naturally occurring structural variants and their synthetic analogs. they happen .or naturally. Synthetic polypeptides can be synthesized, for example, using an automatic polypeptide synthesizer. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides.

"Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogues include those that are bases of unnatural occurrence, nucleotides that attach to bonds with other nucleotides, in addition to the naturally occurring phosphodiester bonds, or which include bases bound through bonds, in addition to the bonds of phosphodiester. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoroamidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAc), and the like. . Such polynucleotides can be synthesized, for example, using an automatic AD synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally not greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), it also includes an RNA sequence (ie, A, U, G, C), wherein "U" replaces "T".

"Detectable part" or a "label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include 2P, Ds, fluorescent dyes, dense electron reagents, enzymes (eg, as commonly used in an ELISA assay), biotin-streptavadine, dioxigenin, "haptens and proteins, for which antisera are available or monoclonal antibodies, or nucleic acid molecules, with a sequence complementary to a target.The detectable part often generates a measurable signal, such as a radioactive, chromogenic or fluorescent signal, which can be used to quantify the amount of the detectable part bound in a sample The detectable part can be incorporated in or bound to a size or probe, in covalent form or - through ionic bonds, van der Waals or hydrogen, for example, the incorporation of radioactive nucleotides , or biotinylated nucleotides, which is recognized by streptavidin.The detectable part may be directly or indirectly detectable. the union of a second part detectable, directly or indirectly, to the first detectable stop. For example, the detectable part can be a ligated compound or a ligation partner, such as biotin, which is a binding partner for streptavidin, or a nucleotide sequence, which is a binding partner for a complementary sequence, to which it can specifically hybridize The binding partner can itself be directly detectable, for example, an antibody can itself be labeled with a fluorescent molecule.The binding partner can also be indirectly detectable, for example, an acid nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule, which can also be detected through hybridization with other labeled molecules of the nucleic acid (See, for example, PD Fahrlander and A Klausner, Bio / Technology (1988) 6: 1165.) The quantification of the signal is achieved, for example, by the scintillation count, the densitometer, or the flow cytometer. "Plurality" means at least two. "Purify" or "purification" means the removal of at least one contaminant from the composition to be purified. The purification does not require that the purified compound be 100% pure. A "ligate" or "ligate compound" is a compound that specifically binds to a target molecule. A "receptor" is a compound that binds specifically to a ligand compound.

"Antibody" refers to a polypeptide ligand compound, substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an epitope (e.g., an antigen). The recognized immunoglobulin genes include the genes of the kappa or lambda light chain constant region, the heavy chain constant region gene, alpha, gamma, delta, epsilon and mu, and the immunoglobulin myriad variable region genes. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments, produced by digesting with various peptidases. They include, for example, the Fab 'and F (ab)' fragments. The term "antibody", as used herein, also includes fragments of antibody produced by the modification of whole antibodies or those synthesized de novo using the methodologies of the invention.

Recombinant DNA They also include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies. The "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain comprising one or more heavy chain constant region domains, CH.l r CH2, CH3, but does not include the heavy chain variable region.

A ligand or receptor compound (eg, antibody) "binds specifically to", or "is specifically immunoreactive with," a compound analyte when the ligand compound or receptor functions in a binding reaction that is determinative of the presence of the analyte in a sample of heterogeneous compounds. Thus, under the conditions of the designated assay (eg, immunoassay), the ligand compound or the receptor is preferably linked to a particular analyte and does not bind in a significant amount to other compounds present in the sample. a polynucleotide specifically binds under hybridization conditions to an analyte polynucleotide comprising a complementary sequence, an antibody binds specifically under immunoassay conditions to an antigen analyte carrying an epitope against which the antibody has been elevated, and an adsorbent It specifically binds an analyte under appropriate elution conditions. "Agent" refers to a chemical compound, a mixture of chemical compounds, a sample of an undetermined composition, a small molecule combinatorial arrangement, a biological macromolecule, a collection of bacteriophage peptides, a collection d exhibition of a bacteriophage antibody (for example, scFV) a display collection of polysome peptides or extract obtained from biological materials, such as bacteria, plasma, fungi, or animal cells and tissues. Appropriate techniques involve the selection of collections of recombinant antibodies in phagocyte or similar vectors. See, Huse t al (1980), Science 246: 1275-1281, and Ward et al (1989) Nature 341; 544-546. The protocol described by Huse is made more efficient in combination with phagocyte display technology. See, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. "Recombinant polynucleotide" refers to a polynucleotide that has sequences that are not joined together naturally. An amplified or assembled recombinant polynucleotide can be included in a suitable vector and the vector can be used to transform a suitable host cell. A host cell comprising the recombinant polynucleotide is referred to as a "recombinant host cell". The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide." A recombinant polynucleotide can serve with a non-coding function (eg, promoter, replication origin, ribosome binding site, etc.) likewise. Suitable unicellular hosts include any of those routinely used in expressing polynucleotides from eukaryotes or mammals, including, for example, prokaryotes, such as E. coli and eukaryotes, including, for example, fungi, such as yeasts; and mammalian cells, which include insect cells (e.g., Sf) and animal cells, such as CHO, Rl.l, BW, LM, African Green Monkey Kidney cells (e.g., COS 1, COS 7 , BSC 1, BSC 40 and BMT 10) and cultured human cells. The "expression control sequence" refers to a nucleotide sequence in a polynucleotide, which regulates the expression (transcription and / or translation) of a nucleotide sequence operably linked to it. "Operationally linked" refers to a functional relationship between two par in which the activity of a party (for example the ability to regulate transcription) results in an action on the other party (for example the transcription of the sequence). Expression control sequences may include, for example and without limitation, promoter (eg, inducible, repressible or constitutive) promoter sequences, enhancers, transcription terminators, a starting codon (i.e., ATG), splice signals for introns, and stop codons. "Expression vector" refers to a vector comprising a recombinant polynucleotide that includes expression control sequences, operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression, other expression elements can be supplied by the host cell or the expression system in vi tro. Expression vectors include all those known in the art, such as cosmidas, plasmids (e.g., uncoated or contained in liposomes) and viruses that incorporate the recombinant polynucleotide. "Coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA, or a mRNA, to serve as models for the synthesis of other polymers and macromolecules in biological processes, which have either a defined nucleotide sequence (ie, a rRNA, tRNA and mRNA) or --- a defined sequence "of amino acids and the biological proper that result from them.So, a gene that encodes a protein if the transcription and translation of mAR "produced by that gene produces the protein in a cell or other biological system." Both the coding strand, whose nucleotide sequence is identical to the sequence of the mAR? and usually provided in the sequence list, as the uncoded cord, used As the model for the transcription, of a gene or cAD ?, can be referred to as the coding of the protein another product of that gene or cAD ?. Unless specified otherwise anera, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleotide sequences that encode proteins and RA can include the introns. The "molecule that absorbs energy" refers to a molecule that absorbs energy from an energy source in a desorption spectrometer, thus enabling the desorption of the analyte from a probe surface. Molecules that absorb energy used in MALDI are frequently referred to as a "matrix." The cinnamic acid, cinnapinic acid and dihydroxybenzoic acid derivatives are frequently used as molecules that absorb energy in the laser desorption of bioorganic molecules.

II. CHROMATOGRAF A OF THE RETENATE The chromatography of the retenate is a method for the multidimensional determination of analytes in a sample. The method involves: (1) selectively adsorbing analytes from a sample to a substrate, under a plurality of different combinations of adsorbent / eluent ("selectivity conditions") and (2) detecting retention of analytes adsorbed by desorption spectrometry. Each selectivity condition provides a first separation and separation dimension of adsorbed analytes from those that do not adsorb. The desorption mass spectrometry provides a second separation dimension and the separation of the analytes adsorbed with each other, according to the mass. Because the chromatography of the retenate involves using a plurality of different selectivity conditions, many separation dimensions are achieved. The relative adsorption of one or more analytes, under selectivity conditions, can also be determined. The multidimensional separation provides both the resolution of analytes and their characterization. In addition, the analytes, thus separated, remain attached in a retentive map, which can be corrected for further manipulation to examine, for example, the structure of the analyte and / or function. Likewise, the adjunct analytes can by themselves be used as adsorbents to bind to other analytes exposed to the substrate. In summary, the present invention provides a fast, multidimensional resolution and high information of the analytes. The method can take several forms. In one embodiment, the analyte is adsorbed to two different adsorbents at two physically different locations and each adsorbent is washed with the same eluent (selectivity threshold modifier). In another embodiment, the analyte is adsorbed in the same adsorbent in two different physical locations and washed with two different eluents. In another embodiment, the analyte is adsorbed to two different adsorbents in different physical locations and washed with two different eluents. In another embodiment, the analyte is adsorbed to an adsorbent and washed with a first eluent, and retention is detected: then, the adsorbed analyte is washed with a second, different eluent, and subsequent retention is detected.

A. METHODS OF CARRYING OUT THE RETENANT CHROMATOGRAPHY 1. Exposure of the Analyte to the Conditions of Selectivity to. Preparation of the substrate When carrying out the chromatography of the retenate, or analyte, which is retained by an adsorbent, it is presented to an energy source on a substrate. A sample containing the analyte can make contact with the adsorbent, before or after this adsorbent is fixed to the substrate, which will serve to present the analyte to the desorption medium. For contact purposes, the adsorbent may be in liquid or solid form (ie, on a substrate or solid phase). Specifically, the adsorbent may be in the form of a solution, suspension, dispersion, water-in-oil emulsion, oil-in-water emulsion, or microemulsion. When the adsorbent is provided in the form of a suspension, dispersion, emulsion or microemulsion, a suitable surfactant agent may also be present. In this embodiment, the sample can make contact with the adsorbent by mixing a liquid sample with the liquid adsorbent. Alternatively, the sample can be provided on a solid support and the contact will be achieved by bathing, soaking or immersing the solid support containing the sample in the liquid adsorbent. In addition, the sample may contact by spraying or washing on the solid support with the liquid adsorbent. In this embodiment, different adsorbents can be provided in different containers. In one embodiment, the adsorbent is provided on a substrate. The substrate can be any material that is capable of binding or retaining the adsorbent. Typically, the substrate is comprised of glass, ceramics, electrically conductive polymers (for example, carbonized PEEK), TEFLON®-coated materials, organic polymers, native biopolymers, metals (for example nickel, brass, steel or aluminum), films, porous and non-porous globules of interlaced polymers ( for example, agarose, cellulose or dextran), other insoluble polymers, or combinations thereof. In one embodiment, the substrate takes the form of a probe or an element presenting a sample, which is inserted into a desorption detector. For example, with reference to Figure 1, the substrate may take the form of a belt. The adsorbent can be nested to the substrate in the form of a linear array of zones, each of which can be exposed to the analyte. Sal strips may be joined together so that the plurality of adsorbents forms an array 30 having discrete zones in defined rows. The substrate may also be in the form of a plate having an array of horizontal and vertical rows of adsorbents, which form a regular geometric pattern, such as a square, rectangle or circle. The probes can be produced as follows. The substrate can be any solid material, for example, stainless steel, aluminum or a silicon wafer. U metal substrate can then be coated with a material that allows the derivation of the surface. For example, a metal surface can be coated with silicon oxide, titanium oxide or with gold. The surface is then derived with a difunctional linker. This linker includes at one end a functional group that can covalently bind with a functional group on the surface. Thus, the functional group may be an inorganic oxide or a sulfhydryl group for gold. The other end of the linker generally has an amino functionality. Useful bifunctional linkers include aminopropyl triethoxysilane or aminoethyl disulfide. Once bound to the surface, the linkers are subsequently derived with groups that function as the adsorbent. In general, the sorbent is added to locations' blinking on the probe. In a type of probe zones of about 3 mm in diameter, they are arranged in an orthogonal arrangement. Absorbents can, by themselves, be part of bifunctional molecules that contain a group reactive with the available amino group and the functional group that acts as the adsorbent. Functional groups include, for example, the normal phase (silicon oxide), rsed phase (C18 aliphatic hydrocarbon), quaternary amine and sulfonate.

Likewise, the surface can be further derivatized with other bifunctional molecules, such as carbodiimide and N-hydroxysuccinimide, creating an activated preform. These preforms can be functionalized with bioorganic adsorbents (eg, nucleic acids, antibodies, other protein ligand compounds). The biopolymers can bind the functional groups on the preforms, through amine residues or sulfhydryl residues. In one embodiment, the adsorbents are bound to entangled polymers (e.g. films) which are themselves bonded to the surface of the probe through the available functional groups. Such polymers include, for example, cellulose, dextran, carboxymethyl-dextran, polyacrylamide and mixtures thereof. The probes with attached adsorbents are easy to use. In another embodiment, the adsorbent is attached to the first substrate to provide a solid phase, such as polymeric or glass beads, which is subsequently placed on a second substrate, which functions as the means to present the sample to the desorption energy of the detector. of desorption. For example, the second substrate may be in the form of a plate, which has a series of cavities in predetermined steerable locations. These cavities function as containers for a first substrate, derived with the adsorbent, for example the polymeric globules derived with the adsorbent. An advantage of this embodiment is that the analyte can be adsorbed to the first substrate in a physical context, and transferred to the substrate that presents the sample for analysis and desorption spectrometry. Typically, the substrate is adapted for use with the detectors employed in the methods of the present invention, to detect the analyte bound to and retained by the adsorbent. In one embodiment, the substrate can be inserted, removably, in a desorption detector, where a source of energy can collide with the area desorbing this analyte. The substrate may be suitable for mounting on a carriage that can be moved horizontally and / or vertically, which moves the substrate horizontally and / or vertically to successive positions, each predetermined adsorbent location on a trajectory for interrogation by the source of energy and the detection of the analyte there united. The substrate may be in the form of a conventional mass spectrometry probe. The strips, plates or probes of the substrate can be produced using conventional techniques. Next, the adsorbent can be directly or indirectly coupled, adapted or deposited on the substrate, before contact with the sample containing the analyte. The adsorbent can be coupled, directly or indirectly, to the substrate by any suitable means of attachment or immobilization. For example, the adsorbent can be coupled directly to the substrate, by deriving the substrate with the adsorbent to directly attach the adsorbent to the substrate through the covalent or non-covalent ligand. The attachment of the adsorbent to the substrate can be achieved through a variety of mechanisms. The substrate can be derived with a completely prepared adsorbent molecule, by attaching the previously prepared adsorbent molecule to the substrate. Alternatively, the adsorbent can be formed on the substrate by having a precursor molecule to the substrate and subsequently adding additional precursor molecules to the growing chain bound to the substrate by the first precursor molecule. This mechanism of construction of the adsorbent on the substrate is particularly useful when the adsorbent is a polymer, particularly a biopolymer, such as a DNA or APJST molecule. A biopolymer adsorbent can be provided by successively adding bases to the first base attached to the substrate, using methods known in the art of oligonucleotide fragment technology. See, for example, U.S. Patent No. 5,445,934 (Fodor et al). As can be seen from Figure 2, a few like two, and many like 10, 100, 1000, 10,000, or more adsorbents, can be attached to a single substrate. The size of the adsorbent site can be varied, depending on the experimental design and purpose. However, it is not necessary to be greater than the diameter of the energy source that collides (for example the diameter of the laser zone). The zones may contain the same or different adsorbent. In some cases, it is advantageous to deliver the same adsorbent at multiple locations on the substrate to allow evaluation against a plurality of different eluents or so that the bound analyte can be preserved for future use or reference, perhaps in secondary processes. By providing a substrate with a plurality of different adsorbents, it is possible to use the plurality of binding characteristics provided by the combination of different adsorbents with respect to a single sample and thus to bind and detect a wider variety of different analytes. The use of a plurality of different adsorbents on a substrate, for the evaluation of a single sample, is essentially equivalent to concurrently conducting multiple chromatographic experiments, each with a different column of chromatography, but the present method has the advantage of requiring only one simple system. When the substrate includes a plurality of adsorbents, it is particularly useful to supply the adsorbents in predetermined steerable locations. By supplying the adsorbents in predetermined steerable locations, it is possible to wash an adsorbent in a first predetermined steerable location, with a first eluent and wash an adsorbent in a second predetermined steerable location with a second eluent. In this way, the binding characteristics of a single absorber for the analyte can be evaluated in the presence of multiple eluents, which each selectively modify the binding characteristics of the adsorbent in a different manner. The steerable locations can be arranged in any pattern, but preferably in regular patterns, such as in lines, orthogonal arrays, or regular curves, such as in circles. Similarly, when the substrate includes a plurality of different adsorbents, it is possible to evaluate a single eluent with respect to each different adsorbent, in order to evaluate the binding characteristics of a given adsorbent in the presence of the eluent. It is also possible to evaluate the binding characteristics of different adsorbents in the presence of different eluents. (1) Incremental or Gradient Adsorbent Surfaces A series of adsorbents, having different binding characteristics, can be provided by synthesizing a plurality of different polymeric adsorbents in the substrate. The different polymeric adsorbents can be provided by attaching a precursor molecule to the substrate, initiating the polymerization reaction and terminating this polymerization reaction in various degrees of complete state for each adsorbent. Likewise, the terminal functional groups in the polymers can be reacted to chemically derive them to various degrees with different affinity reagents (eg, -NH3, or COO.) Upon completion of the polymerization or the bypass reaction, the adsorbents in various degrees of polymerization or derivation, are produced. The various degrees of polymerization or derivatization provide different binding characteristics for each different polymeric adsorbent. This embodiment is particularly useful for providing a plurality of different adsorbents of biopolymers on a substrate. If desired, the polymerization reactions can be carried out in a reaction vessel, rather than in the substrate itself, for example, polymeric adsorbents of various binding characteristics can be provided by removing an aliquot of product from the container. of reaction, as the polymerization / derivatization reaction is proceeding. The aliquots, which have been extracted at various points during the polymerization / derivatization reaction, will exhibit various degrees of polymerization / derivatization to supply a plurality of different adsorbents. The different aliquots of the product can then be used as adsorbents having different binding characteristics. Alternatively, a plurality of different adsorbents can be provided by repeating in sequence the steps of terminating the reaction, removing an aliquot of the product, and restarting the polymerization / derivatization reaction. The products extracted at each termination point will exhibit various degrees of polymerization / derivation and as a result will provide a plurality of adsorbents having different binding characteristics.

In one embodiment, a substrate is provided in the form of a strip or a plate, which is coated with adsorbent, in which one or more bonding characteristics vary in a two-dimensional gradient. For example, a belt having an adsorbent that is weakly hydrophobic at one end and strongly hydrophobic --- at the other end is supplied. Or, a plate is provided which is weakly hydrophobic and anionic in one corner, and strongly hydrophobic and anionic in the opposite corner diagonally. Such adsorption gradients are useful in the qualitative analysis of an analyte. Adsorption gradients can be made by one. controlled spraying application or by the flow of material through the surface in a time-dependent manner, to allow the reaction to be completed in an incremented manner over the gradient dimension. This process can be repeated, at right angles, to provide orthogonal gradients of similar or different adsorbents, with different binding characteristics. The sample containing the analyte can be contacted with the adsorbent, or before or after the adsorbent is placed on the substrate, using any suitable method, which can bind between the analyte and the adsorbent. The adsorbent can simply be mixed or combined with the sample. The sample may be contacted with the adsorbent by bathing or soaking the substrate in the sample, or immersing the substrate in the sample, or spraying the sample onto the substrate, by washing the sample on the substrate, or by generating the sample or analyte in contact with the adsorbent. In addition, the sample may be contacted with the adsorbent by solubilizing the sample in or mixing the sample with an eluent and contacting the solution of the eluent and sample to the adsorbent, using any of the above techniques (i.e., bath, soak, dipping, spraying or washing on it). b. Analyte to Adsorbent Contact Exposure of the sample to an eluent, before binding the analyte to the adsorbent, has the effect of modifying the selectivity of the adsorbent, while simultaneously contacting the sample with the adsorbent. These components of the sample that will bind to the adsorbent and thus be retained, will include only those components that bind to the adsorbent in the presence of the particular eluent, which has been combined with the sample, rather than all of the components that will bind to the adsorbent. in the absence of the elution characteristics that modify the selectivity of the adsorbent. The sample must make contact with the adsorbent for a sufficient period of time to allow the analyte to bind to the adsorbent. Typically, the sample contacts the analyte for a period between about 30 seconds and about 12 hours. Preferably, the sample contacts the analyte for a period between about 30 seconds and 15 minutes. The temperature at which the sample contacts the adsorbent is a function of the particular sample and the selected adsorbents. Typically, the sample contacts the adsorbent under ambient temperature and pressure conditions, however, for some samples, a modified temperature (typically 4 to 37 ° C) and pressure conditions may be desirable, and will be easily determined by the experts in the field. Another advantage of the present invention over conventional detection techniques is that the present invention makes possible numerous different experiments performed with very small amounts of sample. In general, a sample volume containing a few atom atoms up to 100 picomoles of analyte in about 1 to 500 μl is sufficient to bind to the adsorbent. The analyte can be preserved for future experiments, after binding to the adsorbent, because any location of adsorbent that does not undergo the desorption and detection steps of all retained analytes will retain the analyte there. Therefore, in the case where only a very small fraction of sample is available for analysis, the present invention provides the advantage of performing a multitude of experiments with different adsorbents and / or eluents, to be carried out at different times without wasting samples c. Washing of the adsorbent with eluents After the sample is contacted with the analyte, which results in the binding of the analyte to the adsorbent, the adsorbent is washed with the eluent. Typically, to provide a multi-dimensional analysis, each location of the adsorbent is washed with at least one first and second different eluents. Washing with the eluents modifies the population of the analyte retained in a specific adsorbent. The combination of the binding characteristics of the adsorbent and the elution elution characteristics provide the selectivity conditions that control the analytes retained by the adsorbent after washing. Thus, the washing step selectively removes the components of the sample from the adsorbent. The washing step can be carried out using a variety of techniques. For example, as noted above, the sample can be solubilized in or mixed with the first eluent, before contact of the sample with the adsorbent. The exposure of the sample to the first eluent before, or simultaneously with, the contact of the sample to the adsorbent, has, in a first approximation, the same net effect as the binding of the analyte to the adsorbent and the subsequent washing of the adsorbent with the first eluent After the combined solution is contacted with the adsorbent, this adsorbent can be washed with the second eluent or subsequent eluents. The washing of an adsorbent that has the analyte attached can be achieved by bathing, soaking or immersing the substrate, which has the adsorbent and analyte attached, in u eluent; or the rinse, sprayed or laid on the substrate with the eluent. The introduction of the eluent to small diameter areas of the affinity reagent is best achieved by a microfluidic process. When the analyte is bound to the adsorbent in only one location and a plurality of different eluents are employed in the washing step, the information regarding the selectivity of the adsorbent, in the presence of the individual catalyst, can be obtained. The analyte bound to the adsorbent in one location can be determined after each wash with the eluent, following a repeated pattern of washing with a first eluent, desorbing by detecting the retained analyte, followed by washing with a second eluent, and desorbing and detecting the analyte retained. The washing steps followed by the detection desorption can be repeated for a plurality of different eluents, using the same adsorbent. In this way, the adsorbent with the analyte retained in a single location can be reexamined with a plurality of different eluents, to provide a collection of information with respect to the analytes retained after each individual wash. The above method is also useful when supplying the adsorbents in a plurality of predetermined steerable locations, when the adsorbents are all the same or different. However, when the analyte binds the same or different adsorbents in a plurality of locations, the washing step can alternatively be carried out using a more systematic and efficient approach, involving parallel processes. That is, the washing step can be carried out by washing an adsorbent in a first location with the eluent, then washing a second adsorbent with the eluent, then desorbing and detecting the analyte retained by the first adsorbent and then desorbing and detecting the analyte retained by the second adsorbent. In other words, all the adsorbents are washed with the eluent and then the analyte retained by each is desorbed and detected for each location of the adsorbent. If desired, after detection at each adsorbent location, a second washing step for each adsorbent location can be performed followed by the second desorption and detection step. The washing steps of all the adsorbent locations, followed by the desorption and detection of each location of the adsorbent can be repeated for a plurality of different eluents. In this way, the entire array can be used to efficiently determine the character of the analytes in a sample. The method is useful when all locations of adsorbents are washed with the same eluent in the first wash step or when the plurality of adsorbents are washed with a plurality of different eluents in the first wash stage. 2. Detection The analytes retained by the adsorbent, after washing, are adsorbed to the substrate. The analytes retained on the substrate are detected by the desorption spectrometry: desorption of the analyte from the adsorbent and directly detecting the desorbed analytes. to. Desorption methods Desorption of the analyte from the adsorbent involves exposure of the analyte to an appropriate energy source. Usually, this means the shock of the analyte with radiant energy or energetic particles. For example, energy can be light energy in the form of laser energy (eg, UV laser) or energy from a flash lamp. Alternatively, the energy can be a stream of fast atoms. The heat can also be used to induce / help desorption. Methods for desorbing and / or ionizing analytes for direct analysis are well known in the art. One such method is named desorption / ionization of matrix-assisted laser or MALDI. In MALDI, the analyte solution is mixed with a matrix solution and the mixture is allowed to crystallize after it has been deposited on an inert probe surface, trapping the analyte within the crystals can enable desorption. The matrix is selected to absorb the laser energy apparently imparting it to the analyte, resulting in desorption and ionization. In general, the matrix absorbs in the UV range. This MALDI for large proteins is described in, for example, U.S. Patent No. 5,118,937 (Hillenkamp et al) and U.S. Patent No. 5,045,694 (Beavis and Chait). Surface enhanced laser desorption / ionization, or SELDI, represents a significant advance over MALDI, in terms of specificity, sensitivity selection. SELDI is described in U.S. Patent No. 5,719,060 (Hutchens and Yip). SELDI is a solid phase method for desorption, in which the analyte is presented to the energy stream on a surface that increases the capture and / or desorption of the analyte. In contrast, MALDI is a liquid phase method where the analyte is mixed with a liquid material that crystallizes around the analyte. A version of SELDI, named SEAC (Augmented Surface Affinity Capture), involves presenting the analyte to the desorption energy, - in association with an affinity capture device (ie, or adsorbent). It has been found that when the analyte is thus adsorbed, it can be presented to the desorption energy source with a greater opportunity to achieve the desorption of the target analyte. A material that absorbs energy can be added to the probe to help desorption. Then the probe is presented to the energy source to desorb the analyte. Another version of SELDI, named SEND (Surface Augmented Net Desorption), involves the use of a layer of energy-absorbing material, on which the analyte is placed. A substrate surface comprises a layer of molecules that absorb energy, chemically bound to the surface and / or essentially free of crystals. The analyte is then applied alone (ie, net) to the surface of the layer, without being substantially mixed with it. The molecules that absorb energy, as the matrix does, absorb the energy of desorption and cause the analyte to be desorbed. This improvement is substantial, because the analytes can now be presented to the energy source in a simpler and more homogeneous way, because the performance of the solution mixtures and the random crystallization are eliminated. This provides more consistent and predictable results that enable the automatic form of the process. The energy absorbing material can be a conventional matrix material or it can be a matrix material whose pH has been neutralized or brought to a basic range. Molecules that absorb energy can be linked to the probe through covalent or non-covalent resources. Another version of the SELDI, named SEPAR (Union and Photolabile Surface Released Release), involves the use of photolabile binding molecules (unstable to light). A photolabile binding molecule is a divalent molecule having a site covalently bound to a solid phase, such as a flat probe surface or other solid phase, such as a globule, which may be part of the probe, and a second site that it can be covalently bound to the affinity reagent or analyte. The photolabile binding molecule, when both the surface and the analyte are bound, also contains a photolabile bond which can release the affinity reagent or analyte upon exposure to light. This photolabile linkage can be within the binding molecule or at the binding site to either the analyte (or affinity reagent) or the surface of the probe. b. Method for Direct Detection of Analytes The desorbed analyte can be detected by any of several means. When the analyte is ionized in the desorption process, such as laser desorption / ionization mass spectrometry, the detector can be an ion detector. Mass spectrometers generally include means for determining the time of flight of the desorbed ions. This information is converted to mass. However, one does not need to determine the mass of the desorbed ions to solve and detect them; The fact that the ionized analytes collide with the detector at different times provides the detection and resolution of them. Alternatively, the analyte can be labeled as detectable with, for example, a fluorescent part or a radioactive part. In these cases, the detector can be a fluorescence or radioactivity detector. A plurality of detection elements can be arranged in series to completely interrogate the components of the analyte and the function associated with the retentate at each location in the array.

Desorption Detectors Desorption detectors comprise elements for desorbing the analyte form from the adsorbent and elements for directly detecting the desorbed analyte, i.e., the desorption detector detects the desorbed analyte without an intermediate step of capturing the analyte in The detection of an analyte will normally involve the detection of signal strength, which, in turn, reflects the amount of analyte adsorbed to the adsorbent. Desorption can also have other elements, one of which is to accelerate the analyte desorbed to the detector, another is to determine the analyte's time of flight from desorption to detection by the detector. laser desorption / ionization mass, which is well known in the art.The mass spectrometer includes a port a in which the substrate carrying the adsorbed analytes, eg a probe, is inserted. Desorption is achieved by colliding the analyte with energy, such as laser energy. The device may include an element to move the surface, so that any area in the array is carried in line with the laser beam.

The collision of the analyte with the laser results in the desorption of the intact analyte in the flight tube and its ionization. The flight tube generally defines a vacuum space. The plates electrified in a portion of the vacuum tube create an electrical potential that accelerates the ionized analyte to the detector. A clock measures the time of flight and the electronic parts of the system determine the speed of the analyte and convert it into mass.As any person skilled in the art understands, any of these elements can be combined with the other elements described here, and the set of desorption detectors, which employ various means of desorption, acceleration, detection, time measurement, etc.

B. Selectivity conditions An advantage of the invention is the ability to expose the analytes to a variety of different binding and elution conditions, thus providing both the increased resolution of the analytes and the information about them in the form of a recognition profile. As in conventional chromatography methods, the capacity of the adsorbent to retain the analyte is directly related to the attraction or affinity of the analyte for the adsorbent, when compared to the attraction or affinity of the analyte for the eluent or the eluent for the adsorbent. . Some components of the sample may have no affinity for the adsorbent and, therefore, will not bind to the adsorbent, when the sample is contacted with this adsorbent. Due to their inability to bind to the adsorbent, these components will be separated immediately from the analyte to be resolved. However, depending on the nature of the sample and the particular adsorbent used, a number of different components can be attached initially to the adsorbent. 1. Adsorbents Adsorbents are the materials that bind analytes. A plurality of adsorbents can be used in the chromatography of the retenate. The different adsorbents may exhibit excessively different binding characteristics, somewhat different binding characteristics, or subtly different bonding characteristics. Adsorbents that exhibit excessively different binding characteristics typically differ in their bases of attraction or mode of interaction. The basis of attraction is usually a function of chemical or biological molecular recognition. Bases of attraction between the adsorbent and analyte include, for example, (1) a salt-promoted interaction, for example hydrophobic interactions, thiophilic interactions and immobilized dye interactions; (2) hydrogen bonding and / or van der Waals force interactions and charge transfer interactions, such as in the case of hydrophilic interactions; (3) electrostatic interactions, such as interactions of ionic charges, particularly interactions of ionic charges, positive or negative; (4) the ability of the analyte to form coordinated covalent bonds (i.e., the formation of coordination complexes) with a metal ion in the adsorbent; (5) the binding of the enzyme active site; (6) reversible covalent interaction, for example, disulfide exchange interactions; (7) glycoprotein interactions; (8) biospecific interactions; or (9) combination of two or more of the above interaction modes. That is, the adsorbent can exhibit two or more bases of attraction, and thus it will be known as a "mixed functionality" adsorbent. to. Interaction Adsorbents Promoted by Salt Adsorbents that are useful for observing salt-promoted interactions include hydrophobic interaction adsorbents. Examples of hydrophobic interaction adsorbents include matrices having aliphatic hydrocarbons, specifically C1-C18 aliphatic hydrocarbons; and matrices having functional groups of aromatic hydrocarbons, such as phenyl groups. The binding analytes with hydrophobic interaction adsorbents, which include the amino acid residues exposed to uncharged solvents, and specifically the amino acid residues which are commonly referred to as non-polar, aromatic and hydrophobic amino acid residues, such as phenylalanine and tryptophan. Specific examples of analytes that bind to hydrophobic interaction adsorbents include lysozyme and DNA. Without wishing to be bound by a particular theory, it is believed that the DNA binds to the hydrophobic interaction adsorbents for the aromatic nucleotides in the DNA, specifically the purine and pyrimidine groups. Other adsorbents useful for observing salt promoted interactions include thiophilic interaction adsorbents, such as, for example, T-GEL®, which is a type of thiophilic adsorbent, commercially available from Pierce, Rockford, Illinois. The thiophilic interaction adsorbents bind, for example, to immunoglobulins, such as IgG. The mechanism of interaction between IgG and T-GEL® is not completely understood, but the residues of trp exposed to solvent are suspected to play a certain role. A third adsorbent that involves ionic interactions promoted by salt and also hydrophobic interactions, include the interaction adsorbents of immobilized dyes. These immobilized dye interaction adsorbents include immobilized dye matrices such as, for example, CIBACHRON ™ blue, available from Pharmacia Biotech, Piscataway, New Jersey. The interaction adsorbents of immobilized dyes bind generally to proteins and DNA. A specific example of protein that binds to the immobilized dye interaction adsorbent is bovine serum alumina (BSA). b. Interaction Adsorbents Hydrophilic Adsorbents that are useful in observing hydrogen bonding and / or van der Waals forces, based on the hydrophilic interactions, include surfaces comprising the normal phase adsorbents, such as silicon oxide (ie, glass). The normal phase or surface of silicon oxide, act as a functional group. In addition, adsorbents comprising surfaces modified with hydrophilic polymers, such as polyethylene glycol, dextran, agarose or cellulose, can also function as hydrophilic interaction adsorbents. Most proteins will bind to the hydrophilic interaction adsorbents, due to a group or combination of amino acid residues (ie, hydrophilic amino acid residues), which bind through hydrophilic interactions involving the hydrogen bonding. or the van der Waals forces. Examples of proteins that will bind to the hydrophilic interaction adsorbents include myoglobin, insulin and cytochrome C. In general, proteins with a high proportion of polar or charged amino acids will be retained on the hydrophilic surface. Alternatively, glycoproteins with hydrophilic sugar parts exposed on the surface also have high affinity for hydrophilic adsorbents. c. Electrostatic Interaction Adsorbents Adsorbents that are useful in observing interactions of electrostatic or ionic charges include adsorbents such as, for example, sulfate anion matrices (ie, S03) and carboxylate anion matrices (ie, COO), or anions of phosphate (0P03). Matrices that have sulfate anions are negatively charged permanently. However, matrices having carboxylate anions have a negative charge only at a pH above their pKa. At a pH below the pKa, the matrices will exhibit a substantially neutral charge. Suitable anionic adsorbents also include the anionic adsorbents, which are matrices having a combination of sulfate and carboxylate anions and phosphate anions. The combination provides a negative charge intensity, which can vary continuously as a function of pH. These adsorbents attract and bind proteins and macromolecules that have positive charges, such as, for example, ribonuclease and lactoferrin. Without the desire to be bound by any particular theory, it is believed that the electrostatic interaction between an adsorbent and positively charged amino acid residues, which include lysine residues, arginine residues and histidine residues, are responsible for the binding interaction. Other adsorbents that are useful in observing interactions of electrostatic or ionic charges include cationic adsorbents. Specific examples of cationic adsorbents include matrices of secondary, tertiary or quaternary amines. Quaternary amines are positively charged permanently. However, secondary and tertiary amines have charges that are pH dependent. At a pH below the pKa, the secondary and tertiary amines are positively charged, and at a pH above their pKa, they are negatively charged. Suitable cationic adsorbents also include cationic adsorbents, which are matrices having combinations of different secondary, tertiary and quaternary amines. The combination provides an intensity of positive charges, which can be continuously varied as a function of pH. Cationic interaction adsorbents bind to anionic sites on molecules that include proteins that have amino acid residues exposed to the solvent, such as residues of aspartic acid and glutamic acid. In the case of ionic interaction adsorbents (both anionic and cationic) it is often convenient to use a mixed-mode ionic adsorbent, which contains both anions and cations. Such adsorbents provide the continuous buffering capacity as a function of pH. This continuous regulatory capacity makes it possible to expose a combination of analytes to eluents, which have different regulation components, especially in the pH range of 2 to 11. This results in the generation of local pH environments in the adsorbent, which they are defined by proton exchange groups, immobilized, that can be titled. Such systems are equivalent to the solid phase separation technique known as chromatic focus. The isoforms of hormones that stimulate the follicle, which differ mainly in the components of charged carbohydrates are separated into a chromatic approach to the adsorbent. Still other adsorbents that are useful for observing electrostatic interactions include the dipole-dipole interaction adsorbents, in which the interactions are electrostatic, but no formal charge or protein donor or acceptor is involved. d. Covalent Interaction Adsorbent Coordinated Adsorbents which are useful in observing the ability to form covalent bonds coordinated with metal ions include matrices carrying, for example, ions of divalent and trivalent metals. Immobilized metal ion chelator arrays provide immobilized synthetic organic molecules that have one or more electron donor groups, which form the basis of coordinated covalent interactions with transition metal ions. The groups of primary electron donors, which function as ion chelators of immobilized metals include oxygen, nitrogen sulfur. Metal ions bind to immobilized metal ion chelators that result in ion complexes of metals that have some number of remaining sites for interaction with electron donor groups in the analyte. Suitable metal ions include, in general, the ions of transition metals, such as copper, nickel, cobalt, zinc, iron and other metal ions, such as aluminum and calcium. Without the desire to be bound to any particular theory, metal ions are thought to interact selectively with specific amino acid residues, in peptides, proteins or nucleic acids. Typically, the amino acid residues involved in such interactions include histidine residues, tyrosine residues, tryptophan residues, cysteine residues, amino acid residues having oxygen groups, such as aspartic acid and glutamic acid. For example, immobilized iron ions interact with the phosphoserine, phosphotyrosine and phosphothreonine residues in proteins. Depending on the immobilized metal ion, only those proteins with sufficient local density of the above amino acid residues will be retained by the adsorbent, such interactions between metal ions and proteins can be so strong that the protein can not be separated from the complex by conventional means. Human ß casein, which is highly phosphorylated, binds very strongly to immobilized Fe (III). Recombinant proteins that are expressed with a 6-histidine tag binds very strongly to Cu (II) and Ni (II) immobilized. c. Site Interaction Adsorbents Active Enzyme Adsorbents that are useful in observing binding interactions of the enzyme active site include proteases (such as trypsin), phosphatases, nucleases kinases. The interaction is a specific interaction of the sequence of the binding site of the enzyme in the analyte (typically a biopolymer) with the catalytic binding site in the enzyme. Enzyme-binding sites of this type include, for example, the active sites of trypsin that interact with proteins and peptides having lysine-lysine or lysine-arginine pairs in their sequences. More specifically, the soy trypsin inhibitor interacts with and binds to an immobilized trypsin adsorbent. Alternatively, the serine proteases are selectively retained on the immobilized L-arginine adsorbent. f. Adsorbents of Covalent Interaction Reversible Adsorbents that are useful in observing reversible covalent interactions include disulfide exchange interaction adsorbents. Adsorbents - of disulfide exchange interaction include adsorbents comprising immobilized sulfhydryl groups, for example, mercaptoethanol or immobilized dithiothriethol. The interaction is based on the formation of covalent disulfide bonds between the adsorbent and the cysteine residues exposed to the solvent in the analyte. Such adsorbents bind to proteins or peptides having cysteine residues and nucleic acids including bases modified to contain reduced sulfur compounds. Glycoprotein Interaction Adsorbents Adsorbents that are useful in observing glycoprotein interactions include glycoprotein interaction adsorbents, such as adsorbents having immobilized lectins (ie oligosaccharides that carry proteins), an example of which is CONCONVALIN®, which is commercially available from Pharmacia Biotech from Piscataway, New Jersey. Such adsorbents function at the base of the interaction involving the molecular recognition of the carbohydrate moieties in the macromolecules. Examples of analytes that interact with and bind to glycoprotein interaction adsorbents,. they include glycoproteins, particularly histidine-rich glycoproteins, whole cells and isolated subcellular fractions. h. Adsorbents of Biospecific Interaction Adsorbents that are useful in observing biospecific interactions are generically named "biospecific affinity adsorbents". The adsorption is considered Biospecific if selective and the affinity (equilibrium dissociation constant, Kd) is at least 10"3 M up to (eg, 10" 5M, 10"7M, 10" 9M). Examples of Biospecific affinity adsorbents include any adsorbent that specifically interacts with and binds to a particular biomolecule. Biospecific affinity adsorbents include, for example, immobilized antibodies that bind to antigens; immobilized DNA that binds to DNA binding proteins, immobilized DNA and RNA substrates or inhibitors that bind to proteins and enzymes; immobilized drugs that bind to proteins that are linked to drugs; immobilized ligand compounds that bind to receptors; immobilized receptors that bind ligated compounds; Immobilized RNA that bind to DNA and RNA binding proteins; immobilized avidin or streptavidin that bind to biotinylated and biotinylated molecules; membranes of immobilized phospholipids and vesicles that bind to lipid-binding proteins. Enzymes are useful adsorbents that can be modified in the analyte adsorbent. The cells are useful as adsorbents. Their surfaces present complex binding characteristics. Adsorption to cells is useful for identifying, for example, ligand compounds or signaling molecules that bind to surface receptors. Viruses or phagocytes are also useful as adsorbents. Viruses frequently have ligand compounds for cell surface receptors (for example, gpl20 for CD4). Likewise, in the form of a phagocyte display collection, phagocyte coat proteins act as agents to test binding to targets. Biospecific interaction adsorbents depend on known specific interactions, such as those described above. Other examples of biospecific interactions for limes can be used adsorbents, will be readily apparent to those skilled in the art and are considered by the present invention. In one embodiment, the biospecific adsorbent may also comprise an auxiliary or "assistant" molecule, which does not participate directly in binding to the target analyte. i. Degrees of Union Specificity By exposing adsorbents that have different modes of interaction, the components of a sample can be roughly divided based on their interaction with the different adsorbents. Thus, the attraction of the analyte for adsorbents having different modes of interaction provides a first separation parameter. For example, by exposing a sample containing the analyte to a first adsorbent, with a base of attraction involving hydrophobicity and a second adsorbent with a base of attraction involving the ionic charge, it is possible to separate those analytes from the sample, which bind to a hydrophobic adsorbent and separate those analytes that bind to an adsorbent that has the particular ionic charge. Adsorbents having different bases of attraction provide resolution of the analyte with a low degree of specificity, because the adsorbent will bind not only to the analyte, but to any other component in the sample which also exhibits an attraction for the adsorbent, for the Same basis of attraction. For example, a hydrophobic adsorbent will bind not only to a hydrophobic analyte, but also to any other hydrophobic component in the sample; a negatively charged adsorbent will bind not only to a positively charged analyte, but also to any other positively charged component in the sample, etc. The resolution of the analytes based on the attraction of the analyte to the absorber can also be refined by exploiting the binding characteristics of the relatively intermediate specificity or altered force of attraction. The resolution of the analyte on the basis of the binding characteristics of the intermediate specificity can be achieved, for example, using functionally mixed adsorbents. Once the resolution of the analyte is achieved with a relatively low specificity, the binding characteristics found attract the analyte of interest can be exploited in combination with a variety of other binding and elution characteristics, to remove even the unwanted components and thus solve the analyte. For example, if the analyte binds to hydrophobic adsorbents, the analyte can also be resolved from other components of the hydrophobic sample, providing a functionally mixed adsorbent, which exhibits as a base of attraction a hydrophobic interaction and also exhibits a second base of different attraction. The functionally mixed adsorbent can exhibit hydrophobic interactions and negatively charged ionic interactions, in order to bind the hydrophobic analytes that are positively charged. Alternatively, the functionally mixed adsorbent can exhibit hydrophobic interactions and the ability to form coordinated covalent bonds ----- with metal ions, in order to bind hydrophobic analytes that have the ability to form coordination complexes with metal ions in the adsorbent . Still other examples of adsorbents that exhibit the binding characteristics of intermediate specificity will be readily apparent to those skilled in the art, based on the description and examples set forth above. The resolution of analytes on the basis of the binding characteristics of intermediate specificity can also be refined by exploiting the binding characteristics of relatively high specificity. The relatively high specificity binding characteristics can be exploited,. using a variety of adsorbents, which exhibit the same base of attraction, but a different attractive force. In other words, although the basis of attraction is the same, further resolution of the analyte from the other components of the sample can be achieved by using adsorbents having different degrees of affinity for the analyte. For example, an analyte that binds to an adsorbent based on the acid nature of the analyte can also be resolved from other acidic components of the sample, using adsorbents that have affinity for analytes in the specific ranges of the acid H. Thus, the analyte can be solved by using an adsorbent attracted to the pH sample components of 1-2, another adsorbent attracted to the pH sample components of 3-4, and a third absorber attracted to the pH sample components. from 5-6. In this way, an analyte having a specific affinity for an adsorbent, which binds to the analyte with a pH of 5-6, will be resolved from the sample components of pH 1-4. Adsorbents of increasing specificity can be used by decreasing the range of attraction, that is, the difference between the binding characteristics of adsorbents, which exhibit the same base of attraction.

A first analyte adsorbed to a primary adsorbent can, by itself, have adsorbent properties. In this case, the analyte first adsorbed to a substrate can become a secondary adsorbent to isolate secondary analytes. In turn, the retained secondary analyte can function as a tertiary adsorbent to isolate a tertiary analyte from a sample. This process can continue through several interactions. 2. Eluentes Eluenes, or wash solutions, selectively modify the absorption threshold between the analyte and the adsorbent. The ability of an eluent to desorb and elute a binding analyte is a function of its elution characteristics. Different eluents may exhibit very different elution characteristics, somewhat different elution characteristics or subtly different elution characteristics. The temperature at which the eluent is brought into contact with the adsorbent is a function of the particular sample and the selected adsorbents. Typically, the eluent is contacted with the adsorbent at a temperature between 0 and 100 ° C, preferably between 4 and 37 ° C. However, for the same eluents, modified temperatures may be convenient, which will be readily determined by those skilled in the art.

As in the case of adsorbents, eluents that exhibit excessively different elution characteristics will generally differ in their base of attraction. For example, the various bases of attraction between the eluent and the analyte include the charge or pH, ionic strength, water structure, specific competitive binding reagent concentrations, surface tension, dielectric constant and combinations of two or more of the foregoing. to. Eluents based on pH The eluents that modify the selectivity of the adsorbent based on the pH (ie the charge) include the known pH regulators, acid solutions, basic solutions. By washing an analyte bound to a given adsorbent with a particular pH regulator, the charge can be modified and, therefore, the resistance of the bond between the adsorbent and the analyte, in the presence of the particular pH regulator, can be excited. These analytes, which are less competitive than others for the adsorbent in the pH of the eluent, will be desorbed from the adsorbent and eluted, leaving linked only those analytes that bind the adsorbent more strongly to the pH of the eluent. b. Eluents Based on the Ionic Force Eluents that modify the selectivity of the adsorbent with respect to ionic strength include salt solutions of various types and concentrations. The amount of salt solubilized in the eluent solution affects the ionic strength of the eluent and correspondingly modifies the binding ability of the adsorbent. Eluents containing a low concentration of salt provide a slight modification of the binding ability of the adsorbent with respect to the ionic strength. Sources containing a high concentration of α provide greater modification of the adsorbent binding ability with respect to ionic strength. c. Eluents Based on the Structure of the Water Eluents that modify the selectivity of the adsorbent by altering the water structure or concentration, include the urea or chaotropic salt solutions. Typically, urea solutions include, for example, solutions ranging in concentration from 0.1 to 8 M. Chaotropic salts that can be used to supply eluents include sodium thiocyanate. Eluents based on the water structure modify the ability of the adsorbent to bind to the analyte, due to alterations in hydration or the binding water structure. Eluents of this type include, for example, glycerol, ethylene glycol and organic solvents. Chaotropic anions increase the water solubility of the non-polar parts, thus decreasing the hydrophobic interactions between the analyte and the absorber. d. Eluentes with Detergent Base Eluens that modify the selectivity of the adsorbent with respect to surface tension and analyte structure include detergents and surfactants. Suitable detergents for use as eluents include ionic and non-ionic detergents, such as CHAPS, TWEEN and NP-40. Detergent-based eluents modify the ability of the adsorbent to bind to the analyte as hydrophobic interactions are modified, when hydrophobic and hydrophilic detergent groups are introduced. The hydrophobic interactions between the analyte and the adsorbent, and within the analyte, are modified and the loading groups are introduced, for example the denaturation of proteins with ionic detergents, such as SDS. and. Eluente Based on Hydrophobicity The eluents that modify the selectivity of the adsorbent with respect to the dielectric constant are those eluents that modify the selectivity of the adsorbent co with respect to the hydrophobic interaction. Examples of suitable eluents that function in this capacity include the organic solvents of urea (0.1-8M), such as propanol, acetonitrile, ethylene glycol and glycerol, and detergents such as those mentioned above. The use of acetonitrile as the eluent is typical in reverse phase chromatography. The inclusion of ethylene glycol in the eluent is effective in eluting immunoglobulins from salt-promoted interactions with thiophilic adsorbents. f. Eluent Combinations Suitable eluents can be selected from any of the above categories or can be combinations of two or more of the above eluents. Eluents comprising two or more of the above eluents are capable of modifying the selectivity of the adsorbent for the analyte based on the multiple elution characteristics. 3. Variability of the Two Parameters The ability to provide different binding characteristics, selecting different adsorbents and the ability to provide different elution characteristics, washing with different eluents, allows the variance of two different parameters, each of which is not capable of performing individually the selectivity with which the analytes bind to the adsorbent. The fact that these two parameters can be widely varied, ensures a greater range of binding attraction and elution conditions, so that the methods of the present invention can be useful for joining and thus detecting many different types of analytes. The selection of adsorbents and eluenes for use in analyzing a particular sample will depend on the nature of the sample and the particular analyte or class of analytes to be characterized, even if the nature of the analytes is not known. Typically, it is advantageous to provide a system that exhibits a wide variety of binding characteristics and a wide variety of elution characteristics, particularly when the composition of the sample to be analyzed is unknown. By the provision of a system exhibiting wide ranges of selectivity characteristics, the probability that the analyte of interest is retained by one or more of the adsorbents will be significantly increased. An expert in the field of chemical or biochemical analysis will be able to determine the selectivity conditions useful for retaining a particular analyte by the provision of a system exhibiting a wide range of binding and elution characteristics and observing these binding and elution characteristics that provide the best resolution of the analyte. Because the present invention provides systems that include wide ranges of selectivity conditions, the determination by the person skilled in the art of the optimum binding and elution characteristics for a given analyte can easily be achieved without the need for undue experimentation.

C. Analytes The present invention allows the resolution of analytes based on a variety of biological properties, chemical or physico-chemical analytes, exploiting the properties of the analyte through the use of appropriate selectivity conditions. Among the many properties of analytes that can be exploited through the use of conditions of appropriate selectivity, is the hydrophobic index (or measurement of hydrophobic residues in the analyte), the isoelectric point (ie, the pH at which the analyte has no charge), the hydrophobic moment (or measure of the amphipathicity of an analyte or the extent of asymmetry in the distribution of polar and non-polar residues), the moment of lateral diplome (or measure of asymmetry in the distribution of charge in the analyte), a factor of molecular structure (which accounts for the variation in the surface contour of the analyte molecule, such as the distribution of volumetric side chains along the skeleton of the molecule), components of secondary structure (for example, propellers, parallel and antiparallel sheets), disulfide band, electron donor groups exposed to solvents (eg His), aromaticity (or measurement of the pi-pi teraction between aromatic residues in the analyte) and the linear distance between charged atoms. These are representative examples of the types of properties that can be exploited for the resolution of a given analyte from a sample by the selection of the appropriate selectivity characteristics in the methods of the present invention. Other suitable properties of analytes that can form the basis of resolution for a particular analyte of the sample will be readily known and / or determined by those skilled in the art, and are considered by the present invention. The method of the invention is not limited with respect to the types of samples that can be analyzed. The samples may be in the solid, liquid or gaseous state, although typically the sample will be in the liquid state. The solid or gaseous samples are preferably solubilized in a suitable solvent to supply a liquid sample, according to the techniques of those skilled in the art. The sample may be a biological composition, non-biological organic composition, or inorganic composition. The technique of the present invention is particularly useful in resolving analytes in a biological sample, particularly fluids and biological extracts; and to resolve analytes in non-biological organic compositions, particularly small molecule, organic and inorganic compositions. The analytes can be molecules, multimeric molecular complexes, macromolecular assemblies, cells, subcellular organelles, viruses, molecular fragments, ions or atoms. The analyte may be a simple component of the sample or a class of structurally, chemically, biologically or functionally related components that have one or more characteristics (eg, molecular weight, isoelectric point, ion charge, hydrophobic / hydrophilic interaction, etc.). ) in common. Specific examples of analytes that can be solved using the methods of retentate chromatography of the present invention include biological macromolecules, such as peptides, proteins, enzymes, polynucleotides, oligonucleotides, nucleic acids, carbohydrate oligosaccharides, polysaccharides, fragments of biological macromolecules discussed above, such as fragments of the nucleic acid, fragments of peptides and protein fragments; biological macromolecule complexes, indicated above, such as nucleic acid complexes, DNA-protein complexes, receptor-ligand complexes, enzyme substrates, enzyme inhibitors, peptide complexes, protein complexes, complexes of carbohydrates and complexes of polysaccharides; small biological molecules, such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids plant growth regulators, phosphate and diphosphonucleoside sugars esters, synthetic small molecules, such as pharmaceutically or therapeutically effective agents, monomers, peptide analogues, steroid analogues, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, amino acid analogs, antibacterial agents, transport inhibitors, surface active agents (surfactants), mitochondrial and chloroplast functional inhibitors, electron donors, carriers and acceptors, synthetic protease substrates, substrates for phosphatases, substrates for esterases and lipases and reacted proteins modification; and synthetic polymers, oligomers and copolymers, such as polyalkylenes, polyamides, poly (meth) acrylates, polysulfones, polystyrenes, polyethers, polyvinyl ethers, polyvinyl esters, polycarbonates, polyvinyl halides, polysiloxanes, POMA, PEG, and copolymers of either of two or more of the above.

III. INFORMATION PROCESS The detection of analytes adsorbed to an adsorbent, under particular elution conditions, provides information about analytes in a sample and their chemical character. The adsorption depends, in part, on the binding characteristics of the adsorbent; The analytes that bind to an "" adsorbent have the characteristic that makes the union possible. For example, molecules that are cationic at a particular pH will bind to an anionic adsorbent under elution conditions that include that pH. The strongly cationic molecules will only be eluted from the adsorbent under very strong elution conditions. Molecules with hydrophobic regions will bind to hydrophobic adsorbents, while molecules with hydrophilic regions will bind to hydrophilic adsorbents. Again, the strength of the interaction will depend, in part, on the extent to which an analyte contains hydrophobic or hydrophilic regions. Thus, the determination that certain analytes in a sample bind to an adsorbent under certain elution conditions, not only solves analyte in a mixture by separating them from each other and from analytes that do not possess the appropriate chemical character for the binding, but also identifies an class of individual analytes or analytes that have the particular chemical character. Collecting information about the retention of analytes in one or more particular adsorbents under a variety of elution conditions provides not only the detailed resolution of analytes in a mixture, but also the chemical information about the analytes, which itself can lead to your identity. These data are referred to as "retention data". The data generated in the retention tests are mostly analyzed with the use of a programmable digital computer. The computer program usually contains a readable medium, which stores keys. Certain keys are dedicated to memory, which include the location of each - feature in a substrate arrangement, the identity of the adsorbent in that characteristic and the elution conditions used to wash the adsorbent. Using this information, the program can then identify the set of features in the array that define certain selectivity characteristics. The computer also contains a key that receives, as input, data on the signal strength of various molecular masses received from a particular steerable location on the probe. These data can indicate the number of analytes detected, which optionally include for each detected analyte the resistance - of the signal and the determined molecular mass. The computer also contains keys that process the data. This invention considers a variety of methods for processing data. In one embodiment, this involves creating an analyte recognition profile. For example, data on the retention of a particular analyte identified by the molecular mass can be classified according to a particular binding characteristic, for example, binding to anionic adsorbents or hydrophobic adsorbents. These collected data provide a profile of the chemical properties of the particular analyte. The retention characteristics reflect the function of the analyte which, in turn, reflects the structure. For example, retention of coordinated covalent metal chelators may reflect the presence of histidine residues in a polypeptide analyte. By using retention level data to a plurality of cationic and anionic adsorbents under elution at a variety of pH levels, it reveals information that one can derive the isoelectric point of a protein. This, in turn, reflects the probable number of ionic amino acids in the protein. Therefore, the computer can include keys that transform the union information into structural information. Likewise, the secondary process of the analyte (for example the modifications after the translation) results in an altered recognition profile reflected by the differences in joints or mass. In another modality, the retention assays are performed under the same sets of selectivity thresholds in two different cell types, and the retention data of the two assays are compared. Differences in retention maps (eg presence or signal strength in any characteristic) indicate analytes that are differentially expressed by the two cells. This may include, for example, the generation of a difference map that indicates the difference in signal strength between two retention tests, * thus indicating which analytes are retained in an increased or decreased form by the adsorbent in the two tests. The computer program may also include keys that receive instructions from a programmer as input. The progressive and logical trajectories for the selective desorption of analytes from predetermined specific locations in the array can be anticipated and programmed in advance. The computer can transform the data into another format for presentation. The data analyzes may include the steps of determining, for example, the signal strength as a function of the characteristic position of the collected data, by removing the "abstentionists" (data deviating from a predetermined statistical distribution) and calculating the affinity of the data. Relative binding of the analytes from the remaining data. The resulting data can be displayed in a variety of formats. In one format, the strength of a signal is displayed on a graph as a function of molecular mass. In another format, called the "gel format", the strength of a signal is displayed along with a linear axis intensity of darkness, which results in an appearance similar to the bands in a gel. In another format, signals that reach a certain threshold are presented as vertical lines or bars on a horizontal axis, which represents the molecular mass. Therefore, each bar represents a detected analyte. The data may also be presented in signal strength plots for a pooled analyte according to the binding feature and / or elution characteristic.

IV. APPLICATIONS OF RETENATE CHROMATOGRAPHY Retenate chromatography involves a combinatorial separation method, which includes the detection and characterization of multiple analytes in parallel. These combinatorial methods have many applications. Such applications include, without limitation, the development of target analyte detection schemes; development of protein purification strategies; protein purification methods; identification of specific phagocytes from a phagocyte display library, which bind to an objective analyte, which includes identification of the target epitope using complementary collections of phagocyte display; the identification of proteins based on the physico-chemical properties of the analyte; the inspection and differential display of proteins of gene expression; the toxicological classification; the simultaneous detection of multiple diagnostic markers, -the discovery of drugs; and the inspection and detection of sets of multimeric proteins translating polynucleotides in vi tro.

A. Methods for Extracting in Sequence Analytes of a Sample The chromatography of the retenate involves the analysis of retention of an analyte under a plurality of adsorbent / eluent conditions. A variant of this method is sequence extraction. In sequence extraction, a sample is not exposed independently to two different selectivity conditions. Rather, the sample is exposed to a first selectivity condition to extract certain analytes from the sample in the adsorbent, and leave the analytes not adsorbed in the eluent. Then the eluent is exposed to a second selectivity condition. This also extracts several analytes from the eluent. Frequently, if the adsorbents in the first and second exposures have different bases of attraction (for example, the normal and hydrophobic phase) the adsorbent will draw a different set of analytes from the eluent. This second eluent is then exposed to a third condition of selectivity, etc. In a method of practicing sequence extraction, the adsorbent is placed at the bottom of a cavity, so that the sample can be mixed on top of it. An eluent is added to the adsorbent and after allowing the binding between the analytes in the sample, the eluent wash is collected. This collected wash is then exposed to a second adsorbent, and the analytes are extracted from the sample by the junction. In one embodiment, the goal of sequence extraction is preparative rather than analytic. More specifically, the goal may be to extract almost all of the desired analyte from a sample. In this case, the sample is usually small, for example a few microliters, in an area of about a few millimeters in diameter. The adsorbents are selected so that they do not absorb an analyte that one does not want to exhaust from the sample. After several interactions, the collected wash is finally exhausted from the unwanted analytes, leaving those desired for subsequent analysis, for example, by desorption spectrometry or traditional chromatography methods. In another modality, the non-retained sample is, by itself, analyzed for the analytes by any analytical technique. Even after a single retention stage, the process allows the materials adsorbed to an adsorbent to be examined and those analytes that do not adsorb.

B. Methods for the Progressive Resolution of Analytes in a Sample An object of the retenato chromatography is the unambiguous resolution of an analyte from a complex sample mixture. This is especially important for the application - in clinical diagnosis, drug discovery and functional genomics. These areas may involve the identification of one or more analytes from a biological sample. This invention provides a method for identifying "conditions of effectiveness with improved resolution for an analyte." The method involves identifying a selectivity condition in which the analyte is retained and, in an iterative process, adding additional binding characteristics or elution characteristics to the analyte. the selectivity condition, which provides the improved resolution of the analyte.

A mass spectrum of a complex sample exposed to a selectivity condition generally includes signals from many components of the sample. The complexity of the signals can interfere with the unambiguous resolution of the analyte. The method for the progressive resolution of an analyte allows one to identify the selectivity conditions with improved resolution of the analyte for the unambiguous detection of an analyte in a sample. A selectivity condition exhibits an "improved resolution" of an analyte compared to another selectivity condition if the analyte signal is more readily distinguishable from the signals of other components. This may include, for example, decreasing the number of analytes bound to the adsorbent, thereby decreasing the total number of signals, or increasing the selectivity of the selectivity condition for the analyte, thereby enhancing the analyte signal compared to other signals. Of course, when the analyte binds exclusively to the substrate, it generates the only analyte signal during detection. Progressive resolution methods involve an iterative process in which the additional selectivity characteristics (binding or elution) are added in sequence to a constant set of known selectivity characteristics retaining the analyte. In a first stage, a series of effectiveness conditions are tested to identify one that retains the target analyte. In a next step, one or more of the selectivity characteristics of the selectivity condition are selected for the constant play for further analysis. A new set of selectivity conditions are generated. Each of the conditions in the new game includes the characteristics selected in the constant game and at least one new condition not in the constant game. For example, if the constant set includes an anionic adsorbent and a low salt eluent, the new condition may involve varying the pH of the eluent. Each of these new variables is tested in the ability to improve the resolution of the analyte and the modified selectivity condition with improved resolution is identified. In a next step, an aggregate selectivity condition, which provides improved resolution, is added to the constant game. The game of the modified constant is tested again in the second way, generating a new set of selectivity conditions that include the characteristics of constant play and a set of new features. Thus, in each stage, the selectivity conditions are selected so that the resolution of the analyte is improved compared to the selectivity condition in a previous step. The method is well described as an example. A cell sample typically contains hundreds or perhaps thousands of proteins. One may wish to obtain the unambiguous resolution of a single target protein analyte in the sample. In a first step, a retention map for the target analyte is developed using a plurality of selectivity conditions. For example, the adsorbents can be an ion exchanger, a cation exchanger, a normal phase absorber and a reverse phase adsorbent. The elution conditions tested in each adsorbent can be at a variety of pH levels, a variety of ionic strengths, a variety of detergent conditions and a variety of hydrophobicity based conditions. For example, four different elution conditions can be tested for each condition. Thus, in this example, sixteen different selectivity conditions were tested in their ability to adsorb the target analyte. From this retention map, one selects at least one selectivity condition under which the target analyte is retained. One can select a selectivity condition under which the objective binds maximally. However, it may be advantageous to select a condition under which the objective does not bind in a maximum manner if this condition of effectiveness is more selective for the objective than the other conditions of selectivity. It is presumed, in this example, that the analysis of the retention map shows that the target is retained by the anion exchange adsorbents at an approximately neutral pH, but is also weakly adsorbed to a hydrophilic adsorbent. A variable or eluent adsorbent of the identified selectivity condition that results in retention of the analyte is then selected for use under all subsequent selectivity conditions. As used here, it will be added to the "set of constants of the selectivity condition". In the next iteration, one tests the ability of the target analyte to bind under a second plurality of selectivity conditions. Each condition of selectivity in the second game includes the elements of the set of constants of the selectivity condition. However, each selectivity also includes another variable - a different adsorbent or eluent added to the selectivity condition. Thus, within the constraints of employing at least the constant game, the second set of selectivity conditions is also selected to be more diverse than the first game. Methods for increasing diversity include, for example, testing finer gradations of an elution condition or different forces of an adsorbent. It may also include, for example, the addition of other selectivity characteristics under selectivity conditions. Continuing the example, the anion exchange adsorbent can be added to the set of constants. This condition is now tested with a greater variety of variables, for example eluents or adsorbents. The eluents to be tested may include a variety of low pH regulators at finer graduations than those tested in the first interaction. For example, the first interaction may have tested regulators with pH 3.0, pH 5.0, pH 7.0 and pH 9.0, and shown that the target link to the anion exchange adsorbent is close to neutral pH. During the second interaction, the regulators tested can be at a pH of 5.0, pH of 5.5, pH of 6.0, pH of 7.0, pH of 7.5 and pH of 8.0. In addition, each of these regulators can also be varied to include other elution characteristics, for example ionic strength, hydrophobicity, etc. The analysis of the second retention map, which results in this stage, will generally allow identifying a condition that provides a better resolution than the selectivity condition identified in the first round. 11.

Again, one of the variables of 'this selectivity condition is chosen and added to the set of constants of the selectivity condition for the subsequent interrogation in the next iteration. Continuing the example, suppose that the condition of selectivity in the second round, which resolves the analyte, better uses a pH regulator of 6.5. This eluent can now be added to the set of constants, which now include an anion exchange resin and a pH regulator of 6.5. In the next iteration, selectivity conditions include this set of constants and another variable .. The variable may be, for example, the addition of a new component to the eluent, such as different ionic forces; or another adsorbent may be added to the mixture, such as the variety of hydrophobic adsorbents mixed with the anion exchange adsorbent; one can vary the density of the anion exchange resin. Again, the selectivity condition is identified from this set which shows improved resolution of the analyte. The process can continue until the analyte is purified to essential homogeneity. In this case, the selectivity condition is specific for the analyte. As you can see, by increasing the number of variables tested in each stage, one can decrease the number of iterations needed to identify an appropriate selectivity condition. C. Method of Preparative Purification of a Analyte In another aspect, this invention provides methods for purifying an analyte. The methods take advantage of the power of the retenate chromatography to quickly identify attractive bases to adsorb an analyte. A first step involves exposing the analyte to a plurality of selectivity conditions and determining retention under the conditions by chromatography of the retentate. This generates a characteristic recognition profile of the analyte. The selectivity conditions under which the analyte is retained are used to develop a protocol for the preparative purification of the analyte. For the preparative purification of the analyte, this analyte is adsorbed in sequence and eluted from a series of adsorbent / eluent combinations which are identified as analyte ligatures. Thus, for example, the recognition map may indicate that the analyte binds to a normal phase adsorbent and a metal chelator. The analyte is then contacted with a first chromatographic column, for example, containing the normal phase adsorbent, which binds to the analyte. Unbound material is washed and separated. Then the analyte is eluted by a sufficiently strict washing. The eluent is then contacted with a metal chelate column, for example, to bind the analyte. The unbonded materials are washed and separated. Next, the bound material, which includes the analyte, is eluted from the metal chelate column. In this way, the analyte is isolated in preparative amounts. A preparative amount of a sample is at least 10 μl, at least 100 μl, at least 1 ml or at least 10 ml. The information generated during the progressive resolution of the analytes can be used to designate protein purification strategies at a higher chromatographic scale (based on elution). The bases of the adsorbent for the attraction, the binding conditions and the elution conditions (ie, the selectivity conditions) for an objective analyte protein, come to be defined by the chromatography of the retenate. This information can save a huge amount of time, energy, and precious analyte, which would otherwise be wasted during the trial and error process of the purification strategy design that now takes place. This section also provides the large-scale purification efforts, made with commercially available adsorbents.

Methods to Obtain Probes for Detection Specific Analytes This information provides probes for the specific detection of one or more analytes by desorption spectrometry, as well as methods for generating these probes. Such probes that resolve the analyte are useful for the specific detection of analytes in diagnostic and analytical methods. The first step in generating a probe to solve one or more analytes is to produce a retention map for the analytes, under a plurality of different adsorbent / eluent combinations. For example, the resolution of the analyte can be determined for four different adsorbents washed with each of the five different eluents. This provides twenty sets of retention data for each of the analytes. The analysis of the resulting retention map will indicate which condition or conditions of selectivity will best resolve the analytes. Preferably, a selectivity condition can be identified that unambiguously solves all the analytes. Then one or more selectivity conditions are selected-for use of the probe that resolves to analyte, so that each of the analytes is resolved in at least one zone of adsorbent. The probe may also contain an adsorbent, which does not bind to the analyte or analytes. This absorbent zone is useful as a control. The zones may include a plurality of adsorbent zones in steerable locations, selected for their ability to retain and resolve the analyte or analytes. In this case, the adsorbents are selected by binding to the analyte under a single eluent condition. This is useful because the entire probe can be washed with only one eluent in the detection process. The retention map generated for a particular analyte can be used to create a customary adsorbent for the analyte. For example, the nature of the adsorbents that retain an analyte indicate a set of bases for the attraction of an analyte. A customary adsorbent can be designed by preparing a multiplex adsorbent that includes elements of adsorbents that supply these bases of attraction. Such customary adsorbent is very selective for the target analyte. One or a few customary adsorbents may be sufficient to generate a recognition map for the analyte. For example, if it is found that under particular elution conditions, an analyte is retained by adsorbents that bind materials that have certain degrees of hydrophobicity, positive charge and aromaticity, one can create an adsorbent customary by design or through the use of combinatorial synthetic strategies that have functional groups that attract each of these three characteristics. The detection of binding to this adsorbent identifies the analyte. Such probes are useful in. detect the analyte or analytes in a sample. The sample is exposed to selectivity conditions and this probe is interrogated by desorption spectrometry. Because the probe solves the analytes, its presence can be detected by searching for the characteristic recognition profile. Such probes are particularly useful in identifying a set of diagnostic markers in a patient sample. In one embodiment, the array is designed to be attached to specific classes of proteins of interest. These include the diagnostic markers as well as the analytes defined by the function. For example, an array can be prepared which is specifically attached to cell surface proteins of a certain class (for example, kinases), transcription factors, intracellular receptors, etc. The adsorbents can be specific for biopolymers, for example, antibodies. In one embodiment, adsorbents are genetic packaging, such as compounds-of protein locus that display phagocytes-for a certain class of proteins. In this case, a phagocyte display collection can be pre-selected with a certain class of molecules, to eliminate phagocytes that bind to that class. Then, phagocytes that have been subtracted from the population are used as adsorbents.

E. Diagnostic Probes and Diagnostic Methods The diagnosis of pathological conditions frequently involves the detection in a patient sample of one or more molecular markers of the disease. Certain conditions can be diagnosed by the presence of a single diagnostic marker. The diagnosis of other conditions may involve the detection of a plurality of diagnostic markers. Also, the detection of several markers can increase diagnostic confidence. Therefore, this invention supplies probes for desorption spectrometry, comprising at least one adsorbent that solves at least one diagnostic marker of a pathological condition. The preparation of such probes involves, first, the selection of markers to be detected. The label can be one for any disease state, for example cancer, heart disease, autoimmune disease, viral infection, Alzheimer's disease or diabetes. For example, the detection of prostate specific antigen (PSA) is highly suggestive of prostate cancer. HIV infection can be diagnosed by detecting the antibodies against the various HIV proteins, such as one of the pl7, p24 or p55, and one of p31, op51 i p66 and one of gp41 or gpl20 / l60. The detection of amyloid-β42 and the tau protein in the cerebrospinal fluid is highly indicative of Alzheimer's disease. Likewise, markers can be identified by methods of this invention, which involve the detection of the presence of an analyte difference in healthy subjects versus subjects with pathological conditions. In a next step, the adsorbents are developed and retain one or more diagnostic markers. Preferably, a single adsorbent is prepared, which solves all the markers. This can be accomplished, for example, by creating an area that contains several antibodies, each of which binds to one of the desired markers. Alternatively, the probe may comprise a plurality of adsorbent zones, each zone capable of resolving at least one target analyte under a selectivity condition. In one embodiment, the adsorbent is a multiple adsorbent comprising ligand compounds that are specific to the labels. For example, the adsorbent may comprise an antibody, a polypeptide ligand compound or a polynucleotide that specifically binds to the target analyte. In one embodiment, the antibody is a single chain antibody, identified by classifying a combinatorial collection., Single chain antibodies are specific for particular markers that can be developed by the classification of phagocyte display collections, or the methods described herein. . In another embodiment, the adsorbent comprises non-organic biomolecular components, which either retain the target analyte specifically or retain the analyte with sufficient specificity for unambiguous resolution by desorption spectrometry. The preparation of adsorbents for the detection of specific analytes is also described here. Significantly, a single zone of adsorbent used in these methods need not be specific for a single analyte and, therefore, need not require the specific affinity mediated by a biopolymer, between the target and the adsorbent. The affinity detection methods have depended mainly on the specific binding between a biopolymer and a target. These include, for example, the specific affinity of an antibody for a protein, a polynucleotide for a complementary polynucleotide or a lectin for a carbohydrate. Such specificity is necessary because the meaning of the detection was indirect: the objective or was identified; a label, often attached to the adsorbent, was identified. Therefore, the more specific the adsorbent, the less likely that contaminants will bind to the adsorbent and interfere with specific detection. Nevertheless, desorption spectrometry results in the direct detection of an analyte. Therefore, the presence of contaminants will not interfere with the specific detection unless the contaminant signal overlaps the target signal. Diagnostic methods involve, first, selecting a sample of the patient to be tested. The sample can be, for example, a tissue, blood, urine, excrement or other body fluid (lymphatic, cerebrospinal, intraarticular, etc.). The sample is then exposed to a substrate containing the diagnostic adsorbents, under conditions to allow retention of the diagnostic markers. The adsorbent is washed with an appropriate eluent. Then the markers are detected (for example, solved) by desorption spectrometry (for example, mass spectrometry). This invention also provides sets for specific detection of diagnostic markers, including: (1) a substrate for use in desorption spectrometry, comprising at least one adsorbent in at least one steerable location, which resolves at least one marker of diagnosis, under a selectivity condition comprising the adsorbent and an eluent and (2) the eluent or instructions for the preparation of the eluent. Upon exposure of the sample to the adsorbent and washing with the eluent, that is, by executing the selectivity condition, the analyte is sufficiently purified or specifically bound for resolution by desorption spectrometry.

F. Methods for Identifying Proteins In another aspect, this invention provides a method to aid in the identification of a protein. The method "involves determining the match parameters for the physico-chemical characteristics of a protein analyte, using the chromatography of the retenate and looking for a protein database to identify proteins that have the matching parameters." Derivation of the physical information -chemistry based on the retention characteristics discussed above.The database will typically provide the de-amino acid sequence and / or the nucleotide sequence that encodes the amino acid sequence of each protein.Structural features, such as molecular mass , hydrophobicity, pH, mass of fragments, etc., are easily derived from this information.An analyte protein will share any structural characteristic with only a subset of proteins in the database.Therefore, identity candidates were found by classifying the proteins according to the characteristics is structures shared with the protein analyte. Thus, in view of the inaccuracy, the degree of specificity or level of confidence inherent in identifying one or more of the physico-chemical properties of the reference, one can not expect the proteins in the database to match perfectly in all the characteristics Of the reference. Therefore, match parameters can be established to identify, for example, the narrowness of fit between the characteristics of the protein analyte and the characteristics of the reference polypeptide in the database. As our identification of genes in the genome increases, the chance that any protein analyte in the database will exist as a reference polypeptide also increases. Therefore, this method enables you to quickly solve a protein of interest in a sample, obtain the structural information about the protein and then use this information to identify the protein.

G. Methods of Assembling Multimeric Molecules The ability of adsorbents to attach to desired molecules is useful in constructing multimeric molecules and evaluating the compounds that perform their assembly. One unit of the multimeric molecule binds to an adsorbent. Then it is exposed to a sample that contains another unit of the multimeric molecule. The exposure can be performed under a variety of conditions to test binding parameters. The binding of a subunit to the multimer can be inspected by desorption spectrometry. Then, a subsequent subunit can be tested for union in the same way. The methods of drug classification, described here, are useful for testing agents in their ability to interfere with assembly. Therefore, an analyte in one stage of the process becomes an absorbent in the next stage.

H. Methods of Performing Enzyme Assays This invention also provides methods for carrying out enzyme assays. Enzyme assays generally involve exposing a sample, which is to be tested, with an enzyme substrate, under conditions in which the enzyme is active. After allowing the enzyme to act on the substrate, an enzymatic reaction product is detected. In quantitative tests, the amount of the product is determined. This amount is usually compared to a control or a standard curve, thus providing a quantity of the activity of the enzyme in the sample.

This invention provides methods for detecting an enzyme, including detecting an amount of the activity of the enzyme, in a sample. The method takes advantage of the fact that the activity of an enzyme often produces a product whose mass is different from the original substrate. In the method, the solid phase is prepared, which comprises an adsorbent that binds to the substrate. A quantity of the substrate is attached to the adsorbent. Then the adsorbent is exposed to the sample under conditions and for a time that allows any enzyme to act on the substrate, then any bound material is detected by the desorption spectrometry.The detection of an analyte that has a characteristic of molecular mass of the product of enzyme activity, provides an indication of the presence of the enzyme. The strength of the signal will be a function of the amount of enzymatic activity in the sample.

I. Methods to Identify Analytes That Are Express Differentially Among Biological Materials In another aspect, this invention provides methods for identifying organic biomolecules, particularly proteins, which are differentially expressed between two or more samples. "Differential expression" refers to differences in the quantity or quality of an analyte between two samples. Such differences will result in any stage of protein expression from transcription through modification after translation. The methods taking advantage of the extraordinary resolution power and the sensitivity of the retentate chromatography. First, the recognition profiles using the same set of selectivity conditions are prepared for analytes from the two biological samples. The greater the number of selectivity conditions used, the higher the resolution of analytes in the sample and, therefore, the greater the number of analytes that can be compared. Then, the recognition maps are compared to identify analytes that are differentially retained by the two sets of adsorbents. Differential retention includes quantitative retention. This indicates, for example, the regulation above and below the expression. Differential retention also includes qualitative differences in the analyte. For example, differences in the modification after translation of a protein can result in differences in recognition maps, which can be detected as differences in binding characteristics (for example, if the protein is glycosylated, it will bind differently to lectin adsorbents) or differences in mass (for example, as a result of differences in cleavage after translation). The analysis can be carried out by a programmable digital computer. The method is particularly useful in detecting gene that is differentially expressed between two cell types. The two types of cells can be normal versus pathological, for example cancer cells or cells at different levels of them at different stages of development or differentiation, or in different parts of the cell cycle. However, the method is also useful in examining two cells of the same type exposed to different conditions. For example, the method is useful in the classification of toxicology and test agents for the ability to modulate the expression of the gene in a cell. In such a method, one biological sample is exposed to the test agent, and the other cell is not. Then, the retentive maps of the samples are compared. This method may indicate that a protein or other biomolecule is increased or decreased in expression, or is changed in some way based on different retention characteristics or different mass. Using information about the physico-chemical properties of differentially expressed proteins, obtained from retention maps, the identity candidates for these proteins can be determined using the methods described herein. This method is useful in identifying diagnostic markers of diseases. Proteins that are differentially expressed in a sample from a patient or a diseased cultured cell, compared to normal cells or samples, can be diagnostic markers. In general, it is better to compare samples from a statistically significant patient population with normal samples. In this way, the information can be grouped to identify diagnostic markers common to all or a significant number of individuals that exhibit the pathology.

I. Increased Sensitivity by Catabolic Signal Amplification The sensitivity of detecting the differential presence (eg, resulting from differential expression) of large proteins, in a complex mixture, can be significantly increased by the fragmentation of the large protein in minor pieces and detecting the minor pieces. The increased sensitivity is due to several factors. FirstWhen all proteins in a sample are fragmented, for example, by enzymatic digestion, large proteins will probably produce larger fragments compared to small proteins. Second, the general sensitivity of the desorption spectrometry is greater in the smaller molecular masses than in the larger molecular masses. Third, the fragmentation of a protein increases the number of signals from that target, thus increasing the probability of detecting that target. Fourth, the fragmentation of a protein increases the probability of capturing and, thus, detecting at least a fragment of the protein. Fifth, if a protein occurs differentially in two samples, then increasing the number of signals from that protein, the difference in the amount is more likely detected. Likewise, the method is counterintuitive. Generally, one seeks to decrease the complexity of an analyte mixture before analysis. Fragmentation increases complexity. Therefore, in one embodiment of this invention, the sensitivity of detecting an analyte is increased by converting the analyte to a fragment of a smaller molecular mass, prior to detection. Fragmentation can be achieved by any means known in the art. For example, protein analytes can be fragmented using endoproteases. Carbohydrate analytes can be fragmented using glycosidases. Nucleic acids can be fragmented using endonucleases. The sample may be subjected to fragmentation before or after attaching to the adsorbent.

J. Methods for Idfying League Compounds for a Recipient Functional trajectories in biological systems often involve the interaction between a receptor and a ligand compound. For example, the binding between transcription activation often involves the anterior binding of a ligand compound with a transcription factor. Many pathological conditions involve the abnormal interaction between a receptor and its ligand compound. The disruption of the binding between a receptor and a ligand compound is a frequgoal of drug discovery. However, the idty of a ligand compound for a receptor is often unknown; the receptor is an "orphan" receptor This invon provides a method for using retenate chromatography to idfy ligand compounds for receptors The method involves attaching a receptor to an adsorb then a suspect sample contains a compound of ligand for the receptor is exposed to the attached receptor under an appropriate elution condition to bind between the receptor and the ligand compound, then the ligand compounds that have bound to the receptor are detected by the desorption spectrometry. This method is derived, in part, from the sensitivity to desorption spectrometry, to detect small amounts of material adsorbed to an adsorbThe binding of the receptor to the adsorbrequires idfying an adsorbthat retains, and preferably, specifically binding the receptor. Methods for idfying adsorb that specifically bind to a protein are described here In one method, the adsorbit comprises an antibody specific for the receptor. In another embodim the receptor is produced as a recombinant fusion protein that includes a part for specific binding. For example, the receptor can be fused with the Fc portion of an antibody. Such portions bind to protein A, which can be incorporated into an adsorb The sample tested in the presence of a league compound is at the discretion of the practitioner. For example, if the receptor is a nuclear receptor, the sample can be a nuclear extract. If the receptor is a cytoplasmic receptor, the sample may be a cytoplasmic extract. If the receptor is a cell surface receptor, the sample can be fluid from the surface to which the cell is exposed, for example, the serum of an epithelial cell surface receptor. The sample will generally be incubated with the recipi under physiological conditions, for a sufficitime to allow binding, for example at 37 ° C, for several hours. Then, the unbonded material is washed and separated. This method can easily idfy ligamcompounds, which convonal techniques require months to idfy. The chromatography of the rette allows the parallel processing of samples in several adsorbzones. Therefore, this method may involve testing a plurality of differsamples for the presence of a ligand compound, as well as testing a single sample under a plurality of incubation and elution conditions. By determining the mass of the idfied ligand compound and the various physicochemical properties, the ligand compound can be positively idfied, using the information from the genome databases. In another embodimof this method, a set of probes is prepared, "which has been exposed to and has attached proteins from a cell.This probe is useful, by itself, as a secondary probe to idfy cell molecules, which binds to the attached molecules After preparing a map of the retenate from the probe, this probe is secondarily exposed to the test material, usually under less stringconditions than those used to prepare the secondary probe, and the blinking locations analyzed. which are recy attached to the probe are then attached to the molecules already attached.

K. Method for the Discovery of Drugs The identification of molecules that intervene in the binding between a receptor and its ligand compound is an important step in the development of drugs. That intervention provides a method of classifying compounds for their ability to modulate the binding between an adsorbent and an analyte (eg, an adsorbent-receptor and an analyte of the ligand compound) by exposing an adsorbent and analyte to a test compound and detecting the binding between the adsorbent and the analyte by desorption spectrometry. The rapid classification of combinatorial collections for drug candidates requires the ability to expose the target interactions to thousands of drugs is to identify the agents that interfere with or promote the interaction. The chromatography of the retenate makes it possible to attach a member of a pair of ligand / receptor compound to a substrate and use it as a secondary adsorbent. Then, after exposing the member to his partner and the agent, one can determine the desorption spectrometry when and to what extent the couple has joined. Advantages of the retenato chromatography in the classification methods include the ability to specifically attach the receptor to a substrate through an adsorbent, the ability to rapidly deploy the receptor over many zones of adsorbent for the parallel process, and the speed of production, which is possible by reading the results through. the desorption spectrometry. 1. Classification Test The method involves supplying an adsorbent; contacting the adsorbent with the target analyte, in the presence and absence of the agent, under one or more selectivity conditions, and determining whether the amount of binding with and without the agent. The amount of binding is determined by the chromatography of the retenate (for example, by the preparation of a recognition profile). The experiment can be carried out with a control, in which no agent is added, or a control in which a different amount or type of agent is added, and the amount zero is determined by extrapolation. A statistically significant difference in the amount of binding (p < 0.05) indicates that the binding modulates the test agent. This method is particularly useful in the classification of analytes (for example proteins) as target drug candidates. After the development of the protein retention map or the recognition profile of the serum or some other type of target cell, the agent is exposed to the arrangement of analytes retained in its blinking locations. After allowing the union, the unbound agent is eluted or washed and separated. Those analytes that retain the binding agent, under the specified selectivity conditions, are directly identified by desorption mass spectrometry, because the agent itself appears as a new component in the retention map (ie, the agent is desorbe and detect directly). This method is particularly useful for classifying drug candidates, both agonists and antagonists, for their ability to bind analytes or to modulate one or more biological processes. 2. Receptor and Ligated Compound The adsorbent and the target analyte do not need to be coupled in a specific binding. However, in particularly useful methods, the adsorbent and the target analyte are a ligate / receptor compound pair. In one embodiment, the pair * of ligand / receptor compound are a hormone and a cell surface receptor or an intracellular receptor. The adsorbent can be a whole cell or a cell membrane, in the case of a receptor bound to a membrane. A protein receptor or other drug target candidate can be used as an adsorbent. to classify combinatorial drug collections. Hundreds or thousands of drug candidates can be applied to a single type of receiver or to a steerable location. After the removal of unbound or weakly bound drug candidates (ie, the agents) the binding agents are detected e. identify by desorption spectrometry. In another embodiment, the adsorbent is an enzyme that binds and modifies the target substrate. The agents are classified in their ability to modulate the enzymatic transformation of the analyte. For example, enzymatic activity can be detected because the recognition profile of an analyte can differ from that of the product of the activity of the enzyme. Differential retention * indicates that the agent alters the junction. The receptor / ligate compound can be retained on the substrate in a variety of ways. In one method, the receptor / ligand compound is directly retained by a non-specific adsorbent. In another method, the adsorbent is specific for the receptor / ligand compound. For example, the adsorbent may contain an antibody specific for the receptor / ligand compound. This receptor / ligand compound can be a fusion protein in which the fusion part specifically binds to the adsorbent, for example, in the manner that an Fc fragment binds to protein A. In one method, a generic package , such as a phagocyte, from the phagocyte display collection, having on its surface a polypeptide, which binds specifically to the receptor / ligand compound, binds to the substrate. The ligand compound is captured by the polypeptide. Similarly, the adsorbent can be an analyte already attached to the substrate, ie it can be a secondary adsorbent, a tertiary adsorbent, etc. This invention provides a particularly useful method for evaluating the direct as well as indirect consequences of the drugs (or other agents) that bind to the target. The detection of one or more analytes in a retenate map, generated from the proteins of a target cell type, can be altered due to the action of the agent (for example, the drug candidate) in 1) the binding protein itself objective, 2) some other analyte (not the drug binding protein), or 3) a gene expression (up or down regulation). It is the power of high resolution and generation of information from the chromatherapy of the retenate that is used to detect these changes, that is, the induced drug differences in the generic retento map or the recognition profile, observed with and without the drug, which makes this method one of the most powerful tools available for proteomics, functional genomics, drug discovery, therapeutic drug surveillance and cynical diagnostics. 3. Test Agents A test agent that is to be classified in its ability to modulate protmosin expression was administered to a test animal or to cultured cells, in vivo. The selection of the agent to be tested is left to the discretion of the practitioner. However, due to their varieties and ease of administration as pharmaceuticals, small molecules are preferred as test agents. to. Chemistry The agent to be tested can be selected from a number of sources. For example, combinatorial collections of molecules are available for classification. Using such collections, thousands of molecules can be classified for regulatory activity. In a preferred embodiment, the high throughput classification methods involve supplying a collection containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical collections" are then classified into one or more assays, as described herein, to identify those members of the collection (particularly chemical species or subclasses) that exhibit a desired characteristic activity. The compounds, thus identified, can serve as conventional "guide compounds", or they can be used themselves as potential or actual therapeutic agents. The preparation and classification of combinatorial chemical collections is well known to those skilled in the art. "Such combinatorial chemical collections include, but are not limited to, collections of peptides (see U.S. Patent No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-492, 'Houghton et al (1991) Nature, 354: 64.88). Peptide synthesis is not by any means the only approach devised and intended for use with the present invention. Other chemical treatments can be used to generate a diversity of chemical collections. Such treatments include, but are not limited to: peptides (PCT Publication No. WO 91/19735, December 26, 1991), encoded peptides (PCT Publication WO 93/20242, October 14, "1993), bio-oligomers randomized (PCT Publication WO 92/00091, January 9, 1992), benzodiazepines (U.S. Patent No. 5,288,514), diversomers, such as hydantoins, benzodiazepines, and dipeptides (Hobbs et al. (1993) Proc. Nat. Acad. Chem. Soc. 114: 6568), non-peptide peptidomimetics with scaffold of beta-D-Glucose (Hirschmann et al, (1992) J. Amer. Chem.

Soc.114: 9217 -9218), analogous organic synthesis of collections of small compounds (Chen et al (1994) J. Amer. Chem.

Soc. 116: 2661), oligocarbamates (Cho et al. (1993), Science 261: 1303) and / or peptidyl phosphonates- (Campell et al. (1994) J. Org. Chem. 59: 658 (See, generally, Gordon et al., (1994-) J. Med. -Chem. 37: 1385, collections of nucleic acids, collections of nucleic acids and peptides (see, for example, U.S. Patent No. 5,539,083), antibody libraries (see, eg, Vaughn et al (1996) Na ture Biotechnology 14 (3): 309-3124) and PCT / US96 / 10287), carbohydrate collections (see, for example, Liang et al (1996) Sci ence, 274: 1520-1522 and US patent, No. 5,593,853) and collections of small organic molecules (see, for example , benzodiazepias, Baum (1993) C & EN, January 18, page 33, isoprenoids, U.S. Patent No. 5,569,588, thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974, pyrrolidines, U.S. Patent No. 5,525,735, and 5,519,134; morpholino, U.S. Patent No. 5,506,337, benzodiazepines, 5,288,514, and the like). The devices for the preparation of combinatorial collections are commercially available (see, for example, 357 MPS, 390 MPS, Advanced Chem Tech. Louisville KY, Symphony, Rainin, Woburn, MA, Applied Biosystems 433A, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).

L. Methods for Generating Uniting Agents Specifically to an Analyte This invention provides methods for generating agents, for example single chain antibodies, which specifically bind to an objective analyte. These agents are useful, for example, as specific diagnostic agents for attaching targets in the study of ligand / receptor compound interactions. The method is particularly useful for generating agents against targets that can only be isolated in such small amounts, that it is not possible or practical to generate antibodies by immunizing an animal. The method involves the steps of supplying a substrate that has an attached objective; provide a display collection of genetic packages that displays the agents to be classified; expose the collection to the target to specifically retain genetic packages through interaction with the target and detect genetic packages retained by desorption spectrometry. These steps can be conducted in parallel for a large number of adsorbent-analyte candidates within complex populations, without loss of transfer and ambiguities associated with separate selection and detection procedures, including off-line amplification and labeling strategies associated with indirect detection elements. 1. Substrate Supply The first step of the method involves the provision of a substrate comprising an adsorbent that will serve as a target for a polypeptide agent of an exhibit collection to be classified. In one embodiment, the substrate is provided with the objective adsorbent already attached. In another embodiment, the substrate is provided by supplying a substrate having an adsorbent that binds to an objective analyte, exposing the adsorbent to the analyte under elution conditions to allow retention of the analyte, and using the target adsorbent as the target. for the exhibition collection. In one embodiment, the objective is differentially expressed between two types of cells that are compared. For example, targets may be derived from differentially expressed mRNA or may be differentially expressed polypeptides. Methods of identifying such proteins differentially expressed by the methods of retentate chromatography were described above. Once a protein analyte, differentially expressed, is identified, one can develop a selectivity condition that unambiguously solves this analyte. More preferably, retention of the analyte is specific or exclusive. The methods for the progressive resolution of analytes, described above, make it possible to identify the conditions of selectivity that produce the binding specifically of an objective analyte from a complex sample. In one embodiment, the attached target can be modified, for example, by exposure to an enzyme. Alternatively, the method may start at the mRNA or EST lid. In this method, the mAR? or EST, differentially expressed, are identified by routine methods. Then, these molecules are transcribed and moved in vi tro and in si tu on an adsorbent to be attached. For example, a substrate for desorption spectrometry, which has a plurality of adsorbent zones, is prepared. The substrate is covered with a cylindrical tube, which creates a cavity with the adsorbent at the base of the same. In the cavity, one places reagents for transcription and translation in vi tro of differentially expressed mRNA. (usually in the form of the cAD?). (For methods, see, for example, Sambrookl et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,? Y. (1989) and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds. (Current Protocols, a partnership between Greene Publishng Associates, Inc., and John Wiley S Sons, Inc.)). The translation of the mAR? or EST produces a polypeptide that is adsorbed. The cylindrical tube is removed and the adsorbent zones are washed with an eluent, in order to "identify a selectivity condition that retains the analyte of the polypeptide. 2. Provision of the exhibition collection The second stage involves the supply of an exhibition collection. This display collection is comprised of genetic packages which exhibit on their surfaces multiple classes of combinatorial collections of peptides ("polypeptide agents"). However, single chain antibodies are attractive because they can be used in subsequent immunoassays. Many kinds of exhibition collections and their forms of use are known in the art. A basic concept of display methods is the establishment of a physical association between a ligand compound of the polypeptides to be classified and a recoverable polynucleotide, which encodes this polypeptide. This physical association is provided by a multimeric molecular complex, in this case the genetic package, for example the phagocyte particle, which exhibits a polypeptide as a part of a capsid that encloses the phagocyte genome which encodes the polypeptide. The establishment of a physical association between the polypeptides and their genetic material makes it possible to classify the simultaneous mass of very large numbers of genetic packages carrying different polypeptides. These genetic packages that exhibit a polypeptide with affinity to an objective link to the target and these packaging are enriched by the affinity classification to the target. The identity of the polypeptides displayed from these packages can be determined from their respective genomes. Using these methods, a polypeptide identified as having a binding affinity for a desired target can then be synthesized by volume by conventional means.

The genetic packages most frequently used to display collections are those of bacterifages, particularly filamentous phagocytes, and especially phagocytes M12, Fd and Fl. Most of the work has inserted collections "encoding polypeptides that are to be displayed in either Gil or GVIII of these phagocytes that form a fusion protein." See, eg, Dower, WO 91/19818, Devlin, WO 91/18989; MacCaffert, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII). See also Cwirla et al., Proc. Nati. Acad. Sci, USA 87, 6378 -. 6378 -6382 (1990); Devlin et al. , Science 249, 404-406 (1990), Scott & Smith, Science 249, 386-388 (1990); Ladner et al., US 5,223,409 and Ladner et al, US 5,571,698. Such a fusion protein comprises a signal sequence, usually from a protein secreted in addition to the protein that covers the phagocyte, a polypeptide to be exhibited and any of the protein of gene III or gene VIII or its fragment. Exogenous coding sequences are often instated at or near the N-terminus of gene II or gene VIII, although other insertion sites are possible. Some vectors of filamentous phagocytes have been planned to produce a second copy of either gene III or gene VIII. In such vectors, the exogenous sequences are inserted in only one of the two copies. The expression of the other copy effectively dilutes the proportion of the fusion protein incorporated in the phagocyte particles and it may be advantageous to reduce the selection against polypeptides harmful to the growth of phagocytes. The display of antibody fragments on the surface of viruses that infect bacteria (bacteriophages or phagocytes) makes it possible to produce human FVs with a wide variety of affinities and kinetic characteristics. In another variant, the exogenous polypeptide sequences are cloned into fagemid vectors, which encode a phagocyte coat protein and phagocyte packing sequences, but which are not capable of replication. The phagemids are introduced into cells and are packaged by infection with a phagocyte aid. The use of the fagemid system also has the effect of diluting the fusion proteins formed from the coating protein and the polypeptide exhibited with wild-type copies of the coating protein expressed from the helper phage. See, for example, Garrard, WO 92/09690. Eukaryotic viruses can be used to display polypeptides in an analogous manner. For example, the display of human heregulin fused to gp70 of murine Maloney leukemia virus has been reported by Han et al., Proc. Nati Acad. Sci. USA 92.9747-9751 (1995). The spores they can also be used as duplicable genetic packages. In this case, the polypeptides are displayed from the outer surface of the spore. For example, spores of B. subtilis have been reported as adequate. The coating protein sequences of these spores are provided by Donavan et al., J. Mol. Biol. 196, 1-10 (1987). The cells can also be used as duplicable genetic packages. The polypeptides to be displayed are inserted into a gene encoding a cellular protein, which is expressed on the surface of the cells. Bacterial cells include Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vbrio cholerae, Klebsiella pneumonia, Neisseria gonarrhoeae, Neisseria meningi tidis, Bacteroides nodosus, Moraxella bovis and especially Escherichia coli, are preferred. The details of the outer surface proteins are discussed by Ladner et al., US 5,571,698 and Georgiou et al., Nature.

Biotechnology 15, 29-324 (1997) and references cited therein. For example, the lamB protein of E. coli is adequate. 3. Classification of the exhibition collection The third stage involves classifying the exhibition collection to identify a league compound that specifically binds to the goal. The substrate that carries the objective is exposed to the exhibition collection, which exhibits polypeptide agents under appropriate elution conditions for specific binding between the polypeptide and the target molecule. Genetic packages that have agents that recognize the target binding to this goal have already been attached to the substrate. Removal of unbound particles and retention of bound particles results from exposure to the elution condition. A population of genetic packaging, in this case phagocyte M13, representing approximately 1011 plaque forming units (pfu) per ml, is introduced to a substrate with a targetable array of target binding adsorbents. (for example, proteins). At contact, a selectivity condition, optimized for the target adsorbent (i.e. the modifier or eluent of the selectivity threshold) is chosen, such that only a small subset of total phagocyte genotypes are selectively retained, preferably less than 5-10. . Note that the unbound phagocyte (ie, the phagocyte not bound to the target adsorbent) and the phagocyte loosely bound to the target adsorbent are removed by exposure to eluents that alter almost all interactions of the more selective target analyte-adsorbent. Those polypeptides that exhibit phagocytes with the hit affinity for the target adsorbent are selectively retained. 4. Detection of genetic binding packaging containing agents that specifically bind to the target In the fourth stage, the binding of genetic packaging to the target is detected by desorption spectrometry. For example, M13 phagocyte has thousands of copies of a single coat protein. When the phagocyte hits a laser in the desorption spectrometry, the coating protein becomes discharged and can be detected. In this way, one can determine if the collection contains a phagocyte that has an agent that binds to the target. In order to have genetic packaging for subsequent analysis, the classification step can be performed in parallel to different locations in a probe, or the substrate can have a sufficiently large physical dimension so that the laser does not dislodge all the genetic packages bound together to the surface. This method is particularly potent because even a few phagocytes attached to the analyte can be detected. In the case of M13, the preferred detection method is to monitor, by desorption spectrometry, the appearance of the coat protein of gene VIII as a "marker" protein signal. In this way, we have detected "positive" target adsorbents with a few, for example 5, phagocyte particles attached (number of phagocyte particles estimated by the calculation of known dilutions). Other phagocyte markers, in order of preference, include gene V, gene X and gene III (including their fusion products). After detection of those adsorbent locations with the hit affinity adsorbents, ie those locations within the array with minor phagocytes retained after exposure to high selectivity conditions (ie to say, strict eluentes), the union package can now be used as a point of "jump and separation for other uses.

. Isolation of the genetic package In one embodiment, the method also involves the isolation of the genetic package for further analysis. This analysis can involve the reproduction of genetic packaging and isolate the polynucleotides from it. The isolated phagocyte is reproduced by usual methods. For example, the retained phagocyte may be exposed to a biological amplification vehicle, eg, E. coli, plus nutrient media, for the growth of genetic packaging for subsequent analysis. Simple clones can also be tested for their ability to bind to the analyte retained on the substrate. 6. Sequence of nucleotides that encode the polypeptide agent The formation of the nucleotide sequence encoding the polypeptide agent of a linked genetic package provides information for producing the polypeptide agent. The sequence may involve isolating the genetic package from the adsorbent, reproducing it, isolating the polynucleotide and forming the nucleotide sequence by any available resource. In another method, genetic packaging can be reproduced in-itself by contacting the substrate with appropriate materials, such as cells subject to infection by the genetic package. In another modality, the sequence is performed in si tu. The method may involve lysis of the genetic packaging and amplification of the nucleotide sequence by any known resource, for example by PCR. Several different genetic packages may have been linked to different epitopes available on the surface. In this case, one can alter the elution conditions so that only one phagocyte class binds to an epitope. 7. Production of polypeptide agent A next valuable step involves producing the polypeptide agent. The isolated agent can be used, for example as an adsorbent, for specific detection of the target in diagnosis or for the study of ligand / receptor compound interactions. In one method, production of the polypeptide involves first forming the nucleotide sequence encoding the polypeptide. The amino acid sequence can be derived from the nucleotide sequence. The sequence can be achieved by the method described above. This sequence can be the basis of the recombinant or chemical synthesis of the polypeptide agent. In another method, the polypeptide can be produced by the reproduction of the genetic package. This is particularly effective when the genetic package contains many copies of the polypeptide agent. Genetic packaging can be reproduced in or after isolation. A method for producing the polypeptide recombinantly can be carried out as follows. The nucleotide sequence encoding the polypeptide is formed in sequence or isolated by any means, as discussed. Then, the nucleotide sequence is included in an expression vector. This expression vector contains an expression control sequence operably linked to the nucleotide sequence encoding the polypeptide. The expression vector can then be used to express the polypeptide agent recombinantly by means well known in the art. It will be understood that the target may contain more than one epitope. Therefore, the method can produce more than one polypeptide agent specific for the target. The specific agents of the target can then be used as adsorbents for probes used in clinical diagnostics or for drug discovery. That is, because such probes contain in their surface agents that specifically bind to the target, they can be used to isolate the target of complex mixtures, such as biological samples, and to detect the target by desorption spectrometry. Also, because the interaction between the agent and the target can be biospecific, it will probably imply a greater affinity between the two in comparison with an adsorbent developed by the progressive resolution method, described above. 8. Epitope isolation of a target peptide In one version, this method allows one to isolate the peptide epitopes from an objective analyte. The method uses an approach similar to an "anti-idiotypic". In summary, the epitopes of an objective analyte are classified with, for example, a collection of phagocyte display. The isolated phagocyte contains, for example, simple chain antibodies that recognize the epitopes of the analyte. These phagocytes are used, in turn, to classify a second exhibition collection. The phagocyte from the second collection that binds to the single chain antibodies of the first contains the polypeptides exhibited that resemble the structure of the epitope recognized by single chain antibodies. In one embodiment of this method, a nucleotide sequence encoding a polypeptide agent that binds the target analyte is used to produce the M13 phagocyte, in which the agent is displayed as a fusion with gene VIII. Thus, this phagocyte has a covering with hundreds of copies of the target peptide on its surface. This phagocyte is then attached to the adsorbent. This union can be achieved through, for example,. a ligand compound that binds to gene III, or this gene III can be modified to include a receptor for a ligand compound on the substrate. The phagocyte is then exposed to a second display collection. The genetic packaging of the collection that binds to the attached phagocyte is detected and isolated as described above. Preferably, the second display collection contains a mass label of some kind so that its gene VIII protein can be distinguished from gene VIII of the package attached to the substrate. A) Yes, the identification of a substance as an "analyte target", or as an adsorbent, could depend on whether the ligand substance is used, subsequently, for binding to another substance. As can be seen, the ability to attach a substance to an already attached substance can continue, as the methods can identify the conditions that selectively remove the terminally bound substance.

E J E M P L O S The following examples are presented in the form of illustration and in no way limitation. In the following examples, the following products and terms are used. Egg white lysozyme (1 μl diluted to 10 picomoles / μl of water), is available from Sigma Chemical Company, St. Louis, MO. "Human serum" refers to a composition of 1 μl of human serum diluted 1 to 20 mM of sodium phosphate buffer, 0.5 M NaCl, pH 7.0. As used herein, "mg" means milligrams; "ml" means milliliters; "μl" means microliters; "cm" means centimeters; "nm" means nanometers; "M" means molar; "mM" means millimolar; "min" means minutes; "%" or "percent" is the percentage by weight, unless otherwise specified: "NaCl" means sodium chloride; "TFA" means trifluoroacetic acid.

I. PROTOCOLS FOR THE CHROMATOGRAPHY OF THE RETENATE The following protocols are examples of procedures for carrying out the chromatography of the retenate.

A, Protocol for Retempt Mapping (with the use of the Chromatographic Series Array 1. Sample Treatment Dilute the biological sample (eg, serum, urine, cell extract or cell culture medium) in 0.01% Triton XlOO in HEPES or 20 M Na phosphate, pH 7.2. Centrifuge to clarify the sample, if necessary. 2. Application of the Sample Add the sample (1-5 μl) to an area of the Normal Anionic phase or TED-Cu (II) absorber arrangement. For a pre-moistened, hydrophobic adsorbent arrangement, "each zone with 0.5 μl of acetonitrile containing 0.5% TFA, add the sample to the area before the acetonitrile dries, allow the sample to concentrate (almost dry) on area. 3. Washing a. Arrangement of anionic adsorbent Wash zone 1 with 20 mM HEPES or Na phosphate, pH 7.2. Add the first 2 μl of wash solution to the area before the sample dries completely. Allow the solution to be washed to rest on the area for at least 15 sec. Pipette in and out of the area 10 times. Remove the first wash completely, repeat with the second wash of 2 μl of the solution. Wash zone 2 with 0.2 M NaCl in 20 mM Na phosphate, pH 7.2, as before. Wash zone 3 with 1 M NaCl in 20 M Na phosphate, pH 7.2 as before. Wash zone 4 with 20 mM TrisHCl. pH of 8.5 as before. Wash zone 5 with 0.1 M Na acetate, pH 4.5 as before. Wash zone 6 with 0.05% Triton XlOO in 20 m HEPES or Na-Phosphate, pH 7.2 as before. Wash zone 7 with 3 M urea in 20 mM HEPES or Na phosphate, pH 7.2 as before. Wash zone 8 with 10% acetonitrile in water, as before. Wash the entire arrangement completely with water. Air dry the fragment.

Add 0.3 μl of the Energy Adsorbing Molecule (saturated solution, prepared in 50% acetonitrile, 0.5% trifluoroacetic acid). Air dry the fxagmento. Analyze the protein retained in each zone with the laser desorption mass spectrometer / ionization time of flight. b. TED-Cu (II) adsorbent buffer Wash zone 1 with 5 mM HEPES pH 7. Add the first 2 μl of wash solution to the area before the sample dries completely. Allow the solution to be washed to rest on the area for at least 15 sec.

Pipette in and out of the area 10 times. Remove the first wash completely, repeat with the second wash of 2 μl of the solution. Wash zone 2 with 20 mM Na phosphate, 0.15 M NaCl, pH 7.2, as before. Wash zone 3 with 20 mM Na phosphate, 0.5 M NaCl, pH 7.2 as before. Wash zone 4 with 0.1 M Na acetate, pH of 4. 0 as before. Wash zone 5 with 0.05% Triton XlOO in 20 m Na phosphate, pH 7.2 as before. Wash zone 6 with 3 M urea in 20 mM Na phosphate, pH 7.2 as before.

Wash zone 7 with 1% TFA, as before. Wash zone 8 with 30% isopropanol: acetonitrile (1: 2) in water, as before. Wash the entire arrangement completely with water. Air dry the fragment. Add 0.3 μl of the Energy Adsorbing Molecule (saturated solution, prepared in 50% acetonitrile, 0.5% trifluoroacetic acid). Air dry the fragment. Analyze the protein retained in each zone with the laser desorption spectrometer / ionization time of the flight mass. c. TED-Cu (II) adsorbent arrangement Wash zone 1 with 20 mM Na phosphate, 0.5 M NaCl, pH 7.2. Add the first 2 μl of wash solution to the area before the sample dries completely.

Allow the solution to be washed to rest on the area for at least 15 sec. Pipette in and out of the area 10 times. Remove the first wash completely, repeat with the second wash of 2 μl of the solution. Wash zone 2 with 20 mM imidazole in 20 mM Na phosphate, 0.5 M NaCl, pH 7.2 as before. Wash zone 3 with 100 M imidazole in 20 mM Na phosphate, 0.5 M NaCl, pH 7.2 as before.

Wash zone 4 with 0.1 M Na acetate, 0.5 M NaCl, pH 4.0 as before. Wash zone 5 with 0.05% Triton XlOO in 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 as before. Wash zone 6 with 3 M urea in 20 mM Na phosphate, 0.15 M NaCl, pH 7.2 as before. Wash zone 7 with 1% FA, as before. Wash zone 8 with 10% acetonitrile in water, as before. Wash the entire arrangement completely with water. Air dry the fragment. Add 0.3 μl of the Energy Adsorbing Molecule (saturated solution, prepared in 50% acetonitrile, 0.5% trifluoroacetic acid). Air dry the fragment. Analyze the protein retained in each zone with the laser desorption spectrometer / ionization time of the flight mass. d. Hydrophobic Adsorbent Arrangement Wash zone 1 with 5% acetonitrile in 0.1% of TFA Add the first 2 μl of wash solution to the area before -the sample dries completely. Allow the solution to be washed to rest on the area for at least 15 sec. Transfer with pipette in and out of the po area 10 times. Remove the first wash completely, repeat with the second wash of 2 μl of the solution. Wash zone 2 with 50% acetonitrile in 0.1% TFA, as before. Wash zone 3 with 0.05% Triton XlOO in 20 mM HEPES or Na phosphate, 0.15 M NaCl, pH 7.2 as before. Wash zone 4 with 3 M urea in 20 mM Na phosphate, 0.15 M? ACl, pH 7.2 as before. Wash the entire arrangement completely with water. Air dry the fragment. Add 0.3 μl of the Energy Adsorbing Molecule (saturated solution, prepared in 50% acetonitrile, 0.5% trifluoroacetic acid). Air dry the fragment. Analyze the protein retained in each zone with the laser desorption spectrometer / ionization time of the flight mass.

B. Protocol for the Antigen Antibody Assay; Receptor Assay - Ligament compound (which uses the pre-activated adsorbent arrangement) 1. Immobilization of the Antibody on the Activated Adsorbent Arrangement Place a previously activated adsorbent array on a clean, flat surface. Place the antibody or receptor or control solution on each zone of a previously activated adsorbent array, previously moistened with 0.5 μl of isopropanol (add 1 μl of antibody / area before the isopropanol is dried). Incubate (4 ° C or at room temperature, 2-18 hours) in a humid chamber. Use a pipette to remove the remaining solution from the zones. Block the residual active sites on the zones, adding 1 ml of 1M eta-olamine, pH 7.4 in PBS, especially the fragment and incubate in a humid chamber (room temperature, 30 min.). Wash the fragments twice with 1% Triton X-100 in PBS. Immerse the fragment in about 9 ml of the wash solution in a 15 ml conical plastic tube and oscillate on the top bank shaker for at least 15 minutes. Wash with 0.5 M NaCl, in 0.1 M sodium acetate, pH 4.0 as before. Wash with 0.5 M NaCl, in 0.1 M TrisHl, pH 8.0 as before. Rinse with PBS as before. Then cover the fragment with PBS and store at 4 ° C until use. 2. Binding of the antigen or ligated compound Shake moderately or separate the PBS on the fragment. Add 1-5 μl of sample to each zone. For samples with a very low concentration of antigen or ligand compound, place the adsorbent array in a bioprocessor. Wash the areas in the fragment and the bioprocessor cavities with 200 μl of PBS twice. Add up to 300 μl of sample to each cavity, Seal with adhesive tape. Incubate shaking (4 ° C or room temperature, 1-18 hours). 3. Washing Remove the sample from the zones, wash each zone with 2 μl of 0.1% Triton XlOO in PBS, pH 7.2, twice. Add the first 2 ml of the washing solution to the area. Allow the wash solution to sit on the area for at least 15 seconds. Pipette in and out of the area 10 times. Completely remove the first wash, repeat the second wash with 2 μl of solution. This is followed by washing with 0.5 M NaCl in 0.1 M HEPES, pH 7.4. Wash all the "fix with water completely. 4. Analysis of retained proteins Dry the fragment in the air. Add 0.3 μl of Molecule that Absorbs Energy (saturated solution of Sinapinic Acid or EAMI or CHCA, prepared in 50% acetonitrile, 0.5% trifluoroacetic acid) Air dry the fragment. Analyze the protein retained in each zone with the laser desorption mass spectrometer / ionization time of flight.

II. LISOZIMA RECOGNITION PROFILE A recognition profile for lysozyme was generated using high-resolution resolution retenate chromatography. The profile includes the resolution of the lysozyme with six adsorbents, each under a variety of different selectivity threshold modifiers. The result is 40 different spectrographs that characterize differently the physical-chemical properties of lysozyme.

A. Lysozyme Recognition Profile Using a Hydrophilic Adsorbent Array The lysozyme of the chicken egg white was added to several zones of an adsorbent array of chromatographic series of a silicon oxide adsorbent on a stainless steel substrate. After incubation in a humidity chamber, at room temperature, for 15 minutes, each different zone of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 20 mM sodium phosphate buffer , pH 7.0, 2) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0, 3) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0, 4) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5, 5) 1% TFA, 6) 10% acetonitrile in water, 7) 20% acetonitrile in water, 8) 0.05% Tween 20 in 20 mM of sodium phosphate buffer, 0.15 M NaCl, pH 7.0 and 9) 3 M urea in 20 mM sodium phosphate buffer, pH 7.0. Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process was repeated with a fresh aliquot of washing solution. Next, the zone of the adsorbent was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) were added and allowed to air dry. The array was analyzed with the mass spectrometer, using a nitrogen laser (355 mm) and 60 cm of the flight tube. The data was analyzed by computer and taken to the GRAMS / 32c device (available from Galactc Industries Corporation) for the presentation of superimposed data. Figure 5A shows the composite mass spectrum of a lysozyme recognition profile in a normal phase chromatographic series adsorbent array. The background profile shows the intensity of. the signal of lysozyme retained in the silicon oxide adsorbent after washing with the regulator alone, H of 7. The inclusion of sodium chloride (0.2-0.4 M) in the selectivity threshold modifier decreased the retention of lysozyme . This indicates that the interaction of lysozyme (a basic protein) with a silicon oxide adsorbent (negatively charged, at a pH of 7) involves an ion exchange mechanism. The pH decrease of the selectivity threshold modifier, for example at a pH of 4.5, in the sodium acetate buffer, or < 2 in 1% of TFA, almost completely eliminates the negative charge in the silicon oxide adsorbent, and the lysozyme is no longer retained. Including polarity modulating agents (for example organic solvents (such as acetonitrile) or detergents (such as Tween 20) or urea in the selectivity threshold modifier, also reduces the interaction of lysozyme with the silicon oxide adsorbent. This indicates that the other mechanism of interaction involves a hydrophilic interaction.

B. Lysozyme Recognition Profile Using the Hydrophobic Adsorbent Arrangement Chicken egg white lysozyme was added to several zones of an adsorbent array of chromatographic series of polypropylene adsorbent (C3 hydrophobic), coated on a stainless steel substrate coated with silicon oxide. After incubation in a humid chamber at room temperature for 15 minutes, each different zone of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 0.1% TFA, 2) 10% a-ketonitrile in 0.1% TFA, 3) 20% acetonitrile in 0.1% TFA, 4) 50% acetonitrile in 0.1% TFA, 5) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and 6) 3 M urea in 10 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0 Each wash includes pipetting 1 μL of the wash solution, inside and outside from the area of the adsorbent, three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added, and allowed to air dry. The array was analyzed with a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32c device for the presentation of overlapping data. Figure 5B shows the composite mass spectrum of the lysozyme recognition profile in an adsorbent array of the C3 hydrophobic chromatographic series. The background profile shows the signal intensity of lysozyme retained in the C3 hydrophobic adsorbent, after washing with 0.1% TFA alone. The inclusion of a polarity-modulating agent (for example acetonitrile) in the selectivity threshold modifier decreases the retention of the lysozyme in the C3 hydrophobic adsorbent. The concentration range of acetonitrile for the elution of the lysozyme of the hydrophobic adsorbent C3 is between 20 and 50%.

The inclusion of detergent (Tween 20) or urea in the selectivity threshold modifier did not significantly reduce the retention of. the lysozyme in the C3 hydrophobic adsorbent.

C. Recognition Profile of Lysozyme Using a Phenyl Hydrophobic Adsorbent Arrangement Chicken egg white was added to several areas of a polystyrene (phenyl hydrophobic) adsorbent array coated on the silicon oxide-coated stainless steel substrate. After incubation in a humidity chamber, at room temperature, for 15 minutes, an area of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 0.1% TFA, 2) 10 % acetonitrile in 0.1% TFA, 3) 20% acetonitrile in 0.1% TFA, 4) 50% acetonitrile in 0.1% TFA, 5) 0.05% Tween 20 in 20mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0 and 6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0. Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The ar-reglo was analyzed with a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32c device for the presentation of overlapping data. Figure 5C shows the composite mass spectrum of the lysozyme recognition profile in an adsorbent array of the hydrophobic phenyl chromatographic series. The background profile shows the signal intensity of lysozyme retained in the hydrophobic phenyl adsorbent, after washing with 0.1% FA alone. The inclusion of a polarity-modulating agent (for example acetonitrile) in the selectivity threshold modifier decreases the retention of lysozyme. The concentration range of acetonitrile for the elution of the hydrophobic adsorbent C3 lysozyme is between 20 and 50%, however, when the peak intensities of the lysozyme is retained in C3 and the phenyl surface, they were compared under the same condition of washing 20% acetonitrile, the interaction of lysozyme with the phenyl adsorbent is less strong. The inclusion of detergent (Tween 20) or urea in the selectivity threshold modifier also significantly reduced the retention of lysozyme in the hydrophobic phenyl adsorbent.

D. Recognition Profile of Lysozyme Using an Anionic Adsorbent Arrangement Chicken egg white was added to several zones of an anionic group (S03) adsorbent arrangement (i.e. a cation exchange adsorbent) coated onto the stainless steel substrate coated with silicon oxide. After incubation in a humidity chamber, at room temperature, for 15 minutes, each different zone of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 20 mM Phosphate Regulator sodium, pH 7.0, 2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0, 3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0, 4) 0.4 M of NaCl in 20 mM of sodium phosphate buffer, pH of 7.0, 5) 25 mM of sodium acetate buffer, 0.125 M NaCl, pH 4.5, 6) 0.05% of Tween 20 in 20 mM phosphate buffer sodium, 0.15 M NaCl, pH 7.0 and 7) 3 M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0. Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The array was analyzed with a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32C device for the presentation of superimposed data. Figure 5D shows the composite mass spectrum of the lysozyme recognition profile in an adsorbent array of the cation exchange chromatographic series. The background profile shows the signal intensity of lysozyme retained in the anionic adsorbent, after washing with the pH-only regulator of 7. The inclusion of increasing concentrations of sodium chloride (0.1-0.4 M) in the threshold modifier of selectivity, decreased retention of lysozyme This indicates that the interaction of lysozyme (a basic protein) with the anionic adsorbent involves an ion exchange mechanism.A concentration of 0.4 M NaCl is required to elute lysozyme. The pH decrease of the selectivity threshold modifier at a pH of 4.5 in the sodium acetate buffer did not affect the retention of the lysozyme in a strong anionic adsorbent.The inclusion of polarity modulating agent (e.g. detergent., such as Tween 20 or urea) in the selectivity threshold modifier reduces the interaction of lysozyme with the anionic adsorbent.This indicates that the interaction of a prot The hydrophobic lysozyme with the anionic adsorbent is modulated by the polarity of the eluent.

E. Lysozyme Recognition Profile Using a Cationic Adsorption Arrangement Chicken egg white was added to several zones of an array of cationic group (quaternary amine) adsorbent coated on the stainless steel substrate coated with silicon oxide. After incubation in a humidity chamber, at room temperature, for 15 minutes, each different zone of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 20 mM Phosphate Regulator sodium, pH 7.0, 2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0, 3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0, 4) 0.4 M of NaCl in 20 mM of sodium phosphate buffer, pH of 7.0, 5) 25 mM of sodium acetate buffer, 0.125 M NaCl, pH 4.5, 6) 0.05% of Tween 20 in 20 mM phosphate buffer sodium, 0.15 M NaCl, pH 7.0 and 7) 3 M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0. Each wash includes pipetting 1 μl of wash solution, inside and outside the area of the adsorbent, three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The array was analyzed with a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32c device for the presentation of overlapping data. Figure 5E shows the composite mass spectrum of the lysozyme recognition profile in an adsorbent array of the chromatographic series of cationic adsorbent (anion exchange). The retention of the basic lysozyme protein in the cationic adsorbent is very weak. The effect of modulating the modifiers of the selectivity threshold in the retention of lysozyme is minimal.

F. Recognition Profile of the Lysozyme Using an Arrangement of Immobilized Metal Ion Adsorbent Chicken egg white was added to several areas of an adsorbent arrangement of an immobilized metal adsorbent (iminodiacetate-Cu) coated on the silicon oxide-coated stainless steel substrate.

After incubation in a humidity chamber, at room temperature, for 15 minutes, each different zone of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, 2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, 3) 0.1M sodium acetate buffer, 0.5M NaCl, pH 4.5, 4) 0.05% Tween 20 in 20mM sodium phosphate buffer, 0.15M NaCl, pH 7.0 and 5 ) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The array was analyzed with a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32C device for the presentation of superimposed data. Figure 5F shows the composite mass spectrum of the lysozyme recognition profile in an adsorbent array of the immobilized metal chromatographic series. The background profile shows the signal intensity of lysozyme retained in the immobilized adsorbent of copper ions, after washing with the regulator only with pH of 7 .. The inclusion of competitive binding affinity ligand to histidine (by example imidazole) in the modifier of the selectivity threshold, the retention of lysozyme dased. This indicates that the interaction of lysozyme "(which has a single histidine residue in the sequence) with the immobilized adsorbent of copper ions, implies a coordinated covalent binding mechanism.

Dasing the pH of the modifier of the selectivity threshold to a pH of 4.5 in the sodium acetate buffer also dases the retention of the lysozyme in the immobilized copper adsorbent. It is believed that this is the result of the protonation of the histidine residue in lysozyme, which inhibits coordinated covalent interaction. The inclusion of a detergent (for example Tween 20) did not affect the interaction. The inclusion of urea completely eliminated the retention of lysozyme in the immobilized copper adsorbent.

III. RESOLUTION OF ANALYSTS IN THE HUMAN SERUM We solved the analytes in human serum using a variety of adsorbents and eluents. These results show that the analytes are differentially retained by different adsorbents and that retention chromatography is capable of supplying information in both low and high molecular masses.

A. Recognition Profile of Human Serum Protein Using the Ion Adsorbent Arrangement Immobilized Metals Human serum was added to several areas of an adsorbent array of an immobilized metal ion adsorbent (tris (carboxymethyl) ethylenediamine-Cu), coated on a stainless steel substrate coated with silicon oxide. After incubation in a humidity chamber at room temperature for 15 minutes, each different zone of adsorbent was washed with one of the following eluents (selectivity threshold modifiers): 1) 20 mM Phosphate Regulator sodium, 0.5 M NaCl, pH 7.0, 2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, 3) M sodium regulator, 0.5 M NaCl, pH 4.5, 4) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and 5) 3M of urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0. Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The array was analyzed with a mass spectrometer using a nitrogen laser (355 nra) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32c device for the presentation of overlapping data. Figures 6A and 6B show the composite mass spectrum at a low and high molecular mass of the serum protein recognition profile in the adsorbent array of the immobilized metal chromatographic series. The background profile shows the serum proteins retained in the immobilized copper adsorbent, after washing with a regulator only of pH 7. The inclusion of the competitive binding affinity ligand of histidine (for example imidazole) , or the detergent (for example, Tween 20) or the urea in the selectivity threshold modifier, or the pH decrease of the selectivity threshold modifier to 4.5, differentially increased or decreased the retention of different components of the complex mixture of proteins in the same adsorbent.

B. Recognition Profile of Human Serum Protein Using a Plurality of Different Adsorbents Human serum was added to several zones of an array of adsorbents of the following different adsorbents-1) hydrophobic C, 2) phenyl hydrophobic, ) exchange of anions 4) exchange of cations, and 5) immobilized metal (tris (carboxymethyl) ethylenediamine-Cu) Each adsorbent was coated on a stainless steel substrate coated with silicon oxide. After incubation in a humidity chamber, at room temperature, for 15 minutes, each zone of adsorbent was washed with 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, as the selectivity threshold modifier. Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The array was analyzed with a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32c device for the presentation of overlapping data. Figures 7A and 7B show the composite mass spectrum of the serum protein recognition profile in various adsorbents of the adsorbent array of the chromatographic series. The use of only one selectivity threshold modifier in a plurality of different adsorbents (which have different binding characteristics) differentially increases or decreases the retention of different components of the complex mixture of proteins in the different adsorbents.

IV. RESOLUTION OF PREVIOUS PERIOD CHILD ANALYTIC ANALYTS We solved analytes in urine from a previous period using a variety of adsorbents and eluents. These results show that because the adsorbents retain the analytes differentially, the use of several adsorbents provides greater resolution ability. They also show the ability to identify adsorbents that preferably retain specific analytes, which is useful for developing purification schemes.

A. Resolution of Analytes in the Urine of Infancy of Previous Period, which Uses a Variety of Adsorbents and the Same Eluente (Water) Prior period child urine (2 μl) was added to several zones of a carbonized PEEK polymer substrate, coated with the following different adsorbents: 1) C8 hydrophobic (octyl sepharose-, available from Sigma) , 2) phenyl hydrophobic (phenyl sepharose, available from Sigma), 3) anion exchange (Sepharose Q, available from Sigma), 4) cation exchange (Sepharose S, available from Sigma), 5) immobilized metal (IDA) Cu, Chelation Sepharose, available from Pharmacia) and 6) immobilized metal (tris (carboxymethyl) -ethylenediamine-Cu sepharose After incubation in a humid chamber, at room temperature for 15 minutes, each zone of adsorbent was washed with water as the selectivity threshold modifier Each wash includes pipetting 1 μl of wash solution into and out of the adsorbent zone three times.This is repeated with a fresh aliquot of wash solution. 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5 TFA) was added and allowed to air dry The array was analyzed with a laser desorption mass spectrometer / time ionization flight from Hewlett Packard ( Model 2 030), which uses a nitrogen laser (355 nm) and a 150 cm flight tube. The data was analyzed by the HP MALDI TOF software (program) and taken to the GRAMS / 32c device for the presentation of superimposed data. Figures 8A and 8B show the composite mass spectrum in low and high molecular masses of the protein recognition profile of the child's urine in the various absorbers of a chromatographic series. The use of a single selectivity threshold modifier (ie, water) in the various adsorbents (each with different binding characteristics) differentially increases or decreases the retention of the different components of the complex protein mixture as in the different adsorbents.

E. Resolution of Analytes in Child's Urine of Previous Period, Using a Hydrophobic Phenyl Adsorbent, Indirectly Coupled to the Substrate, and Three Different Eluents Child urine from the previous period (2 μl) was added to several zones of a carbonized PEEK polymer substrate, coated with a hydrophobic phenyl adsorbent (phenyl Sepharose, available from Sigma). After incubation in a humid chamber, at room temperature for 15 minutes, each zone of adsorbent was washed with one of the following eluents (threshold selectivity modifiers): 1) water, 2) 2M urea in 20 mM of sodium phosphate buffer, 0.15 M NaCl, pH 7.0 and 3) 0.1% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0. Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times. This process is repeated with a fresh aliquot of washing solution. Next, the adsorbent zone was washed with 1 μl of water twice. An aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and allowed to air dry. The array was analyzed with a laser desorption mass / ionization time-of-flight spectrometer from Hewlett Packard (Model 2030) using a nitrogen laser (355 nm) and a 150 cm flight tube. The data was analyzed by the HP MALDI TOF software (program) and taken to a GRAMS / 32c device for the presentation of superimposed data. Figures 9 shows the composite mass spectrum of the protein recognition profile in the hydrophobic phenyl adsorbent of a chromatographic series. The application of several eluents, which have different elution characteristics in a single adsorbent, differentially increases or decreases the retention of different components of the complex protein mixture. One of the components (marked by *) was selectively retained in the hydrophobic phenyl adsorbent, when 0.1% Tween 20 in PBS was used as the eluent.

V. IDENTIFICATION OF PROTEINS IN A CULTURE MEDIUM OF TWO DIFFERENT LIVING CELLS This example illustrates the identification of proteins that are differentially expressed in cells with the adsorbent array: Chromatographic Series. Two different cancer cell lines of breast cancer were cultured for the same period of time, in a constant composition culture medium. After concentration with a filtration unit, an aliquot of 1 μl of each culture medium was added to several areas of an adsorbent array (Ciphergen Biosystems, Inc., Palo Alto, CA) of immobilized metal adsorbent (tris). (carboxymethyl) ethylenediamine-Cu) coated on a stainless steel substrate coated with silicon oxide. After incubation at room temperature in a humidity chamber for 15"minutes, an area of adsorbent was washed with any of the following eluents (threshold selectivity modifiers): 1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, 2) 20 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, 3) M sodium regulator, 0.5 M NaCl, pH 4.5, 4) 0.1% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, 5) 3 M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, and 6) 1% TFA Each wash includes pipetting 1 μl of the wash solution into and out of the adsorbent zone three times, this process is repeated with a fresh aliquot of wash solution. Next, the adsorbent zone was washed with 1 μl of water twice, an aliquot of 0.3 μl of sinapinic acid (5 mg / ml 50% acetonitrile). : 0.5% TFA) was added and allowed to air dry. The array was analyzed with a laser desorption mass spectrometer / ionization time-of-flight, using a nitrogen laser (355 nm) and a 60 cm flight tube. The data was analyzed by computer and taken to a GRAMS / 32c device (Galactic Industries Corporation) for the presentation of superimposed data. Figure 10A shows the composite mass spectrum of the secreted protein recognition profile of the cell line cell 1 in an array of chromatographic series adsorbent of immobilized metal (Cu). The application of the various eluents of different selectivity thresholds in a single adsorbent, differentially increases or decreases the retention of different components of a complex protein mixture as the cell culture medium. Figure 10B shows the mass spectrum composed of recognition profiles of secreted proteins from cells of both cell lines in an array of immobilized metal (Cu) chromatographic series adsorbent. The same eluent, 0.1% Tween 20 + 3 urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, was used in washing and separating non-retained materials. The ridge marked 7532 Da is the major ridge retained in the secreted protein of cell line 1, which is not expressed in cell line 2. Figure 10D shows the mass spectrum composed of recognition profiles of the secreted protein of cell lines. Cell line 1 in an array of chromatographic series adsorbent immobilized metal (Ni). Using the same eluent, 0.1% of Tween 20 + 3 M of urea in lOmnM of sodium phosphate regulator, 0.5 M of NaCl, pH of 7.0, but using an adsorbent of different surface interaction potential (ie Ni metal). immobilized vs. immobilized Cu metal), the crest of 7532 Da is the only protein retained among all the secreted proteins of cell line 1. The insert shows the same mass spectrum on an expanded scale. The highest peak of 3766 Da is the doubly charged species of the same protein. Figure 10D shows the mass spectrum composed of the recognition profiles of the secreted protein of cells of cell line 1 in an array of immobilized metal chromatographic series adsorbents (Ni), before (lower profile) and after (upper profile) diion of trypsin in si tu. The peptide map generated for a pure protein is a trace of that protein and can be used for identification.

SAW. COMPARISON OF CHROMATOGRAPHY OF THE RETENATO WITH L ELECTROFORESIS DE GEL 2D An advantage of di-retene chromatography is the ability to quickly resolve analytes in a variety of dimensions, which results in the high content of information about a variety of physicochemical characteristics. In contrast, 2D gel electrophoresis provides resolution in only two dimensions. Figure 11 shows a protein recognition profile of infant urine from the previous period, in the hydrophobic phenyl adsorbent of a chromatographic series. The application of several adsorbent eluents provided multiple-dimensional information. The use of different selectivity conditions differentially increases or decreases the retention of the various components of a complex protein mixture (such as precursor child's urine), which results in the detailed resolution of the analytes. In contrast, Figure 12 shows a two-dimensional separation of proteins in the infant urine from the previous period, according to the pl and the molecular mass. The gel provides information about two "dimensions, only, compared to the six dimensions used as adsorbents in the chromatography of the retene.The zones do not resolve well as by mass spectroscopy and resolution at very high and very low molecular masses. It is limited.

VII. EXTRACTION IN ANALYTIC SEQUENCE OF A SAMPLE The analytes can be extracted in sequence from a sample by the serial exposure of the sample to a selectivity condition, followed by the collection of the non-retained sample. A mutant lysate of a decisive type was prepared Hemophilus in 10% glycerol 50 mM EDTA. After centrifugation, the supernatant was diluted 1: 3 in 0.01% Triton XlOO in 25 mM HEPES, pH 7.4. An aliquot of 2 μl of the diluted sample was added to an anionic site zone of the adsorbent array. After incubation at room temperature for 30 minutes, the sample remaining at the anionic site was transferred to an adsorbent array area at a normal phase site. The zone of the anionic site was washed with 2 μl of 0.01% Triton XlOO 25 mM HEPES twice. Each wash was accomplished by pipetting the wash solution in and out of the area by ten times. The washes were combined with the sample initially added in the normal phase zone. After incubation at room temperature for 30 minutes, the sample remaining at the normal phase site was transferred to an area of the Ni (II) site of adsorbent array. The site area of the normal fas was washed with 2 μl of a phosphate buffered saline, twice. Each wash was accomplished by pipetting the wash solution into and out of 1 zone for ten times. The washings were combined with the sample initially added in the Ni (II) zone.

After the sample was concentrated to near dryness in the Ni (II) zone, the unbound analytes were recovered by washing with 2 μl of 100 mM imidazole in the phosphate buffered saline, twice. Each wash was accomplished by pipetting the wash solution in and out of the area for ten times. The washes were transferred to an area of the hydrophobic aliphatic site of the adsorbent array. The sample was allowed to concentrate almost to dryness on the hydrophobic site, the unbound analytes were removed by washing with 2 μl of 5% acetonitrile in 0.1% trifluoroacetic acid twice. Each wash was accomplished by pipetting the wash solution in and out of the area by ten times. Each zone of normal anionic phase, Ni (II), and hydrophobic site was washed with 2 μl of water to remove the remaining regulator .. An aliquot of 0.3 μl of a solution of synaptic acid in 50% acetonitrile, 0.5% of trifluoroacetic acid was added to each zone. The analytes retained at each site were analyzed by the laser desorption mass spectrometer / ionization flight time. Figures 19A and 19D show the retention map of the Hemophilus lysate in the adsorbent array. Multiple crests in the mass range of 3000 to 25,000 Da were observed in the adsorbents. Note that each absorber showed different retention for each of the analytes in the sample.

VIII. PROGRESSIVE RESOLUTION OF AN ANALYZE By adding new elution binding characteristics to a selectivity condition that solves an analyte, one can develop a selectivide condition that provides improved analyte resolution. In this example, a sample was attached to a Cu (II) adsorbent and exposed to a first eluent and two second eluents. The second eluents differed from the first by the addition of another elution condition. Each added condition improved the resolution of the analyte. The silvestr type stationary phase lysate Hemophilus, prepared in 10% glycerol, was diluted 1: 1 and mM sodium phosphate, 0.5 M sodium chloride, pH d 7.0. After centrifugation, an aliquot of 150 μl of the supernatant was incubated in each zone of the adsorbent array at the Cu (II) site in a bioprocessor. After mixing cold for 30 minutes, the sample was removed. Each zone was washed with a different lysate. A first zone was washed with 150 μl of 20 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0. A second zone was washed with 150 μl of 0.05% Triton XlOO in addition to 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0. A third zone was washed with 150 μl of 100 mM imidazole, in addition to 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0. Each wash was accompanied by incubating the washing solution with the zone for 5 minutes, with mixing. The washing was repeated twice. Each zone was washed with water to remove the detergent and regulator. The adsorbent array was removed from the bioprocessor. An aliquot of 0.3 μl of sinapinic acid solution in 50% acetonitrile 0.5% trifluoroacetic acid was added to each zone. The analytes retained in each zone were analyzed with the laser desorption mass spectrometer / ionization flight time. Figures 20A-20C show the retention map of the Hemophilus lysate in the Cu (II) site of the adsorbent array, after washing under the three elution conditions described above. Multiple crests in the mass range of 2000 to 18000 Da were observed. The protein marked with an "*" was only a minor component in the retention map of Figure 20A. When the selectivity condition was modified by the addition of a detergent, Triton XlOO (Figure 20B), to the same regulator, the same "*" protein was retained better than the other analytes and resolved better. When the selectivity condition was modified by the addition of an affinity displacer, imidazole, to the same regulator (Figure 20C), the "*" protein was highly resolved from the other analytes on the retenate map. This strategy of progressive identification of selectivity conditions with improved resolution for an analyte can be adopted to develop a method for the preparative purification of this protein from the total Hemophilus lysate.

X. DIFFERENTIAL EXPRESSION OF AN ANALYZE: DISCOVER THE MARKER PROTEIN a. Human serum An aliquot of 0.5 μl of normal and diseased human serum was diluted with an equal volume of 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0. Each was applied to a different zone at a Cu (II) site of adsorbent arrangement. After incubation at 4 ° C for 1 hour, each zone was washed with 2 μl of 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0, twice. Washed house was accompanied by pipetting the wash solution in and out of the area for ten times. Each zone was thoroughly washed with 2 μl of water to remove the remaining regulator. An aliquot of 0.3 μl of the sinapinic acid solution in 50% acetonitrile 0.5% trifluoroacetic acid was added to each zone. The analytes retained in each zone were analyzed with the spectrometer of. laser desorption mass / ionization flight time. The proteins marked with "*" in Figure 21D are present in significantly larger amounts in the diseased serum than in the normal serum. The results illustrate a method to discover the markers of diseases that can be used in clinical diagnosis.

B. Mouse Urine An aliquot of 1 μl of urine from the normal mouse, sick or treated with a drug, was applied to a different area of a Cu (II) site of adsorbent array. After incubation at room temperature for 10 minutes, each zone was washed with 2 μl of 100 mM imidazole in 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0, twice. Each wash was accompanied by pipetting the wash solution in and out of the area ten times. Each zone was finely washed with 2 μl of water to remove the remaining regulator. An aliquot of 0.3 μl of sinapinic acid solution in 50% acetonitrile 0.5% trifluoroacetic acid was added to each zone. The analytes retained in each zone were analyzed with a laser desorption mass / ionization time-of-flight spectrometer. The maps of the urine renate of the normal mouse (control), sick and treated with drugs, are shown in Figure 1. An analyte was found to be present in a much larger amount in the urine of the sick mouse (middle panel), the same analyte was not found in the urine of the normal mouse (upper panel) and was found in the urine of the mouse treated with drug in a very small amount (bottom panel). This analyte can be used as a potential marker of disease. To illustrate the feasibility of a quantitative diagnostic assay, the area under the protein crest of the retained marker was calculated and shown in the table. A clear quantitative difference was observed between the urine of sick and drug-treated mice. To compensate for the experimental variability, an internal standard analyte was used. The peak area of the normalized disease marker (ie, the peak area of the marker divided by the crest area of the internal standard) for each urine sample was presented in the background panel. There is at least a tenfold reduction of the urine marker of the patient after treatment with the drug.

Human Urine Urines from normal humans and from cancer patients were diluted 1: 2 in 0.01% Triton XlOO in a phosphate buffered saline solution. An aliquot of 1.5 μl of normal or patient human urine was applied to a different zone of an aliphatic hydrophobic site of adsorbent array, previously wetted with 0.5 μl of isopropanol / acetonitrile (1: 2) 0.1% trifluoroacetic acid. After incubation at 4 ° C for 30 minutes, each zone was washed with 2 μl of 50% ethylene glycol in 10 mM TrisHCl, 0.05 M NaCl, pH 7.5, twice. Each wash was accompanied by pipetting the wash solution in and out of the area ten times. Each zone was finally washed with 2 μl of water to remove the ethylene glycol remanent and regulator. An aliquot of 0.3 μl of sinapinic acid solution in 50% acetonitrile 0.5% trifluoroacetic acid was added to each zone. The analytes retained in each zone were analyzed with the laser desorption mass spectrometer / ionization time of flight. The urine retenate maps of four cancer patients and a normal human are shown in Figure < 23A. Multiple protein ridges were retained in the hydrophobic site of the adsorbent array after washing with 50% ethylene glycol in Tris / NaCl buffer. To map potential disease markers, map differences were projected between the individual patient's urine and normal urine. Each bar in the projection of difference above the baseline represents an analyte present in a larger amount in the patient's urine. (Figures 23B-23D). The variation in patterns of difference of patient maps reflect individual fluctuations in a population. However, an analyte of around 5000 Da (marked with *) and a group of analytes of around 7500 Da (marked with *) was found to be consistently present in larger amounts in all patients, therefore, they can be identify as potential markers of disease.

X. CAPTURE OF FAGOCITS OF THE EXHIBIT COLLECTION OF FAGOCYTES Viruses adsorbed to the surface of a protein fragment can be detected by desorption spectrometry. Antibodies against viral coat proteins, used as adsorbents, can capture viruses. An obj ective protein used as an adsorbent can capture phagocytes that exhibit a single chain antibody against the target.

A. Sensitivity of detection of the Antibody of Phagocyte display by the Adsorbent Substrate The M13 phagocyte (1012 particles / ml) in the growth medium was serially diluted in 0.01% Triton XlOO in 25 mM HEPES, pH 7.4. An aliquot of 0.25 μl of each diluted phagocyte suspension was added to an area of an aliphatic hydrophobic site of adsorbent array. An aliquot of 0.3 μl of CHCA in 50% acetonitrile, 0.5% trifluoroacetic acid, was added. The samples were analyzed by the laser desorption mass spectrometer / ionization time of flight. Gene VIII protein of M13 s phagocyte detected with high sensitivity in the array. Figures 24A-24E. A detectable signal (signal / noise> 2) was obtained when the phagocyte suspension was diluted 10,000,000 times.

B. Identification of Phagocyte M13 by the Arregl Adsorbent Serum anti-M13 rabbit (Stratagene) s immobilized on Hyper D Protein A (BioSepra) and washed with a phosphate buffered saline, pH of 7 m, extensively. An aliquot of 1-10 μl of M13 phagocyte suspension (1012 particles / ml) in the growth medium was incubated with 1 μl aliquot of immobilized anti-M13 antibody at 4 ° C overnight. After washing with 0.05% Tween 20 of a phosphate buffered saline solution, pH 7, and then with water to remove the detergent and regulator, an aliquot of the captured phagocyte was analyzed by the laser desorption mass spectrometer. flight time of the ionization, in the presence of sinapinic acid.

The anti-M13 antibody control showed only the antibody signal (charged in a simple and double manner). Figure 25A. When the M13 phagocyte was captured by the antibody, the most easily identifiable protein crests of the phagocyte are the Gene VIII protein and the Gene III protein fused with a single-chain antibody. Figure 25B. Since Gene VIII protein from phagocyte M13 was detected with high efficiency by the method, it can be used as a sensitive monitor of phagocyte capture. C. Specific capture of M13 phagocyte exhibiting the single chain antibody The HIV-1 Tat protein (McKesson BioServices) was coupled to a previously activated substrate. After blocking with ethanolamine, the array was washed with 0.005 Tween 20 in phosphate buffered saline, p 7, and then 0.1% BSA in phosphate buffered saline, pH 7.0. A serial dilution of the single chain antibody exhibiting the M13 phagocyte against the Tat protein was incubated with the Tat protein adsorbent array at 4 ° C overnight. A negative control of a serial dilution of the M13 phagocyte, which did not exhibit the single chain antibody against the Tat protein, was also incubated with the Tat protein adsorbent arrangement in the same manner. The arrays were washed with 0.05% d Tween 20 in phosphate buffered saline, followed by 1 M urea in the phosphate buffered saline, pH 7.0, and finally with water to remove the urea regulator. An aliquot of 0.3 μl of CHCA in 50% acetonitrile 0.5% trifluoroacetic acid was added. The retained phagocyte was analyzed by the mass spectrometer of laser desorption / time of flight of the ionization. A specific binding of the single-chain antibody exhibiting the M13 phagocyte against the Tat protein was observed in a concentration-dependent manner (solid line). Figures 26A-26D. Non-specific binding by a non-specific M13 phagocyte was minimal in the adsorbent array (line in dashes). These results illustrate a very sensitive method of detecting a phagocyte that contains a gene encoding a single chain antibody that specifically recognizes an objective analyte.

XI, CLASSIFICATION TO DETERMINE IF A COMPUEST INHIBITS THE UNION BETWEEN THE RECEIVER AND THE COMPOUND OF LEAGUE The methods of this invention can be used to determine whether a test agent modulates the binding of a ligand compound to a receptor. In this example, we show that retentate chromatography can detect the inhibition of binding between "TGF-β and the bound TGF-receptor, used as an adsorbent by the TGF-free receptor." The recombinant Fc receptor TGF-β (R &D, Minnesota) bound specifically to an Adsorbent Protein G arrangement. TGF-β (R &D, Minnesota) was serially diluted in a conditioned cell medium (2.5 concentrate) and incubated with the array of the G-protein adsorbent from the Fc receptor at 4 ° C overnight.Other set of TGF-β diluted serially in the cell conditioned medium was incubated with the adsorbent array of Protein G of the Fc receptor, in the presence of a modulating agent In this illustration, the modulating agent was the free TGF-β receptor After incubation under the same conditions, the fragments were washed with 0.05% d Triton XlOO in PBS and then 3 M reactive in PBS. aliquot of 0.3 μl of sinapinic acid was added to each zon and analyzed by laser d desorption mass spectrometry / ionization flight time. Figure 27 shows the specific binding of 1 μg / ml of TGFβ to the adsorbent array of Protein G of the Fc receptor (solid line). Few or no proteins in the cellular conditioned medium were found to bind. Figur 27B shows the specific binding of 100 ng / ml of TGF-β to the adsorbent array of Protein G of the F receptor (solid line). When incubation of TGF-β the adsorbent array of Protein G of the Fc receptor, in the presence of a modulating agent (TGF-free receptor), the binding was completely eliminated when there was 100 ng / ml of TGF- ß (Figure 27A, dashed line) and only one binding trace where there was 1 μg / ml of TGF-β (Figure 27B, dashed line). In this illustration, the modulated agent (the same receptor) has a specific binding affinity for the ligand compound, thus offering a very effective composition of the binding event of the target analyte. In the other cases, the ratio of the binding of the target analyte to the adsorbent, in the presence and absence of modulating agent, gives an indication of the effectiveness of the modulating agent.

XII. POWER OF RESOLUTION OF THE CHROMATOGRAPHY OF RETENATE This example demonstrates the ability of deltetete chromatography, with its parallel process of a sample under different selectivity conditions, to resolve proteins in a sample. The lysate of Hemophilus influenzae e was prepared % glycerol. After centrifugation, the supernatant was diluted 1: 3 in 0.01% Triton XlOO in 25 m HEPES, pH 7.4. An aliquot of 2 μl of the diluted sample was added to an area of the cationic site of adsorbent array. After incubation at room temperature for 30 minutes, the zone was washed with 25 mM HEPES, pH 7.4. A second aliquot of μl of the diluted sample was added to an area of the aliphatic hydrophobic site of the adsorbent array. After incubation at room temperature for 30 minutes, the area was washed with water. A third aliquot of 2 μ of the diluted sample was added to an area of the Cu (II) site of the adsorbent array. After incubation at room temperature for 30 minutes, the zone was washed with 0.05% Triton XlOO in a phosphate buffered saline, pH 7.4. An aliquot of 0.3 of a solution of sinapinic acid in 50% acetonitrile 0.5% d trifluoroacetic acid was added to each zone. The analytes retained in each site were analyzed with the laser desorption mass / flight time spectrometer of the ionization. The results are shown in Figures 28-31. The total retained analyte count was around d 550. The result illustrates a method for combinatorial separation, which includes the separation and detection of multiple analytes in parallel.

XIII. ASSEMBLY IN SEQUENCE OF MULTIMERIC STRUCTURES This example illustrates a method of constructing a secondary adsorbent in a primary adsorbent. This secondary adsorbent then acts as a specific adsorbent for an objective analyte. An aliquot of 0.5 μl of the GST fusion receptor, diluted in 10 mM Tris, 100 mM sodium chlorur. 0.4% NP40, pH 7.2 were added to a zone d a normal site of the adsorbent array. The solution allowed to concentrate in the area until almost dryness. The zon was washed with 2 μl of 10 mM Tris, 50 mM sodium chloride, pH 7.2, three times. Each wash was achieved by transferring the wash solution by pipette inside the area for five times. The area was finally washed with 2 μl of water twice, to remove the remaining regulator. An aliquot of 0.3 μl of a solution of sinapinic acid in 50% acetonitrile, 0.5% d trifluoroacetic acid, was added to the zone. The retained GST fusion receptor was analyzed with the laser desorption mass / ionization flight time spectrometer. (Figure 32). An aliquot of 0.5 μl of the GST fusion receptor in 20 mM Tris, 100 mM sodium chloride, 0.4% NP40, pH 7.2, was added to an area of a normal adsorbent array site. A sample containing only the GST protein (without receptor) was applied to another zon as a negative control. The solution was left to concentrate in the area until it was almost dry. 0.5 μl of 10 m Tris, 50 mM sodium chloride, pH 7.2, was added to each zone. The solution was removed using a pipette after 10 seconds of standing at room temperature. An aliquot of 1 μl of a solution containing a ligand-specific compound in a collection of other ligand compounds was immediately added to each zone. The adsorbent array was incubated in a humidity chamber at room temperature for 1 hour. Each zon was washed with 2 μl of 30% isopropanol: acetonitrile (1: 2) in water, twice. Each wash was achieved by pipetting the wash solution into the area out of the area ten times. An aliquot of 0.3 μl of the solution of the -cyano-4-hydroxycinnamic acid in 50 of acetonitrile, 0.5% of trifluoroacetic acid, the zone was added. The ligand compound captured on the s receptor was analyzed with the laser desorption mass / ionization flight time spectrometer. Figure 33A shows the binding of a specific ligand compound out of a collection of 96 other ligand compounds to the GST fusion receptor, which was captured at a normal adsorbent array site. Figure 33B shows that there is no binding of the ligand compound to the GST protein alone (without receptor) captured in the same array, the lime serving as a negative control of the experiment. The present invention provides novel materials and methods for the chromatography of the retenate. While specific examples have been provided, the foregoing description is illustrative and not restrictive. Many variants of the invention will be apparent to the experts on the subject of a specification review. The scope of the invention, therefore, should be determined not with reference to the foregoing description, but with reference to the appended claims together with their full scope or equivalents. All publications and patent documents cited in the application are incorporated by reference in their entirety for all purposes and to the same extent as if each of the publications or patent documents is individually denoted. For its citation of the various references in this document, the Requesters do not admit that any particular reference is a "prior art" in its invention.

Claims (30)

R E I V I N D I C A C I O N S

1. A method of preparing a substrate for detecting at least one analyte in a sample, this method comprises the steps of: a) exposing the sample to at least two different selectivity conditions, each condition of selectivity is defined by the combination of a adsorbent and an eluent, to allow retention of the analyte by the adsorbent; b.) identify by the desorption spectrometry at least one selectivity condition, under which the analyte is retained; and c.) preparing a substrate, comprising at least one adsorbent of an identified selectivity condition.

2. The method of claim 1, wherein the identification step comprises identifying at least one selectivity condition under which a plurality of analytes are retained.

3. The method of claim 1, wherein the step of preparing comprises preparing a substrate including a plurality of adsorbents, which retain the analyte under an elution condition as a multiple adsorbent.

4. A method for progressively identifying a selectivity condition with improved resolution for an analyte in a sample, comprising the steps of: a.) Identifying a selectivity condition that retains an analyte in a sample by: (i) exposing a sample to a set of selectivity conditions, each selectivity condition is defined by at least one binding characteristic and at least one elution characteristic; (ii) detect the analyte. retained under each condition of selectivity by desorption spectrometry; e (iii) identifying a selectivity condition that retains the analyte; and b.) identify a selectivity condition with improved resolution for the analyte, by: (i) selecting at least one binding characteristic or an elution characteristic, from the identified selectivity conditions, and adding it to a constant set of characteristics of selectivity; (ii) exposing the sample to a modified set of selectivity conditions, in which each selectivity condition, in the modified set, comprises (1) the selectivity characteristics in the constant set and (2) a characteristic of ligature or characteristic of elution, that is not in the constant set; and (iii) identifying a selectivity condition from the modified set, by the desorption spectrometry, which retains the analyte with improved resolution, compared to a previously identified selectivity condition.

5. The method of claim 4, further comprising the step of repeating step (b) at least once.

6. The method of claim 5, comprising repeating step (b) until a selectivity condition that retains only the target analyte of the sample is identified.

7. A substrate "for desorption spectrometry, comprising an adsorbent from a selectivity condition identified to resolve an analyte by the method of claim 4.

8. The substrate of claim 7, in the form of an assembly further comprising an eluent of the selectivity condition or instructions on using the eluent in combination with the adsorbent.

9. A method for determining whether an analyte is differentially present in a first and second biological sample, comprising the steps of: a.) Determining a first retention map for the analyte, in the first sample, by at least one selectivity condition; b. ) determining a second retention map for the analyte in the second sample for the same selectivity condition; and c. ) detect a difference between the first and second retention maps; whereby, a difference in retention maps provides a determination that the analyte is differentially present in a first and second samples.

10. The method of claim 9, wherein the first biological sample is derived from a healthy subject and the second biological sample is from a subject suffering from a pathological condition.

11. The method of claim 9, wherein the biological sample comprises first and second cell extracts.

12. The method of claim 9, wherein the retention map comprises a plurality of selectivity conditions.

13. The method of claim 9, comprising, prior to the detection step, the step of converting the analyte into at least one fragment, whose molecular mass is less than the mass of the analyte.

14. The method of claim 9, wherein the step of detecting a difference is enhanced in a programmable digital computer.

15. The method of claim 9, for determining whether an agent alters the expression of a protein in a biological sample, further comprising the step of administering the agent to a first biological sample, but not to a second biological sample.

16. The method of claim 10, wherein the sample is selected from the group consisting of blood, urine, serum and tissues.

17. The method of claim 10, further comprising identifying an analyte, which is present in a larger amount in the second biological sample, than in the first biological sample, whereby the analyte is identified as a candidate of a diagnostic marker for the pathological condition.

18. The method of claim 11, wherein the first cellular extract is derived from a healthy cell the second cellular extract is derived from a cancer cell.

19. A method for diagnosing in a subject a disease characterized by at least one diagnostic marker, comprising the steps of: a.) Supplying a substrate for use in desorption spectrometry, comprising at least one steerable location, each steerable location comprises an adsorbent that resolves at least one of the diagnostic markers, under an elution condition; b.) exposing the substrate to a biological sample from the subject, under the condition of elution, to allow retention of the diagnostic marker; and c.) detecting the diagnostic marker retained by the desorption spectrometry; whereby the retained diagnostic marker provides a diagnosis of the disease.

20. The method of claim 19, wherein the diagnosis involves the detection of a plurality of diagnostic markers and the steerable locations comprise adsorbents that resolve the plurality of diagnostic markers.

21. A device for detecting an analyte in a sample, comprising: (2) a substrate for use in desorption spectrometry, including at least one steerable location, each steerable location comprising an adsorbent that resolves an analyte under a selectivity condition , which includes the adsorbent and an eluent, and (2) the eluent or instructions to expose the sample to the selectivity condition.

22. The team of claim 21 for the diagnosis of a disease, wherein this at least one analyte is at least one diagnostic marker for the disease.

23. The kit of claim 22, wherein the disease is characterized by a plurality of diagnostic markers and the substrate comprises a plurality of steerable locations, each steerable location comprising an adsorbent that resolves at least one of the diagnostic markers.

24. The kit of claim 23, wherein at least one adsorbent is a multiple adsorbent, comprising adsorbent species that each retain at least one diagnostic marker.

25. The kit of claim 23, wherein at least one adsorbent does not comprise a biopolymer.

26. The kit of claim 23, wherein at least one targeting site comprises a ligand compound, specific for a diagnostic marker.

27. The kit of claim 26, wherein the ligand compound is an antibody.

28. A substrate for desorption spectrometry, comprising at least one. adsorbent in at least one steerable location, wherein this at least one adsorbent resolves a plurality of diagnostic markers for a pathological condition from a patient sample.

29. The substrate of claim 28, wherein at least one adsorbent does not comprise a biopolymer.

30. The substrate of claim 28, wherein an adsorbent resolves the plurality of diagnostic markers.