JP2005503537A - High throughput integrated system for biomolecular analysis - Google Patents

Abstract

The present invention provides selective recovery and enrichment of specific biomolecules from biomaterials for subsequent high performance analysis, target protein quantification, target biomolecule variants (eg, splice variants, point mutations and post-translational modifications). ), Elucidation of their properties, analysis and identification of ligands that interact with target biomolecules, and high throughput of large samples using a single unified, economical, multiple parallel processing platform Provide an integrated system for screening. A preferred embodiment of the integrated system is an automated system comprising a molecular trap such as an affinity microcolumn, a derivatization target for a mass spectrometer, a mass spectrometer capable of multiple sample injection, and a processing / data analysis interaction database. Including the device. The present invention also includes methods and processes for using individual elements as well as integrated systems in biological applications. Furthermore, preferred embodiments of the present invention achieve high throughput analysis for multiple separate devices and / or sample preparation and / or processing.

Description

【Technical field】
[0001]
This application is a continuation of both pending provisional applications 60 / 262,530 and 60 / 262,852 filed on January 17, 2001.
Field of Invention
The present invention relates to the field of proteomics. More specifically, the present invention is a method and apparatus for rapid identification and characterization of biomolecules recovered from biomaterials. Furthermore, the present invention includes the ability to process multiple heterogeneous samples simultaneously (high throughput analysis).
[Background]
[0002]
Background of the Invention
Recent advances in human genome sequencing have driven biological science to several new and interesting stages of research activity. Proteomics, one of these activity stages, is widely considered to be the next wave of biological research conducted in concert worldwide. Proteomics is the study of gene products (proteins), their diverse forms and interaction partners, and their regulation and processing kinetics (time). In other words, proteomics is protein research that functions in the natural environment, and its overall intent is to further deepen understanding of their biological functions, if not complete. Such studies are essential to understanding the mechanisms behind genetic disorders or the effects of drug-mediated therapy, while at the same time can be a fundamental basis for clinical and diagnostic analysis.
[0003]
There are several inherent challenges in protein analysis. First and foremost, any protein that is considered sufficiently relevant for analysis is present in a complex biological environment or biological material in vivo. The complexity of these biological materials is one challenge: the protein of interest is often present in the material at relatively low concentrations, and other large amounts of biomolecules such as proteins, nucleic acids, carbohydrates, It provides the problem of being obscured by lipids. To solve this fundamental problem, the technology currently used in proteomics is the relatively old-fashioned two-dimensional (2D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), i.e. All the biomaterials are only fractionated first by a technique in which a large number of proteins are simultaneously moved in two dimensions using gel media as a function of isoelectric point and molecular size. In order to move in a predictable manner, proteins are first reduced and denatured, but this process destroys the overall structure of the protein and loses its function.
[0004]
Currently, the latest proteomic techniques also involve the identification of proteins separated using 2D-PAGE. In this method, a gel spot containing the separated protein is excised from the gel medium and treated with a highly specific enzyme (usually trypsin) to fragment the protein. Next, the obtained fragment was subjected to high-precision mass spectrometry using an electrospray ionization method (ESI) or a MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) type mass spectrometer (MS). The Data obtained in the form of the absolute molecular weight of the fragments as well as knowledge about the specificity of the enzyme is used for in silico searching of genomic or protein databases for information related to the empirical data of the fragments. According to analytical methods and search protocols that have been improved over the past seven years, a few proteins whose mass has been measured with high accuracy in order to identify that the gel-separated protein is derived from a gene. It has evolved to the point that only decomposition fragments are needed.
[0005]
However, the identification of the gene that produces the protein of interest is only the first step in the overall many processes that determine protein structure / function. The 2D-PAGE / MS method cannot answer many problems that occur. One major problem is related to the primary structure of the protein. In a routine identification process, at most 50 percent of the protein sequence is examined, but at least 50 percent of the protein remains unanalyzed. If there are potentially many splice variants, point mutations and post-translational modifications in any protein, many variants are present in the protein, many of which cause disease states And modifications will eventually be missed during the identification process. Thus, the protein does not have the sufficient detail structure required to distinguish a mutant with (normal) function from a mutant with abnormal function (causing a disease).
[0006]
Moreover, current identification processes do not provide any method for protein quantification. Protein quantification is a critical component of proteomics because for many disease states, increases or decreases in the concentration of certain proteins and / or their mutants can be causative or indicative. Currently, protein quantification from gels is inaccurate because it was originally performed using a relatively variable staining method. The staining method can be replaced by a method in which ICAT (isotope-coded affinity tags) is combined with quantification of protein fragments obtained by 2D-PAGE by mass spectrometry. However, the ICAT method is still not realistic for the protein variants. This is because protein variants result in protein fragments with altered mass that are excluded from the quantification process. Similarly, other methods such as ELISA (enzyme-linked immunosorbent assay) and RIA (radioimmunoassay) are equally complex to quantify specific proteins in the presence of mutants. Because of the lack of ability to separate the target protein from the variant, these methods will essentially monitor all protein variants as a single compound. Disease is often an error-prone method because it can be caused / indicated by increased concentrations of only a single variant rather than the cumulative concentration of all variants.
[0007]
Furthermore, the 2D-PAGE / MS method does not provide any search for protein-ligand (eg, other proteins, nucleic acids or bio-related compounds) interactions. Since protein denaturing conditions are used during protein separation, all protein-ligand interactions are perturbed and are outside the scope of research using identification methods. Another method focuses specifically on the analysis of protein-ligand interactions. Among these, the most frequently used are the yeast two-hybrid method (Y2H) and the phage display method, respectively, in vivo molecular recognition that induces gene expression producing reporter proteins that exhibit biomolecular interactions. Use the phenomenon or selectively amplify binding partners with high affinity. Other mechanical methods rely on biosensors that utilize universal physical properties or tags (eg, surface plasmon resonance or fluorescence) as detection methods. These methods are generally time consuming and have two major limitations in that the interacting partners drawn from the biomaterial are indirectly detected and do not provide specific or identifying information about the binding partner.
[0008]
Finally, none of the above methods are preferred for analyzing large quantities and high throughput of specific proteins, their variants, and their interaction partners in a large population of experimental materials. All the above methods require several hours (2D-PAGE) to several weeks (Y2H) to be performed on a single sample. Thus, due to the time and expense, application to hundreds to thousands of samples (from hundreds to thousands of individuals) required for proteomics, clinical and diagnostic applications is not possible.
[0009]
To date, there is no universally integrated system capable of high-throughput analysis of proteins for all of the above reasons. Therefore, there is an urgent need for new and novel techniques that can analyze native proteins present in the natural environment. These techniques include the following items: 1) the ability to selectively recover and concentrate specific proteins from biomaterials for subsequent rapid analysis, 2) the ability to quantify target proteins, 3) target protein mutations The ability to recognize the body (eg splice variants, point mutations and post-translational modifications) and their properties, 4) the ability to analyze and identify ligands that interact with the target protein, 5) single Including the ability to perform high-throughput screening of large samples using an economical platform.
[0010]
All publications and patent applications are hereby incorporated by reference as if individual publications or patent applications were specifically and individually incorporated for reference. Although the invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.
Summary of the Invention
The purpose of the present invention is to selectively recover and concentrate specific biomolecules from biomaterials for subsequent high performance analysis, target protein quantification, target biomolecule variants (eg, splice variants, Point mutations and post-translational modifications), elucidation of their properties, analysis and identification of ligands that interact with target biomolecules, high-volume samples using a single, unified, economical, multiplex and parallel processing platform It is to provide an integrated system capable of throughput screening.
[0011]
Other embodiments of the present invention include molecular traps such as affinity microcolumns, derivatized mass spectrometer targets, mass spectrometers capable of multiple sample injection, and processes / to achieve high throughput analysis. It is to provide an automatic device with data analysis interaction database software.
[0012]
Another object of the present invention is to provide individual elements for integrated systems such as molecular traps, derivatization targets and the like.
[0013]
It is a further object of the present invention to provide a high throughput embodiment of the present invention that employs an automated device for continuous preparation as well as parallel processing of large samples simultaneously.
[0014]
Yet another object of the present invention is to provide methods and processes that utilize individual elements and integrated systems in biological applications.
[0015]
Yet another object of the present invention is to provide an apparatus and method for identifying point mutations and variants of an analyte using an integrated system using high throughput analysis.
[0016]
The novel aspects considered characteristic of the invention are set forth with particularity in the appended claims. The invention itself, however, will be best understood from the following description of preferred embodiments of the invention when read in conjunction with the accompanying drawings, together with further objects and advantages, both as to its structure and operation. Unless otherwise stated, the words and phrases in the specification and claims are intended to give their ordinary meaning and conventional meaning to those of ordinary skill in the ordinary applicable arts. Where other meanings are intended, this specification specifically states that a special meaning has been applied to a word or phrase. Similarly, the use of the word “function” or “means” in the “description of the preferred embodiment” is the use of the US Patent Act, Section 112 Section 6 to define the invention. There is no intention to want. Conversely, where the provisions of US Patent Act, Section 112, Section 6 are sought to be carried out to define the invention, the claims are intended to include the phrase “means for” or “for Specifically describing the “step for” and function, such phrases do not enumerate any structure, material or action that supports that function. Even if the claims enumerate "means for" or "steps for" performing a function, if the structure also enumerates any structure, material or action that supports the means of the process, the intent is This is not to enforce the provisions of US Patent Law, section 112, section 6. Further, even though the provisions of 35 USC 112, Section 6 are implemented to define the invention, the invention is limited only to the specific structures, materials, or acts described in the preferred embodiments. In addition to any and all known or later developed equivalent structures, materials or acts, in addition to any further performing the claimed function, in order to carry out the claimed function, As well as any structure, material or action.
DESCRIPTION OF PREFERRED EMBODIMENTS
Selective collection and enrichment of specific biomolecules from biomaterials for subsequent rapid analysis, target protein quantification, target biomolecule variants (eg splice variants, point mutations and post-translation) (Recognition of modifications), elucidation of their properties, analysis and identification of ligands that interact with target biomolecules, and integration for rapid screening of large samples using a single, unified, economical, multiplex and parallel processing platform Provide the system.
[0017]
Preferred embodiments of the integrated system include molecular traps such as affinity microcolumns, processing stations, and derivatized mass spectrometer target arrays that may be deleted in non-preferred embodiments. The integrated system works with a mass spectrometer that can inject single or multiple samples and can use a database of processing / data analysis interactions. The present invention also includes methods and processes that use individual components and integrated systems in biological applications. Furthermore, preferred embodiments of the present invention provide a plurality of individual devices and / or sample preparation and / or processing to achieve high throughput analysis.
[0018]
A key element of the system of the present invention is the isolation or recovery of specific analytes from surrounding biomaterials in a biological sample. This is achieved using a molecular trap. The recovery process in the preferred embodiment of the molecular trap requires that the biological sample be repeatedly flowed through a device having an affinity receptor on a surface with a large surface area. The affinity receptor is selected to capture a specific analyte. In high-throughput embodiments, these molecular traps are formed in affinity microcolumns, which are small columns, thereby placing multiple molecular traps in parallel to capture the minimum physical quantity. In a preferred form of the side-by-side embodiment, multiple molecular traps are contained within unit components such as manifolds or blocks of material. In such a configuration, the manifold has a number of microchannels in which molecular traps are accommodated.
[0019]
The molecular trap process is accomplished by providing sufficient physical contact between the affinity receptor located on the molecular trap and the analyte contained in the biological sample. An affinity receptor captures or isolates a particular analyte using an affinity interaction between the affinity receptor and the particular analyte. After capturing a particular analyte, the remaining or uncaptured compounds are washed with a series of rinses to release them from the molecular trap. The capture and rinse process concentrates certain analytes and reduces the dead volume of the affinity microcolumn.
[0020]
After specific analytes are captured, they are eluted from the molecular trap using a small amount of reagent that can interfere with affinity interactions. The eluted specific analyte is then subjected to enzymatic / chemical using a bioreactive MS target array for mass spectrometry or for further processing, eg, preparation for subsequent mass spectrometry. Stamped directly onto the target platform of mass spectrometry for processing such as modification. Next, automated mass spectrometry is performed on specific analytes or modified fragments detected with high accuracy. Software that can recognize differences between samples or standards is used to assist in the analysis and organization of a large number of samples based on the database. In addition, instead of stamping the eluted specific analyte on the target of mass spectrometry, the molecular trap is used as a component of the sample introduction device such as the needle of an electrospray mass spectrometer, thereby allowing the specific analyte to be analyzed. Can be eluted directly into an electrospray ionization mass spectrometer.
[0021]
The high-throughput embodiment of the present invention uses automated equipment (robotics) that performs the continuous preparation and parallel processing of multiple samples. The use of microcolumns to capture specific analytes allows for the above sequence format. This is ideal for such high throughput because it minimizes the physical quantity and / or area occupied by the microcolumn array. The use of affinity microcolumns with appropriately arranged automated devices allows multiple samples to be prepared and processed simultaneously from start to finish on an integrated platform, thereby enabling high throughput of samples. Specifically, all capture, separation and elution steps are performed in an automated instrument system or a microcolumn controlled by the system. It uses mechanical / physical means (eg, centrifugation, magnetic or vacuum separation) to separate specific analytes from biological fluids, and other affinity capture methods (eg, beads) that are rinsed with a buffer. As opposed to using a medium). In many cases, this physical separation needs to be performed alone and often disturbs the order of the array and disturbs the parallel processing array. Since these mechanical / physical means are not necessary when using microcolumns, the parallel processing arrangement can be used without being disturbed, and the ordered spatial arrangement remains intact during the entire process. It is. Most conveniently, multiple preparations / analyses are used in commonly used spatial arrays, eg, 4-, 8-, 16-, 48-, 96-, 384 or 1536 well microtiter plate formats. It is carried out continuously and in parallel using an automatic device adapted to the above.
Individual components
Sample modification / preparation
In all of the embodiments described below, it is desirable that the biomaterial or target analyte is modified or prepared either before the affinity reaction or after affinity capture, but prior to elution on the target or target array. Examples of modifications or preparations include, but are not limited to, reduction, label or tag addition, in situ digestion, partial digestion / modification on the surface, pH adjustment, and the like.
Molecular trap
The molecular trap in one embodiment of the present invention is a microcolumn apparatus to which an affinity receptor is bound. The molecular trap is chemically modified, such as by treatment with an aminosilanization reagent, and then affinity receptor binding is activated using any one of a number of derivatization schemes. The use of affinity microcolumns overcomes the disadvantages associated with performing affinity capture by other means. Specifically, as described herein, affinity microcolumns are tuned for mass spectrometers that have become available over the last decade. Before the MALDI-TOF and ESI mass spectrometers came out, mass spectrometry of polypeptides (when feasible) required an amount of analyte in nanomolar units and contained bound receptor when isolated by affinity capture. Frequently, the reagent required milliliters and the biomaterial required liters. MALDI-TOF and ESI mass spectrometers range from low sensitivity to sensitivity above femtomole, and the scale of total affinity isolation, including instrumentation, can be reduced to several stages. Thus, the affinity microcolumns described herein have been devised and manufactured to take full advantage of the sensitivity characteristics of recently enabled mass spectrometry techniques.
[0022]
A further embodiment of the present invention is to provide various affinity microcolumns that are particularly suitable for a given biomaterial, as illustrated in FIG. Since not all biomaterials are identical in terms of biomolecule composition and conditions, the affinity reagent derivation scheme behaves differently in each biomaterial. For example, an affinity reagent adapted to recover a particular protein analyte present in plasma does not perform ideally when the same analyte is present in different biomaterials. In addition, when using different buffer compositions and conditions for each biomaterial, a large number of small organic compounds may be retained and subsequently eluted with the analyte of interest, which inhibits the mass spectrometry process. sell. Thus, not only is the specificity to the analyte of interest high and the non-specific binding to other large molecules that may interfere with the characterization of the analyte, but also the basis of mass spectrometry processes such as MALDI or ESI It is necessary to construct an affinity microcolumn for each biological fluid that minimizes the retention of low molecular weight compounds that may interfere with the physical phenomenon that results.
target
Analytes are essentially microeluted after collection from each biomaterial and are “stamped” directly from the affinity microcolumn onto a target or target array that is inserted into the mass spectrometer. In this manner, a spatial array, such as a titer plate, is maintained as it is from the initial multi-sample container to the affinity capture and wash steps and onto the mass spectrometer target.
[0023]
Furthermore, the present invention encompasses the use of targets specifically adapted for mass spectrometers in the automated preparation and analysis of proteins recovered using affinity microcolumns. In order to incorporate automated equipment for high throughput, it is essential that the parallel process be reproducible between nuclear samples and that the position at which the sample is deposited on the target of the mass spectrometer can be controlled in the parallel process. To ensure that these points are incorporated into an automated process, a self-assembled monolayer (SAM) is created on the mass spectrometer target so that the deposition area of the analyte can be controlled. For example, a control region is created on a gold-plated target using a thiol compound or mercaptan compound that is hydrophobic or hydrophilic in nature. Surrounding the hydrophilic SAM with a hydrophobic SAM creates a clear boundary to confine the water-soluble sample (from the affinity microcolumn) in a well-defined area on the target. In this way, the spatial arrangement defined by the parallel automated device is transferred from multiple affinity microcolumns onto a mass spectrometer target that is patterned identically to the spatial sequence used throughout the automated instrument processing. It can be maintained by eluting multiple samples simultaneously (using automated equipment).
[0024]
In other applications, the mass spectrometer target is further made to include a reactive surface capable of processing the analyte. When studying biomolecules using mass spectrometry, it is often necessary to elucidate chemistry and / or enzymology to obtain further details regarding the structure of the analyte. Analysis using specific chemical modification or enzymatic chemical modification in combination with mass spectrometry is particularly important for the purpose of distinguishing analytes, variants and modifications of analytes in the same sample. In addition, the analyte of interest is largely purified by removing potentially interfering types from the solution using removal interactions designed to remove the obstruction while leaving the analyte of interest to be analysed. Preprocessing for mass spectrometry is often of great value. The most efficient means of performing these operations is to use a mass spectrometer target derivatized with reagents or enzymes for special processing functions.
[0025]
In a preferred embodiment, the target or target array is made by first etching the channel around the designated target area using photoresist technology. A gold film is deposited on the etched substrate by conventional electroplating techniques or plasma deposition. This gold film is formed naturally along the etched contour. Depending on the substrate, one or more intermediate layers, such as a nickel intermediate layer, may be required when depositing the gold film on the surface. Next, an activated or activatable reagent such as dithiobis (succinimidylproprionate) (DSP) or a derivative thereof capable of forming or adsorbing a chemical bond to the modified substrate surface is applied to the target region. Combine so that it does not combine to other areas. Any transport solvent is evaporated or removed to produce a dry self-assembled monolayer (SAM) of activated or activatable reagents. As the protective layer, dextran or the like dissolved in a suitable solvent is deposited on the target SAM. If the solvent is DMSO, the DMSO is removed by placing the target in a vacuum. The target (array) is then coated with a hydrophobic reagent such as octadecyl mercaptan dissolved in a solvent that does not dissolve the protective layer. If dextran is the protective layer, isopropanol can be used to dissolve the hydrophobic reagent. Rinsing the target removes any unbound hydrophobic reagent. In examples where the activation reagent is bound to the target region, the activation reagent can be used by removing the protective layer, such as by simply rinsing with DMSO, which can also be used in the protective layer or Any hydrophobic reagents present on the protective layer will also be removed. In examples where an activatable reagent is bound to the target area, the protective layer is removed and then activated using the activating reagent, or a solvent that transports the activating reagent is also used to dissolve the protective layer The reagent can be activated and used by either directly activating it. In the second case, the dissolved protective layer is removed by a subsequent rinse. Finally, a biological or biological reagent such as a polymer, protein, peptide, or enzyme binds to the surface of the target area. Bioagent binding is facilitated by the activating reagent already bound to the target region. If there is an activation reagent coated with a protective layer, remove the protective layer before adding the biological reagent to the target area, or whether the solvent that transports the biological reagent is also used to dissolve the protective layer and bind directly By either of these, a biological reagent can be added to the surface.
[0026]
An advantage of the target or target array manufacturing process is that a target once coated with a protective layer can be stored for a longer period and used at the discretion of the consumer. Another advantage of the target array is that it provides a defined reactive surface for analyte processing as well as impurity handling.
[0027]
In yet another similar embodiment, the surface of the target of one or more mass spectrometers is made of a reagent that has been found to enhance the quality of sample preparation by promoting the crystal formation of the matrix used to perform the MALDI. Derivatize. Such “seeding” of matrix crystals has proven to be very valuable in automating the entire sample preparation process and allows for the production of highly reproducible samples over the entire area of the target.
High-throughput equipment
The individual components described here together form a single integrated system that allows high-throughput analysis of analytes recovered from biomaterials. The basic analysis begins with the verification of the primary structure, ie the sequence of the analyte. A single high precision determination of molecular weight is often sufficient to establish the primary structure of an analyte. If higher accuracy is required to verify the primary structure of the analyte, create a mass map of the analyte (after recovery) using a chemically / enzymatically active mass spectrometer target. Convenient. During such a mapping procedure, the analyte is digested with a highly specific cleavage reagent, resulting in multiple signals when analyzed using mass spectrometry. When viewed as a group, these signals can verify primary structure with greater accuracy and reliability than single mass measurements. In addition, these data can be used to search a database for test mutants, such as splice mutants, that are significantly different from those expected as normal analytes.
[0028]
In a similar embodiment, other variants of the analyte are mapped to clarify the nature, location and origin of the mutation. Analytes and variants present in a single sample are extracted together from the biomaterial using conventional affinity reagents localized in the microcolumn and simultaneously activated by the mass spectrometer activated target or target array Used for mapping. Since many test variants share high homology with normal analytes, many of the mapping signals are common between the analyte and the variant. However, abnormal or mass shifted signals are also present in the mapping data. Using these different data, it is possible to elucidate the mutation position by combining the knowledge of the cleaving reagent with information on the primary structure, tertiary structure, and quaternary structure of the normal analyte. In addition, mutants were generated using knowledge of mass differences between component residues of the analyte (eg, mass differences between amino acids in proteins or nucleic acids in DNA / RNA) and accurate determination of mass shifts. It is possible to determine metastasis. Such analysis is of great value in elucidating, for example, point mutations present in proteins or polymorphisms present in nucleic acids. Similarly, knowledge of the molecular weight of potential modifying groups (eg, glycans, phosphates, methyl, formyl, etc.) is used in combination with mapping data to elucidate the site and nature of the chemical modification of the analyte it can. Finally, a reactive target designed to handle specific modifications is performed in an integrated system by cleaving the modified moiety from the analyte and viewing the subsequent mass shift in the mass spectrum ( Number) can be used to determine.
[0029]
In other embodiments, the present invention is used for high-throughput quantification of specific analytes present in biomaterials. Using this process, the analyte and internal matching (analyte-like species) are recovered from the biomaterial and processed through mass spectrometry. In the same parallel processing operation, the standard sample is analyzed, and a calibration curve is created by equating the analyte signal with the amount of the analyte present in the biomaterial. A calibration curve can then be used to determine that the amount of analyte present in each sample has increased relative to other samples, or can be determined absolutely.
[0030]
In a further embodiment, the present invention is used to determine interaction partners associated with protein-ligand interactions. In essence, affinity microcolumns are derivatized with a ligand of interest with the intention of screening biomaterials for interaction partners. This ligand acts as an affinity receptor that can selectively isolate the analyte from the biomaterial. The isolated analyte is subjected to mass spectrometry for identification. Often, retained analytes are well identified through direct molecular weight determination from mass spectrometry and knowledge of compounds present in biomaterials. As an alternative, a method is provided in which an unknown analyte is chemically / enzymatically digested with an activated target and the resulting fragment (eg, proteolytic fragment) is subjected to mass spectrometry. The accurately determined molecular weight (and knowledge of cleavage specificity) of the fragment is then used to search a genomic or protein database to identify the analyte.
[0031]
In a similar embodiment, protein-ligand interactions are examined by designing affinity reagents that target specific proteins that themselves hold other analytes. In this manner, the protein complex is recovered from the biomaterial by targeting one of those components. Then, using the analysis method described above, the details of the components and properties of the complex are clarified.
[0032]
Specific embodiments according to the invention will now be described in detail. These examples are illustrative only and the invention is not limited to the materials, methods, or apparatus described in these embodiments.
[0033]
Manufacture of affinity microcolumns
Biologically sensitive affinity-ligand microcolumn capable of high throughput for specific or non-specific target analytes with efficient affinity capture, release, and rapid, sensitive and accurate mass spectrometry The indicated formation of preferred embodiments is described below. In the examples below, a number of methods, configurations and device delivery are described to provide a stable configuration in a biologically rich environment.
[Example 1]
[0034]
Preparation of porous glass molecular trap using mold-graphite spray exfoliation
Porous glass molecular traps are standardized in the specifications of commercially available wide-perforated P-200 pipettor tips using stainless steel annealing molds (100-1000 pieces of 0.071 inch (inlet) per mold) 2 ° taper, polished with graphite stripper). Soda lime glass spherical beads (150-200 μm; 75% SiO) ₂ , 15% Na ₂ O and 10% CaO) and annealing is performed by ramping temperatures from 772 ° C. (equilibrium, t = 0) to 800 ° C. (t = 3 min, 1 min equilibration) in an argon-backfill furnace. Achieved. At the end of lamp-annealing, the mold is immediately removed from the oven and the porous glass molecular trap is withdrawn from the mold. This process typically involves a porous glass molecular trap with high flow characteristics and appropriate perforations and tapers for attachment to the inlet of a wide perforated P-200 pipette tip (size of porous glass molecular trap at room temperature: 0. 061 inches (inlet), 0.092 inches (length), 2 degree taper).
[0035]
In a preferred embodiment, a molecular trap is then inserted at the tip of the pipette tip and placed at the bottom (narrow portion). The molecular trap is fixed by applying a sufficient amount of pressure from the tip. The pipette tip can be heated prior to insertion of the molecular trap to aid in the fixation process.
[0036]
Sample mass spectra using affinity microcolumns prepared according to this example are illustrated in FIGS.
[Example 2]
[0037]
Fabrication of porous glass molecular traps using ceramic molds-Pyrex
In order to produce a porous Pyrex glass molecular trap, a ceramic mold is described. The porous glass molecular trap is standardized in the specifications of a commercially available wide-perforated P-200 pipettor chip using a ceramic tempering mold. Zircar type R plates (4 × 6 × 1-1 / 4 inch) were purchased from Zirker (Florida, NY), surfaced with an end mill on the largest flat and finished with a CNC machine (2100 Holes, 0.0625 inch bottom cut, 2 degree taper). Ball milled powdered borosilicate “Pyrex” (size from 4 μm to 300 μm: 81% SiO ₂ 4% Na ₂ O, 0.5% K ₂ O, 13% B ₂ O ₃ And 2% Al ₂ O ₃ 4) were stacked in a furnace and subjected to initial temperature equilibration using a slow temperature ramp (60 minutes) below the Pyrex softening point (816 ° C.). The mold was then allowed to equilibrate for 30 minutes before ramping to about the Pyrex softening point (821 ° C.), which was maintained for about 30 minutes to form a porous glass molecular trap. For concerted heat treatment (using high quality silica glass), the mold is down-ramped to 708 ° C. and at that temperature 2-20 hours (desired etch) before the slow final temperature ramp for extraction. Maintained (depending on quantity). If immediate use is required, the mold is ramped slowly down to an appropriate recovery temperature (generally 300 ° C.) to avoid glass cracking. This process typically involves porous pyrex glass molecular traps with high flow characteristics and appropriate perforations and tapers for attachment to the inlet of wide perforated P-200 pipette tips (size of porous glass molecular trap at room temperature: 0 0.0625 inch (inlet), 0.130 inch (length), 2 degree taper).
[Example 3]
[0038]
Fabrication of porous silica glass molecular traps using ceramic molds-Vycor
In order to produce a porous Vycor glass molecular trap, the ceramic mold described in Example 2 is used. Zirker type-R ceramic mold, powdered “Vycor” (Corning), porous Vycor (Corning), controlled porous glass (Controlled Porous Glass, NJ), depending on the desired flow-through characteristics Size width from 4 μm to 300 μm: 96% SiO ₂ ) And stacked in a furnace, and after undergoing initial temperature equilibration (90 minutes) with a gradual temperature rise to a Vycol softening point (1500 ° C.) or less (90 minutes), the mold is about Vycol softening point (1530 ° C.) The glass was trapped for 30 minutes before ramping to form a porous glass molecular trap. In order to avoid glass cracking, the mold is allowed to cool with a gentle gradient to an appropriate recovery temperature (generally about 300 ° C.). This process typically produces a porous Vycor glass molecular trap with high flow characteristics and appropriate perforations and tapers for attachment to the inlet of wide perforated P-200 pipette tips.
[Example 4]
[0039]
Fabrication of porous silica glass molecular traps using ceramic molds-silica gel or quartz glass
The ceramic mold described in Example 2 is used to produce porous glass molecular traps using silica gel or quartz glass. Zirker type-R ceramic mold, powdered porous silica gel (Sigma Chemical Co.) or quartz glass (Corning Co.) (size width of 4 μm to 300 μm depending on desired flow-through characteristics: 100% SiO ₂ ) And stacked in a furnace, subjected to an initial temperature equilibrium with a gradual temperature rise gradient (90 minutes) to a silica softening point (1550 ° C.) or lower, maintained at equilibrium for 30 minutes, Raised to about Vycor softening point (1585 ° C.) to form a porous glass molecular trap. To avoid glass cracking, the mold is allowed to cool with a gentle gradient to an appropriate recovery temperature (generally about 300 ° C.). This process typically produces a porous Vycor glass molecular trap with high flow characteristics and suitable perforations and tapers for attachment to the inlet of wide perforated P-200 pipette tips.
[Example 5]
[0040]
Etched porous silica molecular trap
Highly porous silica molecular trap formation is achieved by exposing the porous silica molecular trap formed in Example 4 to various electrochemical etching conditions. For example, a porous silica molecular trap is placed in an aqueous HF (25-50%) solution in absolute ethanol and an etching current (120-200 mA / cm ² ) And irradiation (150 mW / cm) ² ) For 1 to 20 minutes to create a silicon hydride surface.
[Example 6]
[0041]
Fabrication of porous powder metal molecular traps using molds
The porous metal molecular trap is standardized in the specifications of a commercially available wide-perforated P-200 pipettor tip. A lip with a reverse slope (ie narrower tip compared to the mold described above) or taper in the same direction and slightly raised around the opening located at the bottom of the mold (for the porous metal exit by the pushpin) A mold is machined containing (because it needs more than full). The mold is supported by a removable bottom plate, filled with powdered metal (eg, brass, copper, silver, gold), and an Arbor press with complementary push pins in each hole, Apply pressure (suitable for forming individual joints between metal particles) to a given / raised powder metal surface. After forming the powder metal molecular trap, the lower plate is removed and a push pin is placed at the narrow end of the hole to release the porous powder metal molecular trap from the mold. Porous powdery metal molecular traps, when annealed in an oven, increase the structural stability and form metal oxides for organic functionalization.
[0042]
It should be recognized that molecular traps can be made from a variety of different materials and / or combinations of different materials and still be within the scope of the present invention. The material must be able to bind affinity reagents to their surface. Or the only requirement is that it must be chemically modifiable to bind to the affinity reagent. The most preferred material or combination of materials is that it has a high surface area or can be modified to increase the surface area.
[0043]
While the above examples utilize the tapered profile of the microcolumn, features such as non-tapered or cylindrical columns can be used and are still within the scope of the present invention. Indeed, in high throughput embodiments, non-tapered bodies are preferred because the tapered bodies slow down the manufacturing process.
Chemical activity of molecular traps
[Example 7]
[0044]
Silane coating / function of porous glass molecular trap
Porous molecular traps formed by Examples 1-6 or other equivalent processes were pretreated by combining various water, mineral acid treatments, concomitant water rinsing, and drying prior to chemical derivatization (silanization). Can be adjusted. For example, a porous glass molecular trap is suspended in distilled or purified water (generally 10 times the volume) at elevated temperatures (typically below the boiling point of water) and the fresh water is exchanged two or three times. (Or without replacement) and leaching by orbital shaking for 1 to 24 hours. All the water is then removed and subjected to the next two acid treatments. First, 1N hydrochloric acid in water is rinsed with water until a neutral (or near neutral) pH is obtained by applying a porous glass molecular trap (10 times) at an elevated temperature for 1 to 2 hours. A 1N nitric acid in water is then carried out as performed in the first acid treatment. The porous glass molecular trap is dried either at room temperature or in a vacuum oven (100 ° C., 1 atmosphere, overnight).
[0045]
The dried / pretreated porous glass molecular trap is then perfused overnight in anhydrous toluene with orbital shaking under a silanization reagent, eg, 10% silanization reagent N- [3- (trimethoxysilyl) propyl. ] Chemically activated by coating with ethylenediamine to produce a functionalized porous molecular trap. The silane-coated porous molecular trap is allowed to cool to room temperature and then rinsed at room temperature until the supernatant is negative for ninhydrin and / or trinitrobenzoic acid (tnba). Other useful silanizing reagents include, but are not limited to, 3- (trimethoxysilyl) propyl acrylate, 3- (trimethoxysilyl) propylamine, N- [3- (trimethoxysilyl) propyl] aniline, N1 -[3- (trimethoxysilyl) propyl] diethylenetriamine, N- [3- (trimethoxysilyl) propyl] ethylenediamine, 3- (trimethoxysilyl) propyl methacrylate, [3- (trimethoxysilyl) propyl chloride] Octadecyldimethylammonium, N- [3- (trimethoxysilyl) propyl] polyethyleneimine, N1- [3- (trimethoxysilyl) propyl] carboxylic acid, aminopropyltriethoxysilane, 3-glycidoxypropyltrimethoxysilane, Thiolpropyltrimethoxysilane, Chlorop Pills trimethoxysilane, octadecyl trimethoxysilane, octadecyl trichlorosilane, O-[2-(trimethoxysilyl) ethyl] O'methyl polyethylene glycol, Shiriruarudedo the like.
[0046]
These reactions can be facilitated by a base catalyst such as using triethylamine (TEA) added to the reaction or by carrying out under reduced pressure (about 1 atmosphere).
Manufacture of micro column housing (pipette)
In a preferred embodiment, when non-tapered molecular traps are used, the micropipette used as the microcolumn housing is modified as follows:
A microcolumn housing such as a micropipette tip is formed by injection molding or other means well known in the art. The distal end of the housing is modified to provide a conventional micropipette with at least one bayonet-like projection. In one embodiment, the protrusion is a continuous shelf according to the inner diameter of the housing end. However, in a more preferred embodiment, the at least one bayonet protrusion is a protrusion or a triangular protrusion, more preferably 3 protrusions, even more preferably 6 protrusions. . Thus, when molecular traps are loaded into the housing, each is supported by at least one insertion protrusion. After the molecular trap has been filled into the housing, the portions of the sides of the housing that are directly adjacent to the molecular trap are pressed inward to some extent with slight frictional contact with the molecular trap. Care must be taken not to over-press to avoid destroying the molecular traps contained within. In a preferred embodiment, the pressing step is accomplished by heating the outer surface of the housing while cooling the internal molecular trap with a flow of cooling gas (preferably an inert gas). The heated housing is then forced inward using a gentle taper in the heated mold, thereby pressing the housing against the molecular trap.
[Example 8]
[0047]
Batch functionalization
Direct batch activation / derivatization of functionalized porous molecular traps
Direct batch activation / derivatization of microcolumns is directed to any reactive chemical group that binds to a typical affinity ligand and provides environmentally stable binding. Batch activation / derivatization methods proceed in numerous ways, primary amines, carboxylic acids, thiols that can be activated by organic and inorganic reagents such as homobifunctional, heterofunctional or polymeric reagents. Activation / conjugation chemistry is targeted for biological modification with groups, sulfhydryls, hydroxyls, allyl groups, azides, aldehydes, hydrazides, maleimides, triazines and the like.
[0048]
This example illustrates one of these methods by glutaraldehyde activation, followed by ligand coupling of primary amine groups with aldehyde groups on the glutaraldehyde-porous molecular trap. First, an amine functionalized molecular trap was prepared using glutaroaldehyde (25 mg in 0.10 M sodium phosphate) using sodium cyanoborohydride (10 mg / mL) mediated coupling (reaction times ranging from 1 hour to overnight). % Solution, pH 7.8, 100 mM NaCl buffer). After rinsing several times with phosphate buffer, the active aldehyde groups on the glutaraldehyde matrix are incubated with the ligand of interest, such as a protein antibody. Uncoupled (excess) ligand is removed by extensive rinsing with HBS buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 0.005% surfactant P20). This derivatization process produces a molecular trap with a high analyte binding force. The molecular trap is then packed into a P-200 pipettor tip.
[Example 9]
[0049]
Batch amine activation, amplification, reactivation and derivatization of functionalized porous molecular traps
Amine → CHO → Polyamine → CHO → Ab
Activation of the microcolumn by amplification allows other methods for handling ligand coupling. Cross-link medium / high molecular weight amine polymers such as polylysine, aminodextran or amine-modified starburst dendrimers to functionalized / activated porous molecular trap surfaces by various appropriate chemistries to create amplified amine surfaces Let A glutaraldehyde-active amine microcolumn is incubated with polylysine in sodium cyanoborohydrate mediated coupling. The polyamine microcolumn is then reactivated with glutaraldehyde, rinsed with water, and conjugated with a protein ligand.
[Example 10]
[0050]
Suppression of non-specific binding by batch surface amine modification activation / derivatization of functionalized porous molecular traps
Amine → CHO → Poly-amine → NHS / EDC → Poly COOH → NHS / EDC →> Ligand
Another approach for ligand immobilization is through coupling of ligand-derived carboxyl groups to surface amines. An effective method of creating an amplified amine surface is to crosslink a high molecular weight polylysine, aminodextran, amine-modified starburst dendrimer or polyacryl hydrazide to a functionalized porous molecular trap surface and then a carboxyl-terminated crosslinker (e.g., By terminal carboxyl generation by various chemistries such as EDC mediated coupling of succinic anhydride or glutaric anhydride) or bisoxirane activation (becomes an epoxide activated surface). These carboxyl microcolumns are used to immobilize low pI protein ligands via EDC / NHS mediated activation / coupling. Specifically, polylysine microcolumns are incubated in bulk of succinic anhydride (or glutaric acid) (100 mg / mL anhydrous in 0.2 M sodium acetate, pH 4-5) to maintain pH by addition of dilute hydrochloric acid. However, the reaction is generally completed in 2 to 4 hours (ie, tnbs and ninhydrinamine test negative). The carboxy microcolumn is then activated with 100 mM NHS and 100 mM EDC in water at room temperature for 10-20 minutes, rinsed with water, and a slight vacuum is applied prior to protein ligand coupling (antibody 1-10 mg / mL). HBS buffer pH 7.5, shaker overnight at 4 ° C.).
Example 11
[0051]
Activation / derivatization of polymer-modified carboxylic acids in batches of functionalized porous molecular traps
Other methods are available for surface amplification with water-soluble carboxyl-containing polymers such as carboxymethyl dextran (carboxymethyl dextran, carboxymethyl agarose, carboxymethyl cellulose, carboxymethyl amylose, polyglutamic acid, polyacrylic acid, carboxyl-modified starburst dendrimers). It is then accompanied by coupling of the ligand via the interaction of primary amine groups and carboxyl groups on the dextran matrix. First, an amine functionalized microcolumn was performed in 100 mM sodium phosphate, pH 4.8, 100 mM NaCl buffer via EDC (1-ethyl-3- (3 dimethylaminopropyl) carbodiimide) mediated coupling. Coupling to 15 kDa carboxymethyl dextran (CMD). After several rinses with phosphate buffer, the carboxyl groups on the dextran matrix are activated with EDC / N-hydroxysuccinimide (NHS; 100 mM each in water) and incubated with a ligand of interest such as a protein antibody. Unbound (excess) antibody was removed by extensive rinsing with HBS buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 0.005% surfactant P20), after which the molecular trap was removed from P- Pack into 200 pipettor chips. This derivatization process produces a more cohesive microcolumn compared to a microcolumn that does not use an amplification layer (CMD matrix). The initial activation step of aminosilanization results in the amine surface (virtually functionalized surface), but increases the packing power away from the binding surface and is uniform throughout the entire microcolumn ( It is preferred to amplify the surface to ensure a homogeneous coating (thus reducing potential interactions that can occur on the glass substrate and cause non-specific binding).
Example 12
[0052]
Activation / derivatization of functionalized porous molecular traps with carboxymethyldextran carbonyldiimidazole (CDI) in batch
As another approach for ligand immobilization, the EDC / NHS activation method of the CMD layer is replaced with the activation of N, N′-carbonyldiimidazole (CDI). This activation method results in the conversion of carboxyl and hydroxyl groups present in CMD into imidazolyl carbamate intermediates that can react with primary amines or sulfhydryls present in affinity ligands. For example, a CMD microcolumn is rinsed with acetone followed by dimethyl sulfoxide (DMSO). Next, a 100 mg / mL DMSO solution of CDI is placed on a CMD microcolumn, evacuated, and this activation proceeds on an orbital shaker for 2 hours to overnight. The CDI activated affinity pipette is rinsed extensively with DMSO followed by acetone to form a CDI activated CMD microcolumn. Importantly, CDI activation media are known to be stable enough to allow monthly storage when given the correct storage conditions (ie (wet) isopropanol or (dry) inert gas or vacuum). It is that. Therefore, CDI can act as a stable activator of the microcolumn, and can be supplied in advance for a long period of time. Alternatively, the CDI activation matrix is incubated with the protein antibody of interest. Unbound (excess) antibody was removed by extensive rinsing with HBS buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 0.005% surfactant P20), after which the molecular trap was removed from P-200. Pack into pipetta chips.
Example 13
[0053]
Batch in situ polymerization or copolymerization by surface polymerization of organic functionalized porous glass molecular traps
In this example, an organofunctionalized porous glass molecular trap (ie, methacryltrimethoxysilanation) is polymerized in situ with free radical initiation. Typical copolymerized monomers used include, but are not limited to, allyl dextran, allyl amine allyl glycidyl ether, acrylic acid, vinyl dimethyl azlactone, which are tetramethylene diamine (TEMED) or N, in water. Typical initiators such as N, N ′, N′-tetramethyl-1,2-diaminoethane and ammonium persulfate can be used to incorporate into a crosslinker such as methylene-bis (acrylamine). The degree of crosslinking and polymer size is controlled by the amount of initiator, polymerization time, and monomers available for termination. This surface reaction occurs with functional groups that are abundant in the polymer matrix covering the molecular trap surface. These groups can then be utilized for activation / conjugation to the ligand or subjected to further activation and amplification prior to activation and ligand coupling.
Example 14
[0054]
Metal chelator modified porous glass molecular trap
Incorporation of metal chelators into porous glass molecular traps is useful for recovery of metal binding biomolecules. The amine or polyamine microcolumn prepared in the previous example is placed in 0.1M phosphate pH 7 and evacuated using a rotary evaporator. The surface of the primary amine was then directly coupled at 20-100 mg / mL to a bifunctional chelator such as ethylenediaminetetraacetic acid dianhydride (EDTA-DA) and diethylenetriaminepentaacetic acid dianhydride (DTPA-DA). Thus forming metal chelate arms (TED and EDTA, respectively) that can bind metals tightly in the coordination of the complex to capture biomolecules.
[0055]
Alternative methods include ethylenediaminetetraphosphate (EDTPA), 1,4,7,10-tetracyclononane-N, N ′, N ″ -triacetic acid (NOTA), 1,4,7,10-tetracyclododecane -N, N ', N ", N'''-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetradecane-N, N ′, N ″, N ′ ″-tetraacetic acid Metal chelator solutions such as (TETA), 1,2-bis (2-aminophenoxy) ethane-N, N, N ′, N′-tetraacetic acid (BAPTA), N, N (biscarboxymethyl) L-lysine For example, using NHS / EDC (100 mM each, 0.1 M phosphate, pH 7) mediation to bind carboxyl groups to localized surface primary amines.
Example 15
[0056]
Pseudobiological ligand formation, preparation of batch dye functionalized porous glass molecular traps
Synthetic dyes are useful affinity ligands due to the advantage of reactivity with a wide variety of biological materials. Reactive dyes including but not limited to triazine dyes (ie, reactive black, reactive blue, reactive brown, reactive green, reactive orange, reactive red, reactive yellow, etc.) are, for example, convenient methods Was used to bind to an amino microcolumn. The target triazine dye, Cibacron Blue (50 mg / mL) was dissolved in distilled water and added to the amino microcolumn in a round bottom flask and allowed to react overnight under mild reflux. After ligand coupling, the pseudo-biological microcolumn was rinsed with water and incubated with 1N salt solution for 30 minutes under gentle reflux. After extensive water rinsing, the pseudo-biological microcolumns were dried either in air or under reduced pressure at 90 ° C.
Example 16
[0057]
Fabrication of batch ion exchange functionalized porous glass molecular traps
Incorporation of ion exchange media into porous glass molecular traps is useful for biomolecule binding, recovery and analysis or washing as a desalting surfactant in the sample (SDS removal). In this case, the amine or polyamine microcolumn is contained, for example, in a dextran ion exchange medium (eg, dextran sulfate, diethylaminoethyl dextran (DEAE-dextran), heparin sulfate, carboxymethyl dextran (product previously described in Example 10)). Activated for the purpose of hydroxyl grouping of carbohydrates, the surface of the primary amine is coupled with a bifunctional (or active) such as triazine, diglycidoxy ether (oxiranins or epoxides) prior to dextran ion exchange carrier coupling. Similarly, an epoxide functionalized microcolumn generated from the initial silanization reaction or copolymerization is used for derivatization.
[0058]
Alternatively, the amine microcolumn is incubated / coupled with an activated dextran carrier. A further example is the use of aldehyde groups on dextran carriers produced by sodium metaperiodate oxidation of dextran (proximity diol) ion exchange carriers. Specifically, dextran ion exchange medium (10-100 mg / mL) was incubated for 2 hours in the dark in 1M sodium metaperiodate (in water), after which the oxidized dextran ion exchange medium was washed with an organic solvent (ethanol / Precipitate in ether) or dialyze overnight in water. The oxidized dextran ion exchange medium is then incubated with an amine or polyamine microcolumn (0.1 M phosphate, pH 7.5).
[Example 17]
[0059]
Protein immobilization on microcolumn
Proteins with binding specificity are professionally treated as receptors and ligands for immunochemistry. These affinity proteins include, but are not limited to, protein A, protein G, avidin reagent and streptavidin reagent. Protein ligands, specifically protein A or G, are porous for capture / conjugation following capture of the terminal constant region (Fc) of the antibody or subsequent cross-linking prior to use as a biomolecule recovery agent Incorporated into glass molecular trap. The avidin / biotin system is used as an alternative linking agent to create a conjugate that has an amplification motif as well as strong binding properties when bound to an antibody or analyte.
[0060]
For example, the amine or polyamine microcolumn prepared in the previous example is placed in 0.1 M phosphate at pH 7 and evacuated using a rotary evaporator. The surface of the primary amine is then activated with glutaraldehyde (25%, 4-24 hours), rinsed, and functional protein reagent (or amine) in sodium cyanoborohydride (0.1 mg / mL) mediated coupling. Or hydrazide modified biotin) (1-10 mg / mL in HBS-EP).
Example 18
[0061]
Protein modification / digestion using microcolumns
Biomolecules with specific modifications or digestibility are used as receptors or ligands in biochemistry. These biomolecules include, but are not limited to proteins, peptides, enzymes, catalytic antibodies and the like.
[0062]
For example, trypsin modified microcolumns are prepared in the previous examples. When activated, trypsin specifically digests at either the arginine point or the lysine point in the amino acid sequence of the protein, such as the analyte protein of interest.
[0063]
Alternatively, other receptors can be used to combine two biomolecules into a single body, such as analyte phosphorylation by immobilized kinases.
Target manufacturing
Currently, target designs used in commercial manufacturing do not fully support the precise and consistent use of arrayed and / or bioreactive targets. Achieving a high degree of reproducibility between samples is very important for these targets.
[0064]
There are at least three target designs that can correct problems encountered when using current design targets. These designs are as follows.
Trapezoidal target
In the first embodiment of the target according to the present invention, there is a “matched set” target in which the amount of analyte can be strictly applied to a limited area and does not have to worry about spreading beyond the active area of the target. A certain amount of analyte will form a meniscus at the top of the platform formed on the target and will not ooze out on both sides of the platform if properly designed. Due to this meniscus action, the analyte typically has a divided volume of about 2-3 μL. This volume is almost 4mm with little fear of overflowing from the enzymatically active area. ² Fully and fully protect the target area. Thus, irregular digestion due to exposure of the analyte to various changes in the amount of enzyme is eliminated by (only) completely protecting the active surface of the target. Furthermore, if the target is placed in a suitably moistened incubator, a volume of about 2-3 μL will not evaporate. Thus, irregular digestion due to sample drying is eliminated by maintaining a constant sample volume on the table.
[0065]
In manufacturing, the platform is processed into a metal substrate target that attaches to a larger target (stainless steel) that is ultimately introduced into the apparatus. The height of the platform is determined empirically by criteria that appear to be the minimum height necessary to avoid analyte diffusion. A height of about 0.5 millimeters is usually sufficient; a height that can be easily introduced into the facilitating region of all MALDI-TOF devices (minimum tolerance of about 2 mm). Thus, the overall dimensions of the table are dimensions that sufficiently limit the object to be analyzed without introducing errors derived from the apparatus into the analysis.
[0066]
The second embodiment uses a thin sized metal target that is attached to the device with a tongue-in-groove design. Due to the thin dimensions of the targets and the need to have grooved mounting brackets, it is not feasible to machine (machine finish) these targets. Thus, a pressing method is used to create the platform (pressing the target from the back). It should be noted that the pressing method requires proper punching of the machine.
[0067]
Suitable substrates are empirically determined for solvent compatibility, gold coating as well as vacuum compatibility used during manufacture. Initially, the substrate target is made from polystyrene that is compatible with the production solvent, is capable of highly uniform sputter coating, and has low outgassing properties. However, other polymeric materials can be used. The advantage of a polymer substrate target is that it can be manufactured in large quantities and inexpensively.
[0068]
The success criteria for this type of target are: 1) accurately limit the sample volume to the exact surface area, 2) do not reduce the performance of the instrument, 3) be easy, mass-produced and cost effective It is to determine the optimal trapezoidal geometry.
Hydrophobic / hydrophilic contrast of target or target array
A second embodiment design for highly reproducible bioreactive targets is to contrast the enzymatic active site with a hydrophobic medium that can limit the amount of analysis to the active region only. In this method, the enzyme is immobilized at a gold / DSP / enzyme spot generally located on top of the hydrophobic target. The simplest way to manufacture the control device is to build it from a hydrophobic material such as Teflon. The gold sample area is placed on the target by a masking / sputter coating process. The gold region is then derivatized using our normal activation / immobilization method. One concern associated with Teflon devices is that they change within the mass spectrometer. Such effects (changes at the sample stage due to ion removal) are always a concern in mass spectrometry designs. MALDI-TOF analysis was performed on a sample target made from a non-conductive material (eg, quartz target or gel), but had little effect from sample loading. These effects are further minimized using the delayed extraction method available in all commercially available MALDI-TOF instrument uses. However, any disruption to the mass spectrometric performance by the target can be removed by such methods as including the electrical binding of the enzymatically active surface in the masking / gold coating process.
[0069]
Another possibility for the control device is to derivatize the metal substrate target to create a hydrophobic surface on which the hydrophilic enzyme active region is built. The manufacturing protocol of this method is more relevant than the previous method. It is necessary to follow a multi-step protocol in which the target is first coated with a hydrophobic medium and then the pattern of sample points is gold coated onto the hydrophobic coating using a masking process. This process consists of 1) Sputter coating of all targets with gold, 2) Activation with DSP (if necessary), 3) Immobilization of hydrophobic compounds, 4) Sputtering the target again with a pattern mask, 5) Fresh With gold activation and enzyme fixation. One example using this process is to gold coat the target and derivatize with 1-octadecanethiol (omit the DSP activation process). This process was used to create a C-18 derivatized surface. These surfaces are highly hydrophobic so that they can be easily observed with polka dots. Once the target is derivatized with a hydrophobic compound, a fresh gold pattern is applied by masking and sputter coating. The fresh gold is then activated and derivatized using, for example, a DSP / dextran / enzyme immobilization method. This process results in a hydrophilic enzyme activity spot surrounded by a hydrophobic medium. To highlight the visual contrast between active and hydrophobic regions, hydrophobic dyes containing a single primary amine and no other chemically reactive groups (eg Fast Violet B, Methylene Violet 3RAX) ) Can be used in place of the C-18 layer. Similarly, other classes of dyes or pigment-based hydrophobic compounds can be used to further create a visual contrast between the active and hydrophobic regions of the target. In a preferred embodiment, hydrophilic mercaptoundecanoic acid dissolved in isopropanol is microdeposited by flash on an arrayed target, dried and quickly covered with octadecyl mercaptan to form a contrasted target array. Contrast can be reversed for hydrophobic analytes or any combination thereof, including mixed SAM applications.
[0070]
The success criteria for this type of target is to find a general derivatization method (for high contrast targets) that is not too labor intensive or costly to prohibit large scale manufacturing.
Individual target (insert)
The design of the third target embodiment is the use of individual areas (inserts) that can be inserted into a substrate attached to a commercial mass spectrometer after digestion. This method is learned from the “matched set” described above. The insert is a pedestal or contrast design, making it economical for derivatization with higher cost enzymes (by using smaller amounts of higher concentrations of reagents). These targets (even when derivatized with low-cost enzymes) are extremely economical in terms of cost in research applications that can be used one by one.
[0071]
For success with this type of target, it is necessary to create a “matched set” design target as an insert that attaches to a target that is acceptable to a commercially available device.
Biochips and small array targets
Another embodiment of the target array is to incorporate a bioreactive surface into / onto an existing chip-type bioanalysis platform. Microchannel devices (chips) are currently used for protein analysis, followed by biomolecule separation (within one area) followed by enzyme treatment (within a second area on the enzymatically active region). As well as an ideal platform for MALDI-TOF analysis directly from the chip.
Other active surfaces, derivatization methods and assays
Amplification medium
In another embodiment, the surface amplification medium is used to increase the activity of the target array or enzyme activity target. One example of amplification is performed using a solution-based polymerization amplification method, a one-step activation coupling, or an in situ induced polymerization event. These modified surfaces exhibit a certain charge difference with carboxylic acid or amine amplifiers (which are themselves active or activatable).
General enzyme immobilization kit
An important product according to the invention is a fully activated general enzyme immobilization kit. The kit includes a number of affinity micropipettes, including affinity microcolumns, activated or activatable targets or target arrays (amplified or non-amplified) and pre-prepared buffers. The purpose of the kit is to eliminate the need for the end user to send the reagent to the manufacturer to immobilize the reagent by derivatizing the target in-house with a unique enzyme.
Ion exchange surface
In yet other embodiments, the target or target array can be derivatized with carboxymethyl dextran (CMD) that can perform a cation exchange process, so that, finally, during protein analysis in the presence of sodium and potassium buffers, Lead to a higher quality MALDI-TOF spectrum. With a CMD target, unwanted cations are removed from the solution and replaced with protons or ammonium ions, depending on the cation used to precharge the target.
Cation exchange (CE) target
The value of the CE surface has already been described; the reduction of unnecessary cations in the buffer is to reduce the heterogeneous signal, thereby increasing the sensitivity of the mass spectrum (signal homogenization). The reduction of alkaline cations by using a CE surface can also improve the homogeneity of the matrix during sample preparation. These properties are useful for MALDI-TOF analysis of both proteins and nucleic acids. In one embodiment of the CE surface, a 500 kDa CMD amplification surface is used. 500 kDa CMD is selected because it has a higher exchange capacity than low molecular weight CMD. About 10 pmoles of CMD is 4mm ² It is estimated that it can be immobilized on the surface of the target. Considering that each component of CMD has about 1,000 valence sites for exchange (about 30% monomer dextran converted to CMD), 10 nanomolar exchange power is 10 mM alkaline salt for the target. Is estimated to be equal to 1 μL aliquot. However, the interchangeability of the CMD target will have to be evaluated to determine the overall effectiveness in a pure sample. They consist of a single short DNA molecule (eg d (T)) mixed under various concentrations (0.1-10 mM) of buffer salt. ₈ ) Is effective in removing alkali metals. DNA is selected for the test assay because of its high tendency to maintain alkali metals as counterions to the negatively charged phosphate backbone.
[0072]
When a standard CMD target is used, it is easy to reach the exchange limit below the concentration range of the buffer used for many biological applications. Stronger CE function (for example, sulfonate groups produced by chlorosulphonic acid treatment of dextran) and methods that can increase surface amplification (application of polymerized resin to produce macroscopic surfaces (10-100 micrometers)) are the target exchange capabilities Can be used to increase the level of harmful effects experienced during analysis of biomolecules present in moderate to high strength buffers.
Anion exchange (AE) target or target array
Anion exchange (AE) targets or target arrays are a further embodiment useful for sample purification by removing various anionic surfactants from solution. Specifically, the presence of sodium dodecyl sulfate (SDS) in protein solutions is always a concern during MALDI-TOF analysis. SDS generally perturbs the crystal formation required for the function of many MALDI matrices and therefore needs to be removed from solution as part of sample preparation. Another anion that may have a deleterious effect on the MALDI process is the phosphate ion.
[0073]
Polylysine amplification surfaces have inherent weak anion exchange properties. They are used for anion removal by analyzing a test mixture composed of about 5 proteins in the presence of various concentrations of SDS. The polylysine surface cannot provide the exchange capacity or strength necessary for removal of SDS from the solution at the relevant concentration (10 mmol). Diethylaminoethyl (DEAE) exchange groups can be used as an alternative to polylysine. Three derivatization schemes are possible to make DEAE. The first possibility is to couple diethylaminoethylamine to 500 kDa CMD using EDC-mediated chemistry or CDI activation. An inevitable limitation of this method is the need for complete saturation of the carboxylic acid group with DEAE. If not all groups are converted to DEAE, the surface will have both (predictable) cation and anion characteristics. The second method is to activate the polylysine surface with 1,5-difluoro-2,4-dinitrobenzene and attach DEAE to the surface via benzyl halide. This has been successfully done using the 1,5-difluoro-2,4-dinitrobenzene method to selectively immobilize peptides via their N-terminus. However, a possible cause of the failure of the method is that during the activation process, polylysine can be severely cross-linked, essentially forming an inert surface for further derivatization. The third chemistry is to attach diethylaminoethylamine to the surface amplified with CDI activated dextran. This method is a modification of CDI-CMD derivatization and should have a large exchange capacity while reducing heterogeneous cation / anion exchange. All of these preparations were tried and evaluated using test peptide mixtures in the presence of SDS.
Enzymes, assays, incubators
enzyme
Other embodiments use combinations of biomolecules to achieve a combined multifunctional operation. For example, the combined profile is a combination of two enzymes, such as alkaline phosphatase and endoprotease, that can be dephosphorylated following biomolecule mapping. Such an application is useful, for example, in elucidating specific phosphorylation sites present in regulatory proteins. Immobilizing phosphatase with various endoproteases achieves the ultimate goal of creating a surface that can be manipulated by pH. Activity can be assessed by assaying small phosphopeptides available from commercial suppliers.
[0074]
Yet another embodiment uses snake venom phosphodiesterase (PD). Targets successfully derived with PD by partial sequencing give a special dimension to the specificity of the primer extension method by PCR used to analyze short DNA fragments containing mutations present in microsatellite DNA. Immobilization of PD to a bioreactive target can be achieved by assessing activity using small oligonucleotides. Studies related to both AP and PD rely on general derivative protocols according to the present invention.
incubator
A common occurrence during proteolytic digestion using bioreactive targets in combination with affinity microcolumns is that the sample tends to dry (on the target or target array) during the digestion process. A modified oven / incubator capable of performing digestion over the temperature range (25-60 ° C.) was made to surround the bioreactive target in a constant humidity environment. Digestion was performed for more than 1 hour using this incubator with little sample loss. The device is small and portable (dimensions: 4 inches x 6 inches x 1 inch; approximately 0.5 lbs weight; 120 V) and operated at a single temperature (40 ° C sufficient for most applications of bioreactive arrays. ), With a “matched set” target, a sample volume of 1-2 μL can be maintained in a time limit of 1 hour and can be used in a robot station.
Example 19
[0075]
Application of biomolecules
Urine beta-2-microglobulin (β ₂ m) Analysis
The porous glass molecular trap produced according to Example 1 above was activated and derivatized in a batch (30-50 per batch) prior to packing into pipettor tips forming an affinity pipette. After acid preparation (0.05M HCl for 1 hour, air dried), the porous glass molecular trap was refluxed with 10% aminopropyltriethoxysilane (Aldrich, Milwaukee, Wis.) In anhydrous toluene for 12 hours. The amine functionalized porous glass molecular trap was then placed in a reaction vessel and equilibrated for 15 minutes in a reaction buffer (100 mM sodium phosphate, pH 4.8, 100 mM NaCl) under slight vacuum. After equilibration, the buffer was loaded with 15 kDa molecular mass carboxylated dextran (CMD, Fluka, Milwaukee, Wis.) And 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide (EDC, Sigma, St. Louis, MO) (reaction) The mixture was replaced with a mixture of 10 mg / mL each in buffer and air was evacuated again from the reaction vessel. The reaction was allowed to proceed for 1 hour (approximately 20 minutes and 40 minutes with two successive additions of EDC to the reaction mixture), then terminated and rinsed. Prior to antibody coupling, the CMD-porous glass molecular trap was vigorously rinsed with 100 mM sodium phosphate, pH 8.0, 0.5 M NaCl. The porous glass molecular trap was then activated with EDC / N-hydroxysuccinimide (NHS, Sigma, St. Louis, Mo.) (100 mM each in water) for 10 minutes, and affinity purified rabbit anti-human β ₂ Incubated with mIgG (DAKO, Carpinteria, Calif.) (0.1 mg / mL in 20 mM sodium acetate, pH 4.7). Unbound antibody was removed by extensive rinsing with HBS buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 0.005% surfactant P20). This manufacturing process yielded an affinity pipette with a binding force estimated at 10-100 pmol with a dead volume of about 1.5 μL. Anti-β ₂ The m affinity pipette was found to be stable and active for a period of at least 3 months (by storage at 4 ° C. in HBS buffer) after antibody immobilization.
Biological fluid
All body fluids were obtained immediately before use; protease inhibitor cocktail (PIC, Protease Inhibitor Cocktail Set III, Calbiochem, La Jolla, Calif.) ₂ m was added immediately to minimize the possibility of proteolysis.
Tear fluid: Human tear fluid is washed with double distilled water (ddH ₂ The rinsing liquid was collected by washing with O). An eye rinse solution of 20 μL was mixed with 180 μL of HBS buffer and used as a stock tear solution. This stock solution was further diluted 10-fold with water (for MALDI-TOF analysis) or HBS buffer (for mass spectrometry immunoassay (MSIA) analysis).
Plasma: 44.7 μL of human whole blood is drawn from a lancet puncture finger under aseptic conditions using a heparinized microcolumn (Drummond Scientific Co., Broomall, Pa.), Mixed with 205 μL of HBS buffer, and 30 seconds Centrifugation (at 7,000 xg) pelleted red blood cells. 50 μL aliquots of supernatant were mixed with 200 μL HBS and the resulting solution was used for MSIA; aliquots were ddH for MALDI-TOF. ₂ Further diluted with O (10 times).
Saliva: Human whole saliva is ddH for preparation for MALDI-TOF or MSIA ₂ Each was diluted 100-fold with O or HBS buffer.
Urine: human urine, ddH ₂ Prepared for MALDI-TOF by 100-fold dilution with O; 2-fold dilution with HBS buffer was used for MSIA.
Mass spectrometric immunoassay (MSIA)
MSIA uses an anti-β using a manual P-200 micropipettor. ₂ It was performed on the biological fluid by repeatedly sucking the fluid through the m-affinity pipette (approximately 20 times). After repeated flow incubation, the affinity pipette is rinsed with 2 mL HBS buffer (by sucking and discarding HBS in 200 μL aliquots) and then ddH ₂ Rinse with 1 mL of O (using the same wash and disposal method). In the final water rinse waste, it was confirmed that all residual water was expelled from the affinity pipette. 3 μL aliquot of matrix solution (1: 2, acetonitrile: ddH ₂ Retention compound by sucking α-cyano-4-hydroxycinnamic acid (ACCA; Aldrich, Milwaukee, Wis.) In O, 0.2% TFA into an affinity pipette (enough to cover the microcolumn) Was eluted from the affinity pipette, at which point the matrix / eluate mixture was deposited directly onto the MALDI-TOF target. MALDI-TOF mass spectrometry was performed using a mass spectrometer. In summary, the apparatus uses a two stage 30 kV (2 × 1 cm; 15 kV / stage) continuous extraction source to accelerate ions to the entrance of a 1.4 m flight tube containing an ion guide cable. Pulsed N ₂ Ions generated using a laser (337 nm) were detected using a hybrid single channel plate / -3.8 kV bias discrete dynode multiplier. The spectra were recorded using an average transient recorder while monitoring individual laser shots using a separate oscilloscope and reducing the laser intensity during capture (when measured).
Quantitative
Internal standard: Horse β ₂ m (Eβ ₂ m) is human β ₂ m (Hβ ₂ m) and high similarity (about 75% sequence homology), Hβ ₂ Mass difference from m (MW _Eβ2m = 11402.9; MW _Hβ2m = 11729.7) was selected as an internal standard for quantification because it is decomposable and readily available. Fresh horse urine was collected (at a local stable) and immediately treated with a protease inhibitor cocktail. Insoluble compounds were removed from urine by cooling overnight (at 4 ° C.) and then centrifuged at 5,000 × g for 5 minutes. The urine was then concentrated 20-fold with a 10-kDa MW cut-off filter, replaced several times (4 filters / 200 mL urine) and rinsed repeatedly with HBS and water. Treatment of 200 mL fresh urine results in 10 mL β ₂ m-rich equine urine was obtained and used as a stock solution for the internal reference solution for analysis about 100 times.
Calibration curve: Hβ ₂ Quantification of m was performed. In summary, the standard is 1.0 mg / L stored Hβ. ₂ m prepared by stepwise dilution to a concentration of 0.1 mg / L (ie, x0.8, 0.6, 0.4, 0.2, 0.1 in HBS); 0.1 mg / L The solution was a stock solution of the same serial dilution spanning the concentration at the second decimal place (0.01-0.1 mg / L). Hβ ₂ A blank solution containing no m was prepared. Samples for MSIA were prepared by mixing 100 μL of each sample with 100 μL of stored horse urine and 200 μL of HBS buffer. MSIA was performed as described above, respectively, and Eβ ₂ m and Hβ ₂ m Both were extracted simultaneously. Ten 65 laser shot MALDI-TOF spectra were taken from each sample and each spectrum was taken from a different location on the target. Care was taken during data acquisition to maintain an ion signal in the upper 50-80% of the y-axis width and avoid saturating individual laser shots. The spectrum is obtained by Eβ ₂ Normalize to m signal, Hβ ₂ The m signal was determined. The integrals of 10 spectra taken for each test standard were averaged and the standard deviation was calculated. Hβ ₂ A calibration curve was drawn by plotting the average value of the standardized integration of each standard against m concentration.
Screening: Urine samples were collected from each person, treated with a protease inhibitor cocktail and cooled to 4 ° C. The urine sample was immediately centrifuged for 5 minutes (at 5000xg) prior to analysis to remove precipitated material. In preparation for MSIA, 100 μL of each urine sample was mixed with 100 μL of stored horse urine and 200 μL of HBS. This process is the same as the process used to create the calibration curve, except that the standard is replaced with a human urine sample. MSIA was performed as described in the standard curve section.
Affinity and pipette evaluation / Biological fluid screening
Affinity pipettes were evaluated by screening biological fluids that were easily obtained in large numbers. The intent of the screen was to measure the degree of non-specific binding to each body fluid and to easily look up other rinsing protocols that reduce the contribution to non-specific binding. FIG. 2a shows the MALDI-TOF spectrum of diluted human tears, the spectrum showing the compound retained in MSIA. High concentrations of protein present in tears dominate the MALDI-TOF spectrum: lysozyme (MW _calc = 14,696; MW _obs = 14,691) and tear lipocalin (MW) _calc = 17,444; MW _obs = 17,440). Other polypeptide signals are likely in the 2-5 kDa range, possibly β ₂ A low intensity signal is observed at m / z = 11,727 Da due to m. The MSIA spectrum is preferentially retained β ₂ m (MW _calc = 11,729; MW _obs = 11,731), and the signals for lysozyme and other non-specific compounds are weakened. FIG. 2b shows MALDI-TOF and MSIA spectra of diluted human plasma. As usually observed during direct analysis of serum or plasma, the MALDI-TOF spectrum is dominated by the signal from albumin. There are other lower m / z signals, but β ₂ No m signal is observed. The MSIA spectrum is preferentially retained β ₂ A strong signal with m and a few other signals with non-specific compounds. FIG. 2 c shows diluted saliva (MALDI-TOF) and saliva proteins retained in MSIA. The MALDI-TOF spectrum shows a number of signals in the 1-18 kDa range where the peptide region is most prominent; ₂ No signal corresponding to m is observed. The MSIA spectrum obtained using the normal rinsing protocol shows that the selectively retained β ₂ Signals by non-specific compounds in m and multiple low molecular mass ranges are shown. Additional rinses with 0.05% sodium dodecyl sulfate (SDS) resulted in HBS rinse and ddH ₂ A second MSIA analysis contained between O rinses was performed (FIG. 2c). Although SDS rinsing did not completely remove the low mass signal, ₂ Their contribution to the mass spectrum was greatly reduced without a proportional decrease in the m signal. FIG. 2d shows the spectrum resulting from the analysis of human urine. The MALDI-TOF spectrum shows multiple signals in the peptide region ₂ There is no m signal. The MSIA spectrum shows β with a few additional signals for non-specific compounds. ₂ The m signal dominates.
[0076]
The porous glass molecular trap used for affinity pipettes worked well in biological fluid screening. Intermediate CMD amplification of amine functionalized porous glass molecular traps provided a large hydrophilic surface with multiple attachment points (carboxylic acid groups) for antibody coupling. As a result, the antibody loading of each affinity pipette ₂ It is just enough to capture the low concentration of m. Alternatively, the hydrophilic surface can be washed free of most non-specific binding compounds by rinsing with an aqueous ionic buffer. With the exception of saliva samples, MSIA ₂ While the signal derived from m was overwhelming, a moderately clear mass spectrum was shown. An SDS wash of the saliva screen improved the spectral quality but did not completely remove all non-specific compounds. In a similar study, these compounds (identified by mass as lysozyme, α-defensin, histatin) were found to have a pI of about 10 and a moderate pH (7.8) and salt of HBS buffer. This suggests retention by charge interaction (with free carboxyl groups) that is not destroyed by the content (150 mM NaCl). Therefore, if the saliva screen appears to be biologically important, other rinse combinations (eg, high salt or various surfactants) need to be examined. However, the presence of non-specific compounds (in any of the samples) ₂ It is worth noting that it has been identified by direct detection at a characteristic molecular mass without interfering with the unambiguous determination of m.
Quantitative
Protein quantification using MALDI-TOF requires the use of internal standards to compensate for sample composition spot-to-spot differences that cause variations in the laser intensity and analyte ion signal. Proteins with characteristics that are different from the protein of interest are MALDI-TOF quantification of affinity recovered species during internal protein quantification, either directly during protein quantification or by addition of internal standard to peptides eluted from bead affinity media. Internal standards that generally behave like the analyte during laser desorption / ionization are generally preferred. This requirement is met in MSIA by selecting internal criteria with sequence homology with the target protein: remodeling by enzymatic / chemical modification of the target protein, cleavage / extension recombinant of the target protein, isotope Rich media (for example, ¹⁵ N or ¹⁸ O) Recombinantly expressed (same) target protein, same protein as various biological species. Once the receptor is able to capture both the target protein and the internal standard, MSIA can design around a single receptor system. Separately, it is possible to envisage two receptor systems where one receptor is used to recover the target protein and another receptor is used to recover the internal reference.
[0077]
The internal standard chosen here is horse β ₂ m (Eβ ₂ m) which has 75% homology with the human counterpart and Hβ ₂ About 300 Da below m (thus both species share similar characteristics and are easily analyzed in the mass spectrum). For example, polyclonal anti-β ₂ mIgG and Hβ ₂ m or Eβ ₂ Even without data on the relative dissociation constant between m and m, preliminary studies have shown that the antibodies exhibit sufficient cross-reactivity to retain both species. FIG. 3a shows Hβ at a concentration range of 0.01 to 1.0 mg / L. ₂ The spectrum showing the MSIA analysis of m standard is shown. Eβ ₂ Each spectrum normalized to m signal is one of 10 65 laser shot spectra taken at each test point. Hβ ₂ 10 standardized Hβ of each standard for m concentration ₂ The m-integral average is plotted, resulting in the calibration curve shown in FIG. 3b. By linear regression fitting of the data, I / O> 3 with a detection limit of 0.0025 mg / L (210 pM) and a limit of quantification of 0.01 mg / L (850 pM), I _Hβ2m / I _Eβ2m = 4.09 [Hβ in mg / L ₂ m] +0.021 (R ² = 0.983). The standard error was about 5% at all points on the calibration curve.
Β in urine samples ₂ Quantitative determination of m
Ten samples were collected from 4 persons: female (31 years old, pregnancy; sample 1 specimen (F31)), male (30 years old; sample 4 specimens (M30) over 2 days), male (36 years old; 2 days) 2 samples (M36)), male (44 years; sample 3 samples (M44) over 2 days). When the samples were taken, all were in good health. FIG. 4 shows the results obtained by MSIA of 10 urine samples. The bar represents the β determined for each sample ₂ m concentration and the insertion spectrum on each bar is Eβ ₂ Each Hβ normalized to m ₂ m signal is shown. The data for 10 samples are 0.100 ± 0.021 mg / L (High = 0.127 mg / L; Low = 0.58 mg / L) β ₂ It shows remarkable agreement with the average concentration of m. Additional analysis was performed on urine samples obtained from an 86-year-old elderly woman (F86) who recently had a kidney infection. Β found in this sample ₂ Because of the significantly higher concentration of m (see insert spectrum), β within the dynamic range of the calibration curve ₂ In order to maintain the m signal, also the β of F86 ₂ In order to accurately establish the m concentration (3.23 ± 0.02 mg / L), it was necessary to quantitatively dilute the urine 10-fold.
Post-translational modification
Mass-selective detection of MSIA can be found in β ₂ Allows discovery and quantification of m variants. Second, during quantitative screening of urine samples, higher molecular mass species (Δm = + 161 Da) ₂ extracted with m. This species is probably β ₂ m glycosylated (one hexose), most notably observed in F86. FIG. 5 shows the overlap of two MSIA spectra taken from urine of F86 (20-fold dilution) and M36 (no dilution; for comparison). Glycosylated beta ₂ The concentration of m is much higher at F86 than at M36. Glyco-beta ₂ The specific cause of the increasing concentration of m is not certain at present.
[0078]
In previous studies, glycosylated Hβ ₂ m is glycosylated Hβ by direct MALDI-TOF analysis of reasonably high-doped serum ₂ The quantification of m was reported. Due to inadequate mass spectral resolution and serum interference, the curve fitting routine may cause multiple Hβ ₂ m-glycobody signal is used to untangle, and then the integral is reconstructed with reasonable linearity (R ² = 0.88) for use in plotting calibration curves ₂ It was standardized to m. However, the data presented here is remarkably good and can clearly support rigorous quantification without the aid of fitting work, but unmodified β ₂ The standard curve plotted for m is the glycosylated β ₂ It is uncertain whether it can be used directly for exact quantification of m. Such correlations include both β ₂ It is necessary that the affinity constant and desorption / ionization efficiency of the m-body are equal. With respect to affinity constants, the affinity purified polyclonal antibody used here has a broad cross-reactivity (Hβ ₂ m and Eβ ₂ as illustrated by the co-extraction of m), so that both Hβ ₂ There is a high probability that m-body is extracted with similar efficiency. Similarly, the addition of a single carbohydrate moiety should not strictly reduce the relative desorption / ionization efficiency. As a result, glycosylated β ₂ The concentration of m-body is the wild type β in urine samples from healthy individuals. ₂ β at 0.072 mg / L at approximately the same concentration as m ₂ It can be estimated using an m calibration curve.
[0079]
Wild type and glycosylated β ₂ Regardless of whether both of m can be quantified using a single calibration curve, the wild-type β determined in MSIA ₂ The concentration of m is strictly wild type β ₂ It is particularly important that it represents only the concentration of m, not the combination of both species. Thus, MSIA has particular advantages over other methods that cannot discriminate between similar forms of the target analyte. So, increased β ₂ m concentration is used as a general indicator of immune system activity, while β ₂ m-glycosylation is associated with more specific diseases (eg, advanced glycosylated end-products associated with dialysis-related amyloidosis) and MSIA involves these independent contributors The result is a more accurate connection of a specific biomarker to a specific disease.
[0080]
The manufacturing process used in this report produced an affinity pipette with a speculative binding force of 10-100 pmol, while having a dead volume of about 1.5 μL. This binding / elution ratio is related to β found in biological fluids. ₂ It was found that this was sufficiently commensurate with the m concentration. In addition, β using an affinity pipette ₂ Efficient capture of m reduced the sampling volume (less than 100 μL of biological fluid). Furthermore, the affinity pipette chemistry used here shows that three of the four biological fluids show little non-specific binding, and even the fourth {saliva) does not introduce analytical interference into the analysis. It was.
[0081]
The quantitative ability of MSIA is clearly illustrated in the present invention. Β examined here ₂ The concentration range of m (0.010 to 1.0 mg / L) ₂ Sufficient to handle m concentration. Good linearity is the width of two 10 units (R ² = 0.983) or more. The selection of an appropriate reference standard is important for accurate quantification, and in this example, β ₂ Satisfied by the use of m-rich horse urine. However, even if equine urine is seen as an ideal background medium (to mimic the true analytical material closer to the buffer), if a large number of samples are analyzed over a long period of time, the future Purified Eβ to ensure consistency in analysis ₂ It is necessary to replace with m.
[0082]
Although the screening studies presented here are not extensive enough to be considered clinical trials, they have been performed directly on biological samples such as urine samples. ₂ Illustrates the usefulness of MSIA in accurately identifying and quantifying m. The four baseline people who provided urine for this project ₂ He was considered healthy because he had no known genetic illness associated with m or had no illness within one month prior to analysis. Β retained from the sample ₂ β with a molecular mass corresponding to the wild-type sequence of the protein by quantitative evaluation of m ₂ m single signal was revealed (experimental error within 0.02%, Eβ ₂ Spectrum corrected internally by using m signal; see FIG. 4). Quantitative analysis within the group was consistent with that found in the control group of other studies and was significantly consistent ₂ m concentration is shown. As a control, urine samples obtained from elderly people with poor health are ₂ The m concentration showed a significant increase (approximately 30 times greater). This estimate can be easily detected in the mass spectrum β ₂ It should be noted that this was done without interference from the high mass mutant of m (FIG. 5). The most reasonable explanation for the observation of two mass-shifted (still related) signals in a mass spectrum is: 1) the presence of a wild-type protein, 2) a genetic polymorphism or a variant present by post-translational modification. . In this specific case, the mutant is β ₂ As the glycosylated form of m, it is most easily identified by a mass shift of +161 Da; mutants due to genetic polymorphism have a single nucleotide polymorphism (ie, [TGG]-[GGG], resulting in Trp- Gly; dm = 129.15 Da) and is essentially excluded because it is larger than the mass shift. However, if mutants were present in the sample that possessed a significant mass shift (> 15 Da) as a result of genetic polymorphism, they were glycosylated β ₂ would have been recognized as m.
[0083]
Finally, in the MSIA, the analysis is performed fairly quickly and relatively easily, so this method is particularly useful for rapid development of analytical methods and analysis of large numbers of samples. The speed of analysis opens up the possibility of developing real-time methods that can be easily modified in incubation and rinsing protocols when analyzing experimental results that have just been completed. Once these methods were developed and optimized, they could be easily applied to biological fluid screening. Here the samples were analyzed at a rate of about 3 species / hour, allowing several analyzes to be performed in a given individual within one day (shown in FIG. 4, M30). This analysis rate was inherently limited by instruments that were not designed to accommodate multiple sample processing-this time, from sample preparation to analysis. However, the use of parallel pipette stations and mass spectrometers that accept multiple samples in one arrayed format can increase this analysis rate to several hundred times a day. We now describe a pipette station that processes 96-well format titer plates to simultaneously prepare, modify, incubate, capture and rinse multiple samples using a composite affinity pipette in this manner. The sample is then eluted onto the same format mass spectrometer target array.
Integrated automatic device
The mass assay system of FIG. 7a is a high throughput of new nascent fluid analytes with affinity microcolumns in a conduit system that includes one or more functional prestations, use stations, analysis stations and poststations. This is an extraction system. The prestation initiates sample information and organizes, prepares, and formulates the component sample array and target for use by the preworkstation and analysis workstation. For incoming biological fluids, sample identification is performed, which allows initial sample parameters such as biological sample type (blood, urine, cell culture, etc.), patient history, pathology, chemical parameter profile (pH, turbidity) Etc.), and a sample classification database for integration with the final output analysis data is provided, facilitating a database of fit and feedback. Through pre-station high-speed liquid manipulation and subsequent labeling and tracking, the identified biological fluid from the sample source is distributed to the individual sample collection arrays that are separated and labeled for tracking (eg, barcode / laser reading). Further, in-line operations such as appropriate dilution or modification (pH, addition of surfactant, etc.) are performed. Another function of the prestation is the preparation and loading of array components. Here, but not limited to, an array of components such as solid components (plastic, glass, metal, etc.), liquid reagents, targets, etc. is formed and / or dispensed into an appropriate array and loaded into the initial workstation. From the prestation, the array of molecular traps is accessed by the start / storage / sample station, the initial station, or relocated to a use station for sample processing.
[0084]
Examples of pre-station processing include, but are not limited to, assembling a porous molecular trap into an affinity pipette, array labeling, sample preparation, and sample modification. These treatments include solid sample arrays (such as plastic microtiters), array labels, chemical / modifying reagents (eg, storage reagents, activation reagents, buffers, surfactants, reducing agents, etc.), rinse solutions (buffers) , Ultrapure water, etc.), desorbing liquid (MALDI matrix, acid, base, surfactant, etc.) and an analysis target.
[0085]
The prestation combines the activation of ligand and analyte by separate microcolumns, sample collection / separation function, and sample transfer / elution function. Preferably, multiple samples are loaded into the prestation and the multiple samples are spatially arranged in alignment with the molecular trap array so that one sample corresponds to a single molecular trap in the molecular trap array.
[0086]
The sample array is automatically relocated from the pre-station to the use station. The station of use is where the samples and specific analytes contained therein are processed. In one embodiment, one end of the molecular trap array is lowered to the sample and this same sample is drawn into each molecular trap. Since each molecular trap has an affinity receptor located on the surface of the molecular trap, drawing a sample into the molecular trap will contact any of the specific analytes sought by the affinity receptor. Each molecular trap in the array can have a different affinity receptor than the affinity receptors of other molecular traps in the array, thus targeting different specific analytes from the same or different media It becomes possible. The sample material can be drawn into the molecular trap once or several times. After sufficient specific analytes have been captured by the molecular traps in the array, the remaining or uncaptured medium is rinsed at least once (although other embodiments may not require a rinsing step) ). After washing away the non-target compounds, they are eluted from the molecular trap by contacting the specific analytes that have been captured with a solution selected to block affinity interactions. The specific analytes that are eluted can then be prepared directly for mass spectrometry or further prepared for processing such as, for example, enzymatic / chemical modification, and subsequently prepared for mass spectrometry. To do. The prestation may consist of multiple locations for chemical modification, molecular traps, functionalization, biological fluid analysis and / or transport. The last specific analytes eluted are relocated to the target array by stamping them on the target array.
[0087]
Referring to FIG. 7b, the preferred embodiment of the use station further includes a microcolumn manifold to which an array of molecular traps is attached. The microcolumn manifold is attached to the head of an automated device that physically moves the array of molecular traps during each processing station. The physical motion of the microcolumn manifold can be right angle (xy) or circular (rotation) behavior. Alternatively, the microcolumn manifold in the use station may be stationary and the processing station may be relocated under the array of molecular traps. Similar to the physical motion of the microcolumn array described above, the physical motion of the processing station can be right angle (xy) or circular (rotation) behavior. In a further embodiment, it is contemplated that both the microcolumn manifold and the processing station are in physical motion together.
[0088]
In a preferred embodiment, the target array is automatically relocated to a storage / loading station that can contain at least one target array. From the storage / loading station, the target array is transferred using an interaction database to an automated mass spectrometer capable of multiple sample entry and automatic processing / data analysis.
[0089]
Automated mass spectrometers are implemented with specific analytes or modified fragments detected with high accuracy. Software capable of recognizing differences between samples or from standards is used to assist in the analysis of a large number of samples and is the basis for establishing new information systems, namely structural function systems, clinical systems, diagnostic systems and biochemical systems Create an appropriate database. Biochemical systems are generally involved in the types of molecules found in biological organs and cover the full range of catabolic and anabolic reactions that occur in biological systems such as enzymatic reactions, binding reactions, signal reactions, and other reactions. including. Other biochemical systems include model systems that mimic specific biochemical interactions. In the practice of the present invention, examples shown within the context of the present invention of interest include, for example, receptor ligand interactions, protein-protein interactions, enzyme-substrate interactions, cell signaling pathways, transport reactions, genotyping, A phenotypic classification.
[0090]
After analysis by mass spectrometry, the target array can be transferred to the post station for sample processing following mass analysis or further analysis.
[0091]
Thus, in one aspect, the invention will be useful for screening ligands or compounds that affect the interaction between a receptor molecule and its ligand.
In-robot sensuality
Example 20
[0092]
Amine activation / derivatization in an automated device for functionalized porous molecular traps
Another method involves automatic device integration in the format described above, in the process, protocols such as glutaraldehyde activation and ligand coupling are used for coordinated activation / derivatization in the automatic device. Here the amine functionalized porous glass molecular trap is dry loaded / installed (manually or mechanically assisted) into a warm P-200 wide bore pipette affinity pipette (Roggins Corp.) and loaded into a plastic 96 rack Is done. This rack is installed on stage 1 of a 6-stage automatic apparatus, loaded with microtiter plates containing various coupling solutions and rinse solutions, connected to a personal computer, and controlled by software. The coupling of glutaraldehyde to the amine microcolumn was from a 96 well microtiter plate in position 2 containing 110 μL of 25% glutaraldehyde solution in 0.10 M sodium phosphate buffer, pH 7.8 per well. , Using 100 repeated aspirations. This initial coupling requires extensive HBS buffer rinsing (110 μL / well) achieved at station 3 (containing a second 96-well microtiter plate, 50 repeated aspirations), thereby speeding up Automatic device-mediated activation coupling (reaction time 10-20 minutes). After the final water rinse at station 4 (approximately 50 repeated aspirations), the activated aldehyde groups on the glutaraldehyde matrix are then station 5 (0.1-1 mg / mL, 55 μL / well, approximately 200 aspirations). Incubated with the protein antibody of interest. Uncoupled (excess) antibody was exhaustively used at station 6 with HBS buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 0.005% detergent P20, 50 repeated aspirations). Remove with a clean rinse.
Example 21
[0093]
Amine activation, amplification, reactivation and derivatization of amine microcolumns in automated equipment
One extension of the method shown above is the automatic device integration of glutaraldehyde activation, followed by amplification in the automated device and reactivation prior to ligand coupling, in a coordinated manner in the automated device. Activation / amplification / derivation. In this example, an aldehyde functionalized porous glass molecular trap (aldehyde microcolumn) is prepared and loaded at position 1. Next, the activated aldehyde groups on the glutaraldehyde microcolumn were incubated with polylysine (30-300 kDa) (110 μL, 1 mg / mL in HBS) loaded at position 2 of the automated instrument stage to form one polymer backbone. From which fresh glutaraldehyde-mediated activation takes place, and the antibody binds to the surface of the amplified microtop.
[0094]
Following this initial coupling step, a thorough rinse at station 3, 50 repeated aspirations of HBS buffer, results in a rapid automated device-mediated activation coupling (reaction time 30 minutes). After the final water rinse at station 4 (approximately 50 repeated aspirations), the activated aldehyde groups on the glutaraldehyde matrix were removed at station 5 (0.1-1 mg / mL, 55 μL / well, 200 repeated aspirations). When incubated with the protein antibody of interest, the MASSAY microcolumn becomes an affinity pipette. Uncoupled (excess) antibody is removed by rinsing thoroughly at station 6 with HBS buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% detergent P20).
[Example 22]
[0095]
Preparation of chemically masked target and microcolumn elution for MALDI-TOF
The arrayed target provides a hydrophilic target that serves to localize the analyte sample sent from the microcolumn under elution conditions. These elution conditions vary depending on the purpose of the analysis, and are commonly used MALDI-TOF matrix (acetonitrile: 0.2% TFA aqueous solution of alpha cyano-4-hydroxycinnamic acid in a ratio of about 1: 2. ), Diluted acid, diluted base, chaotropic agent and the like.
[0096]
In this example, the target surface is coordinated with a hydrophobic background in a chemical mask prepared for the intended analysis, depending on the type of biomaterial used in the assay of interest. It consists of a working contrasting array. Regardless of the purpose, the first requirement is a series of surface washes of rinse and incubation. First, the target surface is washed with a surfactant, rinsed with water, rinsed with methanol, and then incubated in a 10-15% aqueous hydrogen peroxide solution at room temperature (or elevated temperature) for 30 minutes to 1 hour. The cleaned surface is then water rinsed, methanol rinsed and nitrogen dried, and the target area is derivatized with an interesting alkyl mercaptan. For the cation exchange surface, 11-mercaptoundecanoic acid or 3-mercapto-1-propanesulfonic acid is used as a saturated solution in an organic solvent (such as isopropanol). The surface is immediately coated with a saturated solution of octadecyl mercaptan or rinsed with isopropanol followed by methanol and subsequently coated with a saturated solution of octadecyl mercaptan. The chemically contrasted or masked surface is for sample analysis by microcolumn analyte localization and mass spectrometry. These cation exchange surfaces are designed for operation with common biological materials and are particularly useful when assaying urine-derived materials. Masked targets are also made using positively charged functional groups and may be used as anion exchangers.
Example 23
[0097]
High-throughput integrated system for biological samples MALDI-TOF analysis
This example illustrates an integrated system for parallel processing and analysis of nascent biological fluid-related biomolecules while demonstrating the capabilities of a high-throughput integrated system. Shown in FIG. 8 is a beta-2-microglobulin (β) derived from a human plasma sample performed using the integrated system and method of the present invention. ₂ m) High-throughput semi-quantitative analysis. Aliquots of diluted (5-fold) human plasma samples taken from 6 individuals were prepared for parallel screening on 96-well sample plates. 15 μL of plasma aliquot in each well (6 samples randomized on 96-well plates), 7.5 μL of equine plasma (undiluted, equine β ₂ m including MW _{eq. β2m} = 113966.6, MW _{hum. β2m} = 11729.2) and 128 μL HBS (0.01 HEPES, pH 7.4, 0.15 M NaCl, 0.005% (v / v) polysorbate 20, 3 mM EDTA) buffer. Select 8 samples from 96 samples, and add β to 4 wells. ₂ m 10 ^-2 Add 0.5 μL of the mg / mL solution and add the other 4 wells to the same β ₂ 1 μL of m solution was added. Parallel sample processing is 96 anti-β ₂ With simultaneous incubation / capture of 96 samples on m-derivatized microcolumn. Polyclonal anti-β by coupling of carboxymethyldextran (CMD) -EDC mediated antibody to an amino-coated / modified microcolumn ₂ An m microcolumn was made. The captured protein was extracted with a small amount of MALDI matrix (33% (v / v) acetonitrile, α-cyano-4-hydroxycinnamic acid (ACCA) saturated solution in 0.2% (v / v) trifluoroacetic acid). It is stamped on the surface of a MALDI target array composed of a self-assembled monolayer (SAM) that is eluted from a microcolumn and chemically masked, resulting in a target array having a hydrophilic / hydrophobic contrast. Each sample spot on the target array is analyzed using mass spectrometry and the relative β ₂ The amount of m is determined by an automated MALDI-TOF mass spectrometry software routine. The mass spectrum obtained from the high efficiency analysis of 96 samples is shown in FIG. β ₂ The spectrum taken from the sample with the m standard solution added is black.
[0098]
The semi-quantitative data generated by the high-throughput system shown in FIG. 8 will be described using the bar graph display of FIG. Each spectrum shown in FIG. ₂ Standardized to m signal, human β ₂ The standardized integral for the m signal was determined. Total β of spectra obtained from the same individual sample ₂ The m integrated values were averaged and the standard deviation was calculated. Similarly, 10 ^-2 mg / mL β ₂ The integral values for the samples added with 0.5 μL and 1.0 μL of m solution were calculated and averaged. Depicted in this figure is a standardized human β for 6 individual samples and the sample to which the solution was added. ₂ It is the average value of m integral. This bar graph shows β in the sample to which the solution was added. ₂ clearly shows an increase in m concentration, and β in human blood ₂ The value of high-throughput semi-quantitative analysis performed using the systems and methods described in the present invention, in fact confirming that increased concentrations of m are associated with a variety of disease states. .
[0099]
The integrated system and method described within the present invention is also similar to the human blood β of FIG. ₂ m High throughput quantitative analysis. Six plasma samples were prepared as described in FIG. Of the 96 well sample plate, 15 μL of plasma aliquots in 88 wells (6 samples were randomized on 96 well plates). 7.5 μL of horse plasma (undiluted) and 128 μL of HBS buffer were placed. Purified human β ₂ 7.6 × 10 of m ^-4 Prepare a series of dilutions of the mg / mL standard solution (concentration is 7.7 × 10 ^-4 mg / mL to 1.14 × 10 ^-4 used as a sample (15 μL each) in the last row (8 wells) on a 96 well plate. Parallel sampling and MALDI-TOFMS analysis are performed using polyclonal anti-β ₂ Performed as described in FIG. 8 using an m microcolumn. Mass spectra obtained from high throughput analysis of 88 samples and 8 standards are shown in this figure. The spectrum obtained from the standard sample is painted black.
[0100]
FIG. 11b is a calibration curve drawn from the data of FIG. 11a for the standard sample shown in FIG. This calibration curve is derived from β in human plasma samples screened by high-throughput analysis using the integrated system and method described in this invention. ₂ It is made to determine the m concentration. A representative spectrum of data relating to each standard used to create a calibration curve is shown superimposed on FIG. 11a. Each spectrum is converted to equine β ₂ Standardized to m signal, human β ₂ Standardized integration values for the m signal were determined. The integrated values of the five spectra taken for each calibration standard were averaged and the standard deviation was calculated. The calibration (standard) curve is the human β in the standard sample (adjusted by the dilution rate of human plasma) ₂ Plotting was done by plotting the average of the standardized integrated values of each standard against m concentration. The created calibration curve is shown in FIG. 11b. The concentration range is good linearity with a standard deviation <2% over the entire line (R ² = 0.999).
[0101]
FIG. 12 shows a bar analysis of the data shown in FIG. 10 using the standard curve drawn in FIG. 11b. With respect to the 88 samples in FIG. ₂ Standardized to m signal, human β ₂ The standardized integral for the m signal was determined. Human β for the same individual ₂ The m integral was averaged and the standard deviation was calculated. The mean integral value is converted by the equation derived from the standard curve, and human β ₂ m concentration was calculated. The concentration range determined was 0.75 mg / mL to 1.25 mg / mL.
Example 24
[0102]
Combined integrated system method incorporating high-throughput affinity recovery for point mutations and bioreactive array MALDI-TOF analysis
FIG. 13 is a quantitative high-throughput screen of transthyretin (TTR) for post-translational modification (PTM), with point mutation (PM) performed using the integrated system and method described herein. did. Aliquots of diluted (5-fold) human plasma samples taken from 6 individuals were prepared in 96-well plates for parallel screening. Each well contained 15 μL of plasma volume (6 samples were randomized on 96 well plates) and 135 μL of HBS buffer. Parallel sample processing involves the simultaneous incubation / capture of 96 samples on 96 anti-TTR derivatized microcolumns. Polyclonal anti-beta by coupling glutaraldehyde-mediated antibody to amino-coated / modified microcolumns ₂ An m microcolumn was made. The captured protein is eluted from the microcolumn with a small amount of MALDI matrix (saturated ACCA solution) and consists of a self-assembled monolayer (SAM) that is chemically masked to become a hydrophilic / hydrophobic contrast target Stamped on the surface of the MALDI target array. Each sample spot on the target array is analyzed using mass spectrometry, and the relative TTR amount is determined by an automated MALDI-TOF mass spectrometry software routine. The mass spectrum obtained from the high throughput analysis of 96 samples is shown in FIG. In all of the spectra, the TTR signal is accompanied by another signal of higher mass indicating the TTR that has undergone the post-translational process. Also, all spectra obtained from the analysis of a plasma sample showed two more signals about 30 Da higher than the two “original” TTR signals. See FIGS. 15 and 16 for identification of these peaks.
[0103]
FIG. 14 identifies the post-translational modifications and point mutations observed in the high-throughput TTR analysis using the integrated system and method described herein. Representative spectra from two sample analyzes are shown, showing the presence of 2 and 4 signals, respectively. In the upper spectrum, two signals attributed to TTR are observed. The signal is the TTR's theoretically calculated mass (MW _TTR = 13,762) and oxidized TTR variants (TTR) resulting from cysteinylation at Cys10 (leading to a mass shift of +119 Da) _ox ) Corresponds well to that. In the lower spectrum, in addition to the two TTR signals mentioned above, two more peaks are seen at a mass about 30 Da higher than the two “original” TTR signals. See FIGS. 15 and 16 for identification of these two peaks.
[0104]
Subsequent analysis of TTR point mutations was performed using a combination of a derivatized mass spectrometer target array and high throughput affinity recovery in the system platform and method described in the present invention, as shown in FIG. To clarify. The sample used is the same as that used in FIG. TTR of diluted (50 times in HBS) human plasma was captured by a polyclonal anti-TTR microcolumn as described in FIG. Capture protein is eluted with a small amount of 10 mM HCl on a trypsin-bound target containing a buffer-containing target spot (50 mM TRIS buffer, pH 9.5) for sample pH adjustment (buffer exchange) instead of matrix elution did. Shown in this figure is a mass spectrum obtained from the protein eluted from the anti-TTR microcolumn and digested with trypsin at 40 ° C. for 20 minutes. The resulting peptide map from two trypsin digests was trypsin fragment-12 (T ₁₂ ) Has found a mutation. A database search lists two possible TTR mutations in the sequence of this region: Ala109 → Thr [DNA base change is GCC → ACC], Δm = 30.111 Da, Thr119 → Met [DNA base change is ACG → ATG], Δm = 29.992 Da. The identification of the correct mutation is shown in FIG.
[0105]
FIG. 16 is a high resolution reflectron mass spectrometry method used to determine the identity of point mutations detected in the plasma sample analysis shown in FIG. Built in the way. Trypsin digest fragment T in standard (natural) TTR ₁₂ The monoisotopic signal of (104-127) is m / z = 2644.9922, indicating a difference of Δm = 29.988 Da from the monoisotopic signal of the mutant TTR. Thus, the point mutation results in Thr119 → Met, Δm = 29.992 Da. This TTR point mutation becomes a “Chicago prealbumin” mutation, which is a so-called non-amyloid mutation. The results shown in conjunction with FIGS. 13, 14, 15 and 16 are described herein for post-translational modification and identification of point mutations by concerted high-throughput screening analysis of biological samples. Explains the use of the system and method.
Example 25
[0106]
Integrated combinatorial method incorporating high-throughput affinity recovery and bioreactive array MALDI-TOF for translation word modification (PTM) analysis
FIG. 17a is a representative example of a quantitative high-throughput screen for post-translational modifications present in biological fluids performed using the integrated systems and methods described herein. Concerted biological fluid phosphate analysis was performed using a chelator affinity pipette with an alkaline phosphatase (AP) functionalized target array. Here, a chelator affinity pipette was prepared as previously disclosed according to Example 14. Human whole saliva, centrifuged and diluted 10-fold, was analyzed in the sample after incubation with a metal chelator affinity pipette. Analytes captured from metal chelator affinity pipettes elute with the addition of dilute acid to disrupt the chelator / metal / analyte interaction and become hydrophilic / hydrophobic contrast target arrays, or alkaline Buffer exchange was incorporated for phosphatase digestion. Direct analysis of diluted human saliva is significantly devoid of proline-rich proteins of interest. The spectrum in FIG. 17b (2) shows the capture of metal chelator affinity pipettes shown as two phosphate-rich proteins, PRP-1 and PRP-3. As a dephosphorylation mass signal, a small spectrum is seen in FIG. 17b (3) and in full form in FIG. Detection of multiple analytes with partial and complete phosphorylation of phosphoproteins captured / digested from biological fluids for post-translational analysis such as phosphorylation events has been shown.
Example 26
[0107]
Integrated system method by MALDI-TOF analysis for affinity multiple protein interactions required for recovery of nascent protein complexes
In this example, protein-protein interactions are shown that allow for the recovery of natural multiple protein complexes present in biological materials or biological samples. Shown in FIG. 18 is a multiple protein complex between retinol binding protein (RBP) and transthyretin (TTR) in a human plasma sample performed using the integrated system and method of the present invention. is there. Polyclonal anti-RBP affinity pipettes were formed using glutaraldehyde-mediated amine base supported surface coupling. Human plasma was prepared and used as described in the above examples. MSIA shows in vivo affinity recovery of RBP and complex TTR. The multiple protein complex between retinol binding protein (RBP) and transthyretin (TTR) indicates that protein interactions exist within the native protein complex.
Example 27
[0108]
Integrated system method incorporating high throughput multiple analyte recovery and MALDI-TOF analysis
In this example, to illustrate multi-analyte analysis of a human plasma sample performed for rapid qualification and semi-quantification using the integrated system and method according to the present invention, a multi-receptor affinity micro A high-throughput method using a column is described. FIG. 19 shows simultaneous rapid monitoring of relative amounts of multiple analytes. In order to rapidly capture each analyte from human plasma, an amine activated polycunal anti-b2m / CysC / TTR affinity pipette was used as described above. This in turn can be used to quickly monitor biological fluid concentration adjustments and to quantify adjusted protein events from their normalized relative amounts. ₂ Illustrates the use of multiple antibody affinity pipettes for m, CysC and TTR. In addition, the acute phase of viral infection or β ₂ Potential β in fibril formation from m or TTR ₂ Fig. 4 illustrates the use of affinity pipettes in the integrated system and method of the present invention for monitoring m / CysC concentrations.
[0109]
FIG. 20 shows the rapid monitoring of a complete multi-analyte affinity pipette in the integrated system and method of the present invention. To capture each analyte from human plasma (obtained as described in the previous example) ₂ Combinations as well as individual polyclonal antibody affinity pipettes incorporating m, TTR, RBP, cystatin C or CRP were also used. To quickly monitor biological fluid concentration adjustments and, in some cases, to quantify adjusted protein events from their normalized relative amounts ₂ Illustrates another use of multiple / single antibody affinity pipettes for m, CysC and TTR or CRP. In addition, the acute phase of virus infection (such as AIDS) or β ₂ Potential β in fibril formation from m or TTR ₂ Figure 3 illustrates another use of affinity pipettes for monitoring m / CysC concentration.
[0110]
Given the ability to efficiently increase samples, the integration of high-throughput systems for biomolecular mass spectrometry allows for large-scale clinical, diagnostic, and therapeutic efficacy, as well as biological from biological fluids. Increased use in applications requiring both exceptional qualitative and quantitative analysis in biologically important biomolecular analysis.
[0111]
As used herein, “affinity microcolumn” refers to a molecular trap contained within a housing.
[0112]
As used herein, an “affinity reagent” refers to a chemical molecular species that is on the surface of a molecular trap and is chemically activated or activatable to which an affinity receptor can be covalently bound.
[0113]
As used herein, “affinity receptor” refers to an atomic or molecular species that has an affinity for an analyte present in a biological material or biological sample. Affinity receptors can be natural organics, inorganics or biological materials and can exhibit a broad (single analyte target) to narrow (single analyte target) specificity. Examples of affinity receptors include antibodies, antibody fragments, paratopes, peptides, polypeptides, enzymes, proteins, multi-subunit protein receptors, mimics, organic molecules, polymers, inorganic molecules, chelators, nucleic acids, aptamers, and Although the below-mentioned receptor etc. are mentioned, it is not limited to these.
[0114]
As used herein, “analyte” refers to a molecule of interest that is present in a biological sample. Analytes include nucleic acids (DNA, RNA), peptides, hormonal peptides, hormones, polypeptides, proteins, protein complexes, carbohydrates with biological functions, small inorganic molecules or small organic molecules. However, it is not limited to these. Analytes can of course contain sequences, elements or groups that are recognized by affinity receptors, or these recognition moieties introduced into them by extracellular, enzymatic, chemical treatment of cells. Can have.
[0115]
As used herein, “biological material” or “biological sample” refers to a liquid or extract of biological origin. Biomaterials include, but are not limited to, cell extracts, nuclear extracts, cell lysates, secretions, blood, serum, plasma, urine, sputum, synovial fluid, brain-spinal fluid, tears, feces, saliva, membrane extracts And so on.
[0116]
As used herein, “chemical activation” refers to the process of exposing an affinity reagent to a chemical (or light) in order to attack (photoactivate) a chained linker or affinity receptor. Point to. The reagent that can activate the affinity reagent is not limited, and can be an organic reagent, an inorganic reagent, or a biological reagent. It is often beneficial to activate the affinity reagent using multiple steps, including the use of a chained linker. As used herein, “chained linker” refers to affinity reagents and affinity receptors that can be derivatized with high concentrations of affinity receptors and exhibit the desirable characteristics of low binding to non-specific compounds. Refers to a compound that mediates. A chained linker may be active in nature or may require activation for addition. Suitable chaining reagents include, but are not limited to, homo / heterofunctional organics, natural polymers, synthetic polymers, and biopolymers.
[0117]
As used herein, “dead volume” refers to the empty volume within a molecular trap. As used herein, “small dead volume” refers to a range of 1 nanoliter to 1 milliliter.
[0118]
As used herein, “high fluidity” refers to a flow rate of at least 1 microliter per minute. Higher flow rates are considered to fall within the definition of high fluidity.
[0119]
As used herein, a “mass spectrometer” refers to a device that can volatilize / ionize analytes to form gas phase ions and measure their absolute or relative molecular weight. Preferred forms of volatilization / ionization are laser / light, heat, electricity, atomization / spray, etc., or combinations thereof. Suitable forms of mass spectrometry include, but are not limited to, MALDI-TOF MS, electrospray (or nanospray) ionization (ESI) mass spectrometry, quadrupole ion trap, Fourier transform ion cyclotron (FT-ICR), magnetic A mass spectrometer such as a sector or a combination thereof may be used.
[0120]
As used herein, a “mass spectrometer target” or “mass spectrometer target array” refers to, in addition to, one or more analytes for continuous mass analysis. Refers to the device being placed. In general, the target is suitable for many samples depending on the nature of the mass spectrometer and takes various geometrical arrangements, but the target is selected for the mass spectrometer. Suitable materials for constructing the target include metals, glasses, plastics, polymers, multilayers, etc., or combinations thereof.
[0121]
As used herein, “molecular trap” refers to a high surface area material that contains an affinity reagent bound to the surface, and has high fluidity and low dead volume. The combination of high surface area materials can be, but is not limited to, crystals, glass, plastics, polymers, metals, or any combination of these materials. For example, these glasses can be quartz glass, borosilicate, sodium borosilicate, and other useful materials.
[0122]
As used herein, the term “receptor” generally refers to a member of a pair of compounds that specifically recognize and bind to each other. The other member of the pair is called a “ligand” and includes such things as complexes, protein-protein layer interactions, multiple analyte analysis, and the like. Receptor / ligand pairing can include protein receptors, (membrane) and their natural ligands (related or other proteins or small molecules). Receptor / ligand pairs also include antibody / antigen binding pairs, complementary nucleic acids, nucleic acid related proteins and their nucleic acid ligands such as aptamers and their proteins, metal chelators and metal binding protein ligands, pseudodyes and their protein ligands Organic molecules and their interactions, such as hydrophobic patches of biomolecules or with biomolecules, ion exchangers on or with biomolecules and their electrostatic interactions, etc. It is done.
[0123]
As used herein, “automated apparatus” refers to devices and methods capable of non-participating processing of samples. The automated device preferably operates on multiple samples in parallel to maximize the number of samples processed and analyzed within a given time.
[0124]
Preferred embodiments of the invention are described in the drawings and description of preferred embodiments. While these descriptions directly describe the above embodiments, it is understood that one skilled in the art can contemplate changes and / or variations to the specific embodiments shown herein. Any such modifications or changes that fall within the scope of this description are intended to be included therein as well. Unless otherwise noted, it is the intent of the inventors that the terms in the specification and claims are given their ordinary customary meaning to those of ordinary skill in the applicable arts. Preferred embodiments and best mode of the invention known to the applicant at the time of filing are presented and are intended for illustration and description purposes. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teaching. The embodiments best illustrate the principles and practical applications of the invention, and best illustrate the invention in various embodiments and with various modifications to suit particular uses contemplated by other persons skilled in the art. Selected and explained to be available.
[Brief description of the drawings]
[0125]
FIG. 1 is a diagram showing an MSIA method. Analytes are selectively recovered from the solution by repeated flow through a receptor derivatized affinity pipette. After washing the non-specifically bound compounds, the retained molecular species are eluted onto the mass spectrometer target or target array using (in a preferred embodiment) a MALDI matrix. Next, an accurate m / z value of the analyte is detected by MALDI-TOF MS. The analysis is qualitative in nature but can also be quantified by incorporating it into a technique that uses a mass shift variant of the analyte as an internal standard.
Fig. 2 β of biological fluid ₂ -Figure 1 shows MSIA screening for microglobulin. For the sample, the biological fluid is HBS (H for single MALDI-TOF. ₂ Prepared by diluting with O). After washing the affinity pipette with HBS and water, ACCA (ACN: H ₂ O, saturated at 1: 2; 0.2% TFA) is used to elute the compounds retained directly on the mass spectrometer target. (A) Human tears. (B) Human plasma. (C) Human saliva-saliva required an additional rinse with 0.05% SDS (in water) to reduce non-specific binding. (D) Human urine. In all cases, β ₂ m was efficiently recovered from the biological fluid using a flow incubation / rinse technique. β ₂ The measured mass (using external calibration) for m is the calculated value (MW _calc = 11,729.7; MW _tears = 11,735; MW _plasma = 11,734; MW _saliva = 11,742; MW _urine = 11,735) within about 0.1%. Illustrated various biological fluid screening by MSIA for directed, rapid, sensitive and accurate analysis.
Fig. 3a β ₂ It is a figure which shows the quantitative MSIA calibration curve of m. A representative spectrum of data used to create a calibration curve. Human β at a concentration of 0.01 to 1.0 mg / L ₂ m was examined. Horse β ₂ m (MW = 11396.6) was used as an internal reference.
FIG. 3b is a diagram showing a calibration curve created using the data represented in FIG. 3a. Good linearity (R over a range of 2-10 ² = 0.983) with a low standard error (~ 5%). Error bars reflect the standard deviation of the 10-fold 65-laser shot spectrum taken from each sample. These figures show β ₂ Illustrates quantitative MSIA performed with an m-affinity pipette.
FIG. 4 β ₂ FIG. 6 shows quantitative MSIA screening of m. Human urine samples from 5 people were screened over 2 days. The average value measured in healthy people (10 samples; 4 (3 men; 1 woman) age 30-44) was 0.100 ± 0.021 mg / L. For concentrations measured in an 86-year-old woman with a recent urinary tract infection, ₂ m concentration was significantly increased (3.23 ± 0.072 mg / L).
FIG. 5. Glycosylated β in an 86-year-old woman ₂ FIG. 6 is a diagram of MSIA showing dark density increase (dark gray). During MSIA, a second signal is observed at Δm = + 161 Da and glycosylated β ₂ the presence of m. MSIA has two types of β ₂ m body can be sufficiently decomposed, and nascent β ₂ A more accurate quantification of m was obtained, allowing the quantification of glycoproteins. Such discrimination can be attributed to two types of β ₂ This is important in considering that the m-body is derived from (or is a marker of) a different disease. Healthy MSIAs that show little glycosylation are shown for comparison (light gray).
FIG. 6 Defined biological fluid / β ₂ -Surface-based MSIA for microglobulin specificity. Polyclonal anti-beta bound by carboxymethyl dextran amplification ₂ The use of m-affinity pipettes, or amine base support chemistry, allows the differentiation between non-specific binding compounds and specific binding compounds in biological fluid / MSIA. Samples were prepared from biological fluids and used as shown in FIG. (A) Human plasma. (B) CMD amplified β ₂ Human plasma through an m-affinity pipette. (C) amine / glutaraldehyde bond β ₂ Human plasma through an m-affinity pipette. Direct analysis of the human plasma spectrum (upper spectrum) ₂ m mass signal (MW _plasma = 11,734). CMD is an affinity pipette chemical target β ₂ m is amplified, but shows non-specifically bound compounds (middle spectrum). Only in the last case β ₂ m is efficiently recovered from a biological fluid having a low concentration of non-specific binding compound (lower spectrum). This illustrates a preferred aspect in directed analysis of blood-derived biofluid biomarkers using carefully affinity pipettes for specific mass detection.
FIG. 7a is a schematic diagram illustrating an integrated system for high-throughput analysis of biomolecules from biomaterials.
FIG. 7b is an enlarged schematic diagram showing the station used, which consists of a microcolumn integrated automated device with multiple configurations for chemical modification, microcolumn functionalization, biological fluid analysis, migration, etc. Is done.
FIG. 8 β ₂ It is a figure which shows the high throughput semi-quantitative analysis of mMSIA. Β from human plasma samples ₂ m used the integrated system and method described in the present invention.
9 is a graph showing a bar graph analysis of the data shown in FIG. 8. FIG. Each spectrum shown in FIG. 8 is based on the baseline integration and equine β ₂ normalized to m signal, human β ₂ The standardized integral of the m signal was measured. The total β of the spectrum obtained from a sample of the same individual ₂ The m integral was averaged and the standard deviation was calculated. 10 ^-2 mg / mLβ ₂ The sum of samples spiked with 0.5 μL and 1.0 μL of m was calculated and averaged. Normalized human β of samples from 6 and spiked samples ₂ The average value of m integration is plotted in this figure. This bar graph shows β in the spiked sample ₂ The increase in m concentration is clearly confirmed, and β in human blood is associated with various disease states ₂ In confirming the increase in m concentration, the values of high-throughput semi-quantitative analysis performed by the system and method described in the present invention are illustrated.
FIG. 10 shows β from human plasma samples using the integrated system and method described in the present invention. ₂ FIG. 6 is a diagram showing a high-throughput quantitative analysis of m.
FIG. 11a. Β in human plasma samples screened by high-throughput analysis using the integrated system and method described in the present invention. ₂ It is a figure which shows the plot of a calibration curve from the data of the standard sample shown in FIG. 10 in order to determine m density | concentration.
FIG. 11b β in human plasma samples screened by high-throughput analysis using the integrated system and method described in the present invention. ₂ It is a figure which shows the plot of a calibration curve from the data of the standard sample shown in FIG. 10 for the purpose of determining m density | concentration.
FIG. 12 is a graph showing a bar graph analysis of the data shown above using the calibration curve drawn above. Each of the 88 sample spectra in FIG. ₂ normalized to m signal, human β ₂ The standardized integral of the m signal was measured. Human β of the same individual ₂ The m integral was averaged and the standard deviation was calculated. Substituting the averaged integrated value into the formula derived from the calibration curve in FIG. ₂ The concentration of m was calculated for each person. The determined concentration range is 0.75 mg / L to 1.25 mg / L.
FIG. 13: Quantitative high-throughput screening of transthyretin (TTR) for post-translational modification (PTM) and point mutation (PM) using the integrated system and method described in the present invention. FIG.
FIG. 14 shows the identification of post-translational modifications and point mutations observed in high-throughput TTR analysis using the integrated system and method described in the present invention.
FIG. 15 illustrates the identification of point mutations by incorporation of a derivatized mass spectrometer target platform in the systems and methods described in the present invention.
FIG. 16: High resolution reflectron mass spectrometry as part of the integrated system and method described in the present invention to determine the identity of point mutations detected in the analysis of plasma samples shown in FIG. FIG.
FIG. 17a shows biological fluid phosphate analysis-concert of chelator affinity pipette and alkaline phosphatase functionalized target array. (1) Human whole saliva (10 μL diluted 10-fold). (2) EDTA / Ca ²⁺ Sample from (1) passed through an affinity pipette. (3) Elution by addition of 10 mM HCl, stamped on AP-BRP incorporating 50 mM borate buffer and pH = 10 buffer exchange and digested with phosphate for 15 minutes (50 ° C.) sample. (4) The sample of (3) in which digestion was prolonged for 30 minutes. Direct analysis of a 10-fold dilution of human saliva is significantly devoid of the proline-rich protein-1 (PRP-1) and serine-modified phosphate-rich protein of interest.
FIG. 17b: (2) spectrum shows EDTA / Ca of PRP-1 and PRP-3, two phosphate-rich proteins. ²⁺ FIG. 6 shows affinity pipette capture. The dephosphorylation mass signal is clearly visible in trace spectrum (3) and in full spectrum (4). FIG. 6 illustrates detection of multiple analytes by partial as well as complete dephosphorylation of phosphorylated proteins captured / digested from a biological fluid for post-translational analysis (ie phosphorylation events).
FIG. 18 shows MSIA of a multiple protein complex of retinol binding protein (RBP) and transthyretin (TTR). Active amine, polyclonal anti-RBP affinity pipettes were formed by glutaraldehyde mediated amine base supported surface coupling. Human plasma was prepared and used for the analysis of FIG. MSIA shows in vivo affinity recovery of RBP (MW = 21,062 Da) and conjugated TTR (MW = 13,760 Da). Illustrates protein interactions present in natural protein complexes.
FIG. 19 is a diagram showing simultaneous and rapid monitoring of a plurality of analytes related to relative abundance ratios. Active amine, polyclonal anti-β ₂ An m / CysC / TTR affinity pipette is used to rapidly capture each analyte from human plasma (50-fold dilution in HBS). The figure shows β for rapid monitoring of biological fluid concentration modulation and for quantifying modulated protein events from normalized relative abundance. ₂ An example of the use of multiple antibody affinity pipettes for m, CysC and TTR is shown. This figure shows the acute phase of viral infection (such as AIDS), or β ₂ Potential β in fibril formation from m or TTR ₂ One example of the use of an affinity pipette to monitor m / CysC concentration is shown.
FIG. 20 illustrates rapid monitoring of a wide range of multiple analyte affinity pipettes. Individual β ₂ Polyclonal antibody affinity pipettes / combinations incorporating m, TTR, RBP, cystatin C or CRP capture each analyte from human plasma (diluted 50-fold in HBS). This figure shows β for rapid monitoring of biological fluid concentration modulation and, in some cases, for quantifying modulated protein events from normalized relative abundance. ₂ An example of the use of multiple / single antibody affinity pipettes for m, CysC and TTR is shown. This figure shows the acute phase of viral infection (such as AIDS), or β ₂ Potential β in fibril formation from m or TTR ₂ Fig. 6 shows another example of using an affinity pipette to monitor m / CysC concentration.
FIG. 21 shows a mass spectrometry target array. (A) A trapezoidal target capable of confining a sample by a meniscus action. (B) Control design that can confine the sample by hydrophobic / hydrophilic action. (C) Insertion target used for smaller sampling fills, expensive reagents, or sample transfer.

Claims (10)

A high-throughput integrated system for qualitative and quantitative biomolecule analysis,
a) a robotic platform with a plurality of spatially arranged affinity microcolumns;
b) a mass spectrometer target having a spatial arrangement corresponding to the same spatial arrangement as the affinity microcolumn;
c) a mass spectrometer capable of receiving a target having the spatial arrangement. The system of claim 1, wherein the spatial arrangement includes from 4 to 1,536 elements. The system of claim 1, wherein the robotic platform further comprises a plurality of processing stages. The system of claim 1, wherein when the affinity microcolumn receives a specific biomolecule in a biomaterial, the specific biomolecule is recovered by affinity interaction. The system of claim 1, wherein the mass spectrometer target has enzymatic activity. The system according to claim 1, wherein the mass spectrometer is a MALDI-TOF type mass spectrometer. 4. The system of claim 3, wherein at least one of the plurality of processing stages is for selectively isolating specific biomolecules present in a biomaterial using an affinity microcolumn. . 4. The system of claim 3, wherein at least one of the plurality of processing stages is for rinsing the affinity microcolumn to remove nonspecifically retained compounds. 4. The system of claim 3, wherein at least one of the plurality of processing stages is for placing selectively retained biomolecules on a mass spectrometer target. When there are multiple types of biomaterial samples, a plurality of spatially arranged affinity microcolumns and a mass spectrometer target having a spatial arrangement corresponding to the same spatial arrangement as the affinity microcolumns The system of claim 1, wherein the samples are processed at substantially the same time and in parallel.