WO2006026416A1 - Compositions, procedes, systemes, et kits pour purification d'affinite - Google Patents

Compositions, procedes, systemes, et kits pour purification d'affinite Download PDF

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Publication number
WO2006026416A1
WO2006026416A1 PCT/US2005/030380 US2005030380W WO2006026416A1 WO 2006026416 A1 WO2006026416 A1 WO 2006026416A1 US 2005030380 W US2005030380 W US 2005030380W WO 2006026416 A1 WO2006026416 A1 WO 2006026416A1
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WIPO (PCT)
Prior art keywords
peptides
proteins
sample
substrate
mass
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PCT/US2005/030380
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English (en)
Inventor
Gordon Nicol
Barry Boyes
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Agilent Technologies, Inc.
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Publication of WO2006026416A1 publication Critical patent/WO2006026416A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • Proteins regulate biological processes provide the structural components of cells, and control metabolic functions. Protein activity is not always directly correlated with the expression level of a corresponding mRNA transcript in a cell, but is impacted by post-translational modifications, such as protein phosphorylation, processing events (e.g., cleavage) and the association of proteins with other biomolecules.
  • post-translational modifications such as protein phosphorylation, processing events (e.g., cleavage) and the association of proteins with other biomolecules.
  • proteome analysis Quantitative proteomics or proteome profiling is the systematic analysis of all proteins expressed by a cell or tissue with respect to their quantity and identity and form.
  • changes in protein expression may be related to changes in cell states (e.g., a progression from a normal state to a pathological state).
  • proteome profiling requires the analysis of thousands of proteins, high throughput techniques for obtaining information regarding protein identity and quantity in a sample are required.
  • samples have been resolved by two- dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the relative concentrations of proteins of interest determined by generic protein staining and densitometry or fluorescence intensity.
  • 2D-PAGE two- dimensional polyacrylamide gel electrophoresis
  • a serious drawback in this technique is the limited dynamic range of the 2D gel/staining and scanning.
  • 2D-P AGE is difficult to automate and does not lend itself readily to parallel evaluation of large numbers of samples.
  • lower abundant proteins are often outside of the range of detection of 20- PAGE.
  • Mass spectrometry permits a high throughput approach to proteome analysis.
  • Mass spectrometry may be used to evaluate whole proteins, e.g., by surface-enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF); however,
  • MS/MS tandem mass spectrometry techniques
  • protein samples are digested with chemicals or enzymes (e.g., trypsin), and peptides obtained are further fragmented in a mass spectrometer.
  • enzymes e.g., trypsin
  • Referential analysis between MS fragmentation patterns and the in silico generation of theoretical peptide fragments from reverse translation/transcription of genomic data may be used to identify and quantify proteins, since peptides may provide signatures for proteins from which they are derived.
  • ICAT technology e.g., U.S. Patent 6,670,194 by Aebersold, et al.
  • ICATs isotope-coded affinity tags
  • MS/MS mass spectroscopy
  • the ICAT method is based on the modification of cysteine- containing proteins by an iodacetate derivative carrying a biotin label. After enzymatically cleaving modified proteins into peptides, cysteine-modified, biotin- labeled peptides are purified using avidin-coated beads.
  • This affinity purification step reduces the complexity of the original peptide mixture, making the sample more amenable to mass spectrometry.
  • Comparative quantification may be performed by differentially labeling two samples being compared, e.g., with light and heavy isotope labels, such that peptides that are the same chemically (i.e., have the same amino acid sequence) will have different masses, distinguishable by mass spectrometric analysis.
  • the avidin-biotin interaction is relatively strong and allows the sample to be washed so that the peptides that do not bind to the avidin resin will be removed leaving only peptides that contain the cysteine residue that has been derivatized with this reagent.
  • a drawback of the technique is that the avidin-biotin interaction is not completely specific and peptides that do not contain the biotin moiety will also bind to avidin. In addition, due to the strength of the avidin-biotin interaction, disassociation of avidin-biotin complexes is difficult. Furthermore although the avidin-biotin purification step does reduce the complexity of the sample, there are often false positives in the analysis, i.e., peptides are often found that do not contain a cysteine, which are selected due to the non-specific interactions between such peptides and avidin.
  • Another drawback of the ICAT technology is that it limits analysis of proteomes to those peptides that contain a cysteine residue. Cysteines are found in 85% of the proteins in many mammalian species, exhibiting an overall occurrence in the human proteome of about 1.7%. That is, for every 1000 amino acids in the human proteome there are only 17 cysteines. This makes both identification and quantification of proteins difficult.
  • Peptides selected from complex samples using methods of the present invention are enriched for methionine-, cysteine, and histidine- containing peptides, and can be concentrated when released from the Pt-substrate under eluting conditions.
  • Methionine occurs in the human proteome at a rate of 2.4%, and when combined with the cysteines present, the number of peptides per protein that can be selected for analysis should be nearly 3 times greater than for the ICAT reagent. Histidine occurs in the human proteome at a rate of about 2.2%. This increase in protein coverage may be used to provide additional confidence in protein identification, to improve the accuracy of relative quantification, and to detect post-translational modifications that may be present on the selected peptides.
  • the invention provides methods, systems, compositions and kits for affinity purification of proteins and/or peptides. These methods, compositions and kits may be used in proteome analysis, for example, in conjunction with mass spectroscopy techniques.
  • the invention provides a composition comprising a substrate stably associated with a platinum (Pt) coordination compound.
  • the Pt- substrate composition provides an affinity matrix for selectively binding to proteins or peptide fragments thereof which comprise sulfur or an -NH 2 group, an imidazole, or nitrogen group capable of donating electron groups to a Pt compound coordination compound, such as proteins or peptides comprising methionines, histidines, or reduced cysteines.
  • two different samples are affinity purified to obtain two populations of proteins enriched in sulfur and/or nitrogen groups, e.g., to enrich for proteins comprising cysteine, methionine, or histidine.
  • the two different samples may be differentially labeled, e.g., both populations may comprise different types of labels, or one population may comprise labeled proteins, while the other population comprises unlabeled proteins.
  • a label comprises a molecule that alters the mass of proteins or peptide fragments thereof.
  • the label comprises an isotopic label. Proteins may be labeled before, after, or during binding to the affinity column.
  • the label is one that permits discrimination between two identical but differentially labeled peptide fragments of a protein.
  • differentially labeled peptides may generate distinct mass spectra peaks.
  • differentia] labeling is carried out across multiple different samples to generate substantially chemically identical peptides that are distinguishable by mass.
  • the populations may be mixed together to determine ratios of peptides labeled with first and second labels, or ratios of unlabeled and labeled peptides.
  • mass altering labels are selected which are used to differentiate between modified and unmodified forms of proteins.
  • protein modifications alter the mass of peptides.
  • Internal standards representing modified or unmodified proteins may be spiked into protein samples to provide a means to calibrate amounts of modified proteins.
  • two or more protein samples are compared, for example, a protein sample or source from which the sample is obtained which has been exposed to an agent (a drug, carcinogen, potential toxin, potential carcinogen, teratogen, hormone) or condition (e.g. an environment, treatment regimen, temperature, etc.).
  • the protein samples can also be derived from normal cells in different states of differentiation, or derived from normal and diseased cells, or diseased and drug- treated cells or some combination thereof.
  • Samples may be compared by differentially labeling the samples, for example, with first and second mass-altering labels (e.g., heavy and light isotope pairs) and determining the ratio of an amount of peptide having a first label to an amount of peptide having a second label.
  • first and second mass-altering labels e.g., heavy and light isotope pairs
  • proteome samples may be screened with compounds (e.g., libraries of biomolecules), to identify those compounds that are able to produce a desired cell state.
  • the invention provides a system comprising a Pt coordination compound stably associated with a substrate ("Pt-substrate") for binding proteins and/or peptides, and an analysis system for determining at least one characteristic of a protein or peptide eluted from the Pt coordination compound (e «g., such as mass, sequence, quantity, etc.).
  • the system may comprise modular components which may be connectable to each other via interfacing modules.
  • the system further comprises a separation device for separating proteins or peptides eluted from the Pt coordination compound.
  • the system comprises an analysis device for determining at least one characteristic of a protein or peptide eluted from the Pt-substrate column.
  • the analysis device comprises a mass spectrometer.
  • the separation device may be interfaced with the separation device by, for example, an electrospray device. Either or both the Pt- substrate and separation device may be contained within a microfluidic device and may be connected by channels in a microfluidic substrate.
  • the system further comprises a processor for receiving data from the analysis device.
  • the processor sends instructions to one or more system components for controlling a system function.
  • the processor receives data relating to a system function.
  • the processor provides or alters instructions to system components in response to data received from the system.
  • the processor accesses a memory which may be remote from the processor and/or other system components.
  • kits for facilitating methods according to embodiments of the invention.
  • a kit comprises a Pt- substrate composition and one or more reagents, such as a cell lysis buffer, an elution solution for removing a protein or peptide bound to a Pt-substrate, buffers compatible with MS systems.
  • Figure 1 illustrates a reaction scheme for the binding of sulfur-containing peptides with a Pt compound (Compound I) according to one aspect of the invention.
  • Figure 3A shows a Pt coordination compound stably associated with a substrate via a flexible linker (L).
  • Figure 3B shows reactive complexes which may than be attached to a solid support.
  • Figure 4 illustrates a scheme for the reaction of cysteine residues with a methanethiosulfonate (MTS) reagent, methyl methanethiosulfonate, in order to block binding of cysteine to the Pt resin.
  • MTS methanethiosulfonate
  • Figure 5A illustrates a scheme for the reaction of cysteine residues with iodoacetic acid in order to prevent oxidation of cysteine residues via disulfur linkage, thus preserving the ability of cysteine to bind to the Pt resin.
  • Figure 5B illustrates a scheme for the reaction of cysteine residues with vinyl pyridine in order to prevent oxidation of cysteine residues via disulfur linkage, thus preserving the ability of cysteine to bind to the Pt resin.
  • Figure 6 is a schematic demonstrating the steps for how proteins containing methionine, cysteine, or histidine can be prepared for analysis using the methods of the invention.
  • Figure 7 is a schematic demonstrating the steps for how proteins containing methionine or cysteine can be prepared for analysis using the methods of the invention.
  • Figure 8 is a schematic demonstrating the steps for how proteins containing methionine can be prepared for analysis using the methods of the invention.
  • a “set” or “sub-set” of any item may contain only one of the item, or only two, or three, or any multiple number of the items.
  • a “peptide mixture” is typically a complex mixture of peptides obtained as a result of the cleavage of a sample comprising proteins.
  • sample of proteins is typically any complex mixture of proteins and/or their modified and/or processed forms, which may be obtained from sources, including, without limitation: a cell sample (e.g., lysate, suspension, collection of adherent cells on a culture plate, a scraping, a fragment or slice of tissue, a tumor, biopsy sample, an archival cell or tissue sample, laser-capture dissected cells, etc), an organism (e.g., a microorganism such as a bacteria or yeast), a subcellular fraction (e.g., comprising organelles such as nuclei or mitochondria, large protein complexes such as ribosomes or golgi, and the like), an egg, sperm, embryo, a biological fluid, viruses, and the like.
  • a cell sample e.g., lysate, suspension, collection of adherent cells on a culture plate, a scraping, a fragment or slice of tissue, a tumor, biopsy sample, an archival cell or tissue sample, laser-capture dis
  • peptide refers to an entity comprising at least one peptide bond, and can comprise either D and/or L amino acids.
  • a "ligand” is a peptide consisting essentially of about 2 to about 20 amino acids (i.e., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids).
  • Protein means any protein, including, but not limited to peptides, enzymes, glycoproteins, hormones, receptors, antigens, antibodies, growth factors, etc., without limitation. Proteins include those comprised of greater than about 20 amino acids.
  • polypeptide and “protein” are generally used interchangeably herein.
  • proteins or peptides or a sample refers to a sample comprising proteins only, peptides only, or a mixture of proteins and peptides.
  • a peptide fragmentation signature refers to the distribution of mass-to-charge ratios of fragmented peptide ions obtained from fragmenting a peptide, for example, by collision induced disassociation, ECD, LID, PSD, IRNPD, SID, and other fragmentation methods.
  • diagnostic or a “diagnostic signature" of a target protein or target polypeptide is one which is reproducibly observed when a peptide digestion product of a target protein/polypeptide identical in sequence to the peptide portion of a peptide internal standard, is fragmented or which differs only from the fragmentation pattern of the peptide internal standard by the mass of the mass-altering label.
  • a peptide is said to be “isolated” or “substantially purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals.
  • the peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components.
  • a biological fluid includes, but is not limited to, blood, plasma, serum, sputum, urine, tears, saliva, sputum, cerebrospinal fluid, lavages, leukapheresis samples, milk, ductal fluid, perspiration, lymph, semen, umbilical cord fluid, and amniotic fluid, as well as fluid obtained by culturing cells, such as fermentation broth and cell culture medium.
  • a sample of complex proteins may contain greater than about 100, about 500, about 1,000, about 5,000, about 10,000, about 20,000, about 30,000, about 100,000 or more different proteins.
  • samples may be derived from a natural biological source (e.g., cells, tissue, bodily fluid, soil or water sample, and the like) or may be artificially generated (e.g., by combining one or more samples of natural and/or synthetic or recombinant sources of proteins).
  • expression refers to a level, form, or localization of product.
  • expression of a protein refers to one or more of the level, form (e.g., presence, absence or quantity of modifications, or cleavage or other processed products), or localization of the protein.
  • a difference in expression refers to an increase or decrease in expression.
  • a difference may be an increase or a decrease in a quantitative measure (e.g., amount of a protein or modified or processed form thereof) or a change in a qualitative measure (e.g., a change in the localization of a protein).
  • a difference is observed in a quantitative measure, the difference according to the invention will be at least about 10% greater or less than the level in a normal standard sample.
  • the increase may be as much as about 20%, 30%, 50%, 70%, 90%, 100% (2-fold) or more, up to and including about 5-fold, 10-fold, 20-fold, 50-fold or more.
  • a difference is a decrease
  • the decrease may be as much as about 20%, 30%, 50%, 70%, 90%, 95%, 98%, 99% or even up to and including 100% (no specific protein or RNA present).
  • even qualitative differences may be represented in quantitative terms if desired.
  • a change in the intracellular localization of a protein may be represented as a change in the percentage of cells showing the original localization.
  • a diagnostic trait is an identifying characteristic, or set of characteristics, which in totality, are diagnostic.
  • the term “trait” encompasses both biological characteristics and experiences (e.g., exposure to a drug, occupation, place of residence).
  • a trait is a marker for a particular cell type, such as a transformed, immortalized, pre-cancerous, or cancerous cell, or a state (e.g., a disease) and detection of the trait provides a reliable indicia that the sample comprises that cell type or state. Screening for an agent affecting a trait thus refers to identifying an agent which can cause a detectable change or response in that trait which is statistically significant within 95% confidence levels.
  • cancer refers to a malignant disease caused or characterized by the proliferation of cells that have lost susceptibility to normal growth control.
  • Malignant disease refers to a disease caused by cells that have gained the ability to invade either the cells of origin or to travel to sites removed from the cells of origin.
  • a "cancer-specific marker” is a biomolecule which is expressed preferentially on cancer cells and is not expressed or is expressed to a small degree in non-cancer cells of an adult individual.
  • a small degree means that the difference in expression of the marker in cancer cells and non-cancer cells is large enough to be detected as a statistically significant difference when using routine statistical methods to within 95% confidence levels.
  • a correlation refers to a statistically significant relationship determined using routine statistical methods known in the art. For example, in one aspect, statistical significance is determined using a Student's unpaired t-test, considering differences as statistically significant at p ⁇ 0.05.
  • a "diagnostic probe” is a probe whose binding to a tissue and/or cell sample provides an indication of the presence or absence of a particular trait.
  • a probe is considered diagnostic if it binds to a diseased tissue and/or cell ("disease samples") in at least about 80% of samples tested comprising diseased tissue/cells and binds to less than 10% of non-diseased tissue/cells in samples ("non-disease” samples).
  • the probe binds to at least about 90% or at least about 95% of disease samples and binds to less than about 5% or 1% of non- disease samples.
  • isotopic forms refers to multiple versions of a derivatizing agent which are identical structurally but differ in isotopic content.
  • proteome refers to the protein constituents expressed by a genome, typically represented at a given point in time.
  • a "sub-proteome” is a portion or subset of the proteome, for example, the proteins involved in a selected metabolic pathway, or a set of proteins having a common enzymatic activity.
  • a “remote location,” refers to location other than the location at which the affinity purification and/or mass spectroscopy occurs.
  • a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc.
  • “Communicating information” refers to transmitting the data representing that information as signals (e.g., electrical, optical, radio, magnetic, etc) over a suitable communication channel (e.g., a private or public network).
  • a component of a system which is "in communication with” or “communicates with” another component of a system receives input from that component and/or provides an output to that component to implement a system function.
  • a component which is "in communication with” or which "communicates with” another component may be, but is not necessarily, physically connected to the other component.
  • the component may communicate information to the other component and/or receive information from the other component.
  • “Input” or “Output” may be in the form of electrical signals, light, data (e.g., spectral data), materials, or may be in the form of an action taken by the system or component of the system.
  • the term "in communication with” also encompasses a physical connection that may be direct or indirect between one system and another or one component of a system and another.
  • "Forwarding" an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data.
  • a “computer-based system” refers to the hardware means, software means, and data storage means used to analyze the information of the present invention.
  • the minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • the data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.
  • a computer-based system may include one or more wireless devices.
  • Record data, programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art.
  • any convenient data storage structure may be chosen, based on the means used to access the stored information.
  • a variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.
  • a "processor” references any hardware and/or software combination which will perform the functions required of it.
  • any processor herein may be a programmable digital microprocessor such as available in the form of a electronic controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based).
  • a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.
  • a “database” is a collection of information or facts organized according to a data model which determines whether the data is ordered using linked files, hierarchically, according to relational tables, or according to some other model determined by the system operator.
  • an “information management system” refers to a program, or series of programs, which can search a database and determine relationships between data identified as a result of such a search.
  • an “interface on the display of a user device” or “user interface” or “graphical user interface” is a display (comprising text and/or graphical information) displayed by the screen or monitor of a user device connectable to the network which enables a user to interact with a system processor and/or system memory (e.g., including a data base and information management system).
  • a system processor and/or system memory e.g., including a data base and information management system.
  • providing access to at least a portion of a database refers to making information in the database available to user(s) through a visual or auditory means of communication.
  • the term “assessing” and “evaluating” are used interchangeably to refer to any form of measurement, and includes determining if an element is present or hot.
  • the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. Assessing may be relative or absolute. "Assessing the presence of includes determining the amount of something present, as well as determining whether it is present or absent.
  • the term "using” has its conventional meaning, and, as such, means employing, e.g. putting into service, a method or composition to attain an end.
  • a program is used to create a file
  • a program is executed to make a file, the file usually being the output of the program.
  • a computer file is used, it is usually accessed, read, and the information stored in the file employed to attain an end.
  • a unique identifier e.g., a barcode
  • the unique identifier is usually read to identify, for example, an object or file associated with the unique identifier.
  • the invention provides a substrate stably associated with a platinum (Pt) coordination compound.
  • Pt-coordination compounds may be used to affinity purify biomolecules, such as proteins or peptides, comprising a sulfur (S) or nitrogen (N) group in a sample. See, e.g., as shown in Figure 1.
  • a Pt- substrate composition (such as shown in Figure 2) may be used to reduce the complexity of a complex sample of proteins or peptides, such as a proteome or peptidome, by selectively binding to sulfur-containing or nitrogen-containing biomolecules (e.g., such as proteins/peptides comprising methionine-, histidine-, and reduced cysteine- containing proteins/peptides).
  • An affinity-purified population of proteins may comprise natural proteins, synthetic proteins, modified proteins, unmodified proteins, processed proteins or unprocessed proteins, and combinations thereof.
  • the invention describes analysis of proteins and peptides, the analysis of other molecules is also envisioned, for example, the substrate may be used to select nucleic acids, lipids, fatty acids and steroids for further analysis, such as in mass spectrometric techniques.
  • the method comprises the step of providing a sample comprising a plurality of proteins, contacting the sample with a Pt-substrate comprising compounds with affinity for sulfur groups (e.g., such as proteins comprising methionine and reduced cysteine groups) and nitrogen groups (e.g., such as histidine), and eluting proteins bound to the Pt-substrate, to obtain a population of proteins which are enriched for proteins comprising such groups.
  • the sample comprises the proteome of a cell.
  • the sample may be derived from a biological fluid, tissue, population of cells, biopsy, archival sample, environmental sample and the like.
  • proteins are cleaved into smaller fragments (e.g., peptides), before, during, or after contacting the proteins with the Pt-substrate composition.
  • proteins are contacted with one or more cleaving agents to produce peptide fragments having carboxy-terminal lysine or arginine residues.
  • proteins may be treated with trypsin, Lys-C, another protease, or any combination thereof.
  • Proteins subjected to affinity purification may be characterized using a variety of techniques. For example, in one aspect, affinity-purified proteins or peptide fragments thereof are characterized using mass spectroscopy techniques. Proteins contacted with a cleaving agent, before, during or after contacting with a Pt-substrate, generate peptide fragments that may be characterized by multistage or tandem mass spectrometry. Proteins or peptides bound to Pt-substrate compositions are eluted from the Pt-substrate. Where proteins are eluted, these may be contacted with cleaving agents. When peptides are eluted, in certain aspects, these are evaluated by mass spectrometry.
  • the mass of a peptide determined by mass spectrometry is compared to the mass of a known or previously characterized peptide that may be correlated with sequence information for the known or previously characterized peptide.
  • the method further comprises searching one or more sequence databases for the sequence(s) observed for the protein or peptide fragment thereof.
  • At least one coordination site of the platinum compound binds directly or indirectly (e.g., through a linker) to a substrate.
  • a "linker” refers to a bifunctional chemical moiety which comprises an end for stably associating with a substrate and an end for stably associating with the Pt-coordination compound (e.g., able to donate electrons to a coordination site of the Pt coordination compound).
  • a linker when other than a bond, will have from about 1 to 60 atoms, usually 1 to 30 atoms, where the atoms include C, N, O, S, P, etc., and will generally have from about 1 to 12 carbon atoms and may have from about 0 to 8 or more, or about 0 to 6 or more heteroatoms.
  • the atoms are exclusive of hydrogen in referring to the number of atoms in a group, unless indicated otherwise. Additional types of linker molecules are described in, e.g., Backes and Ellman (1997) Curr. Opin. Chem. Biol. 1 :86-93, Backes et al. (1996), J. Amer. Chem. Soc.
  • a linker comprises a group that may undergo a nucleophilic attack, including, but not limited to an ether, carboxylate, succinimide, or other like group.
  • linkers are selected from the group shown in Figures 3 A and 3B.
  • linkers may include oligonucleotides or peptides (e.g., such as polylysine).
  • linkers are cleavable from the substrate, e.g., they may include photocleavable groups, pH sensitive groups, thermolabile groups or sites for enzyme cleavage.
  • linkers are selected which do not alter the ability of the Pt-compound to bind to other ligands.
  • linkers may be selected which exhibit the least amount of non-selective binding to sample, e.g., linkers are inert to the sample components.
  • the linker may be covalently, ionically, or otherwise stably associated with the substrate.
  • Stable associations can include covalent or non-covalent bonds and, and may be direct (i.e., the Pt coordination compound may bind to the substrate via an interaction between a coordination site on the Pt compound and a chemical group or molecule immobilized on the substrate) or indirect (i.e., the Pt compound may bind to a ligand that may bind covalently or non-covalently to a linker molecule which itself forms direct stable associations with the substrate).
  • the linker is covalently immobilized to the substrate.
  • a Pt compound stably associated with a substrate selectively binds to at least about one biomolecule.
  • a biomolecule ligand coordinating the Pt portion of a Pt- substrate composition comprises a suitable electron density for donating to an empty orbital of the Pt group via a sulfur or nitrogen group.
  • the Pt compound may include one or more stabilizing substituents, which are at least substantially stable or unreactive under conditions of storage and/or use of the substrate.
  • the stabilizing substituents may be same or different from one another, and may be selected based upon the desired conditions of use since these substituents may affect the physical and/or chemical properties of the substrate, e.g., hydrophobicity/hydrophilicity, etc.
  • stabilizing substituents are interconnected, together constituting a stabilizing bridge moiety.
  • the stabilizing bridge is at least divalent, occupying two ligand sites with the platinum atom, but may be multivalent, occupying more than two such ligand sites.
  • Aliphatic amine compounds may be used to form stabilizing bridges.
  • ethylenediamine is used to provide a divalent stabilizing bridge.
  • Suitable Pt compounds which can be stably associated with substrates include, but are not limited to those described in U.S. Patent No. 6,338,943, U.S. Patent No. 6,248,531, U.S. Patent No. 6,133,038, U.S. Patent No. 5,985,566, U.S. Patent No. 5,714,327; U.S. Patent No. 5,580,990; EP 0539466; EP 1262778; WO 92/01699; WO 96/35696, and WO 98/15564.
  • substrates include, but are not limited to, gels, fibers, microspheres, spheres, cubes, particles, beads (including porous beads), pellets, planar substrates (e.g., slides, discs, wafers, chips), channels, microchannels, nanochannels, capillaries, walls of containers, membranes, webs, gels, sheets, tubing, spheres, containers, pads, slices, films, plates, slides, strips, plates, disks, rods, particles, beads, and filters.
  • suitable substrates include, but are not limited to, gels, fibers, microspheres, spheres, cubes, particles, beads (including porous beads), pellets, planar substrates (e.g., slides, discs, wafers, chips), channels, microchannels, nanochannels, capillaries, walls of containers, membranes, webs, gels, sheets, tubing, spheres, containers, pads, slices, films, plates, slides, strips, plates, disks, rods, particles, beads, and filters
  • the substrate may be formed of a variety of materials and the size and shape of the substrate is not a limiting feature of the invention.
  • the substrate may be rigid or flexible or semi-flexible.
  • the term "rigid” is used herein to refer to a structure e.g., a bottom surface that does not readily bend without breakage, i.e., the structure is not flexible.
  • the term "flexible” is used herein to refer to a structure, e.g., a bottom surface or a cover, which is capable of being bent, folded or similarly manipulated without breakage.
  • the substrate comprises a flexible web that can be bent 180 degrees around a roller of less than 1.25 cm in radius at a temperature of 20 °C.
  • Rigid solid supports may be made from silicon, glass, rigid plastics, e.g. polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, etc., or metals, e.g. gold, platinum, etc.
  • Flexible solid supports may be made from a variety of materials, such as, for example, nylon, nitrocellulose, polypropylene, polyester films, e.g., polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resin.
  • plasticizers and modifiers may be used with polymeric substrate materials to achieve selected flexibility characteristics.
  • substrates are selected which may be conveniently sorted, e.g., facilitating collection of affinity-purified biomolccules.
  • substrates may be magnetic or magnetizable, such that exposure to a magnetic field may be used to sort or separate Pt-bound substrates complexed to biomolecules or substrates from which Pt-biomolecules have been removed, or Pt-bound substrates from which biomolecules have been removed.
  • the Pt-substrate comprises a label or identifier (e.g., such as a bar code or radio frequency tag) identifying a sample or a container from which the sample is derived.
  • substrate materials are selected which are particularly suited for interfacing with mass spectrophotometers.
  • the solid substrate comprises a chromatographic material, which under specified conditions binds all proteins or peptides comprising sulfur groups (e.g., such as proteins or peptides comprising a methionine or reduced cysteine) and proteins comprising nitrogen groups comprising electrons which can donate to a Pt coordination site, e.g., such as histidine.
  • a substrate according to the invention is suitable for use in SELDI analysis.
  • the substrate comprises a web.
  • a "web" refers to a long continuous piece of substrate material having a length greater than a width.
  • the web length to width ratio may be at least 5/1, 10/1, 50/1, 100/1, 200/1, or 500/1, or even at least 1000/1.
  • the web is flexible and may be spooled past various processing stations, i.e., stations comprising cleaving agents, wash buffers, elution solutions, and the like.
  • the solid substrate comprises a material suitable for use in liquid chromatography, for example, a resin.
  • a resin refers to an insoluble material (e.g., a polymeric material) or particle that allows ready separation from liquid phase materials by filtration.
  • resins may be used for the packing of chromatographic columns. Resins can be used to carry tags and/or tagged peptides. Suitable resins include, but are not limited to, agarose, guaracrylamide, silica based materials, carbon-based materials, carbohydrate-based polymers (e.g., polysaccharide- containing), and the like.
  • the invention provides a method of using Pt-substrate compositions according to the invention for affinity purifying biomolecules comprising sulfur groups or nitrogen groups.
  • the Pt-substrate compositions are used for affinity purifying proteins or peptides comprising cysteines, methionines, and histidines, and modified, processed or derivatized forms thereof.
  • a protein sample is reduced prior to or while contacting with the Pt- substrate.
  • the method comprises the step of providing a sample comprising a plurality of proteins, contacting the sample with a substrate comprising compounds with affinity for sulfur groups (e.g., such as proteins comprising methionine and reduced cysteine groups) and nitrogen groups comprising electrons which can bind to Pt coordination sites (e.g., such as histidine), and eluting proteins bound to the substrate, to obtain a population of proteins which are enriched for proteins comprising such groups.
  • contacting is performed under conditions of low pH (e.g., less than about 5, or less than about 4, less than about 3.
  • contacting at low pH facilitates binding of methionine and reduced cysteine residues, but is suboptimal for binding of histidine residues (see, for example, EPl 262778).
  • contacting is performed at a less acidic pH (e.g., greater than 6, or greater than 7, or about 8).
  • Suitable buffers include those that do not comprise components or atoms that would bind to Pt coordination sites.
  • Samples may be complex samples (e.g., comprising proteomes or peptidomes) or may be samples that have been subjected to one or more previous purification steps.
  • samples may be treated prior to contacting with Pt-substrate columns to remove high abundance proteins such as albumin.
  • Purification may be based on hydrophobicity, size, charge, sequence, and the like, and may be performed using known methods such as SDS-gel electrophoresis, size-exclusion chromatography, isoelectric focusing, capillary electrophoresis and the like.
  • small volumes of sample may be concentrated prior to contacting with Pt-substrate compositions.
  • non-protein and non- peptide components are removed from the sample.
  • Pt-substrate compositions are used to analyze a complex protein sample comprising at least about 20% of total protein coming from a biological sample source, usually at least about 40%, more usually at least about 75%, and generally 90% or more, up to and including all of the protein obtainable from the source.
  • the proteome may be present in a cell, a lysate, a microsomal fraction, an organelle, a partially extracted lysate, biological fluid, and the like.
  • the proteome will be a mixture of proteins, generally having at least about 100 different proteins, usually at least about 1000 different proteins and in most cases, about 5,000 different proteins or more.
  • the sample will have at least about 0.05 mg of protein, usually at least about 1 mg of protein or about 10 mg of protein or more, typically at a concentration in the range of about 0.1-20 mg/ml, preferably, about 0.5-2.0 mg/ml, and most preferably about 1.0-2.0 mg/ml.
  • the sample may be adjusted to the appropriate buffer concentration and pH.
  • Pt-substrate compositions according to the invention bind to proteins/peptides in a manner that is reversible, i.e., when treated with an appropriate liquid-phase reagent (eluting condition), the proteins/peptides will desorb from the Pt-substrate.
  • Suitable eluting conditions include exposure to solutions comprising bcta-mercaptoethanol, sodium bisulfite, molecules comprising or capable of forming CN " groups, thiocyanates (e.g., potassium thiocyanate, guanidinethiocyanate), sodium thiosulphate, sodium metabisulphite, aromatic or aryl cyanides, or a molecule which competes with the bound biomolecule for the Pt coordination site, and the like.
  • thiocyanates e.g., potassium thiocyanate, guanidinethiocyanate
  • sodium thiosulphate sodium metabisulphite
  • aromatic or aryl cyanides or a molecule which competes with the bound biomolecule for the Pt coordination site, and the like.
  • the reduced cysteines can be protected (or blocked) with an appropriate alkylating reagent, such as iodoacetic acid or vinyl pyridine.
  • an appropriate alkylating reagent such as iodoacetic acid or vinyl pyridine.
  • a population of proteins is contacted with a cleaving agent before, after, or during exposure to a cleaving agent.
  • the proteins are exposed to a cleaving agent before, during, or after binding to a Pt-substrate composition but prior to elution of the proteins from the column.
  • a population of peptides bound to the Pt-substrate compositions may be generated or an eluted population of peptides may be generated.
  • Such peptides may comprise at least one methionine, cysteine, or histidine.
  • Methionine-containing peptides may represent N-terminal fragments of proteins.
  • the peptides that do not contain a methionine or a reduced cysteine can be specifically selected and removed, leaving the Met and Cys peptides bound to the substrate.
  • peptides comprising methionine, cysteine, and histidine are selected.
  • peptides comprising methionine and histidine are selected.
  • the cysteines of the peptides are reacted with a reagent which forms disulfide bonds with cysteine, such as a methanethiosulfonate (MTS) reagent (e.g., allyl methanethiosulfonate, 2-(aminocarbonyl)ethyl methanethiosulfonate, 2-(4-aminobenzoyloxy)ethyl methanethiosulfonate, benzyl methanethiosulfonate, butyl methanethiosulfonate, 2-carboxyethyl methanethiosulfonate, decyl methanethiosulfonate, dodecyl methanethiosulfonate, N- ( ⁇ -D-glucopyranosyl)-N'-[(2-MTS) reagent
  • peptides comprising methionine and cysteine are selected.
  • peptide comprising methionine are selected.
  • cysteines of the peptides are reacted with a reagent which forms disulfide bonds with cysteine, such as methanethiosulfonate (MTS), in order to prevent cysteine binding to the Pt-substrate.
  • MTS methanethiosulfonate
  • Suitable cleaving agents include, but are not limited to, enzymes, for example, one or more of: serine proteases (e.g., such as trypsin, hepsin, SCCE, TADGl 2, TADGl 4); metallo proteases (e.g., such as PUMP-I); chymotrypsin; cathepsin; pepsin; elastase; pronase; Arg-C; Asp-N; GIu-C; Lys-C; carboxypeptidases A, B. and/or C; dispase; thermolysin; cysteine proteases such as gingipains, TEV protease, factor Xa and the like.
  • serine proteases e.g., such as trypsin, hepsin, SCCE, TADGl 2, TADGl 4
  • metallo proteases e.g., such as PUMP-I
  • Proteases may be isolated from cells or obtained through recombinant techniques.
  • the cleaving agent is not limited to an enzyme and can be a chemical reagent, for example, cyanogen bromide (CNBr), 2-nitro-5- thiocyanobenzoic acid, N-bromosuccinamide and other reactive halogen compounds, hydroxylamine, 1 -2M formic or acetic acid, periodate oxidation, 2-(2- nitrophenylsulfenyl)-3-rnethyl-3-brornoindolenine or o-iodosobenzoic acid (See, for example, Hermodson et al., "Methods in Protein Sequence Analysis", ed. Elzinga, Humans Press, Clifton, N.J., pp. 313-323, 1982).
  • CNBr cyanogen bromide
  • 2-nitro-5- thiocyanobenzoic acid N-bromosuccinamide and other reactive halogen compounds
  • the cleaving agent may be associated with the substrate.
  • the cleaving agent may be disposed within the pores of substrates comprising porous beads or may be immobilized via a binding partner.
  • Proteins or peptide fragments thereof subjected to affinity purification may be characterized using a variety of techniques.
  • Affinity-purified proteins may be analyzed using a variety of techniques such as by mass spectrometry, including, but not limited to MALDI-TOF-MS, SELDI-TOF-MS, ESI, TOF, ion trap mass spectrometry, ion trap/TOF mass spectrometry, quadropole mass spectrometry, Fourier Transform mass spectrometry, fast atomic bombardment (FAB), plasma desorption (PD), thermospray (TS), and magnetic sector mass spectrometry.
  • FAB fast atomic bombardment
  • PD plasma desorption
  • TS thermospray
  • each protein sample comprises a different label or one sample is labeled while the other is unlabeled.
  • the label is a mass-altering label.
  • the samples may be mixed before or after affinity purification (e.g., see Figures 6-8).
  • the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids.
  • labeled peptides and labeled fragments are readily distinguished from unlabeled peptides/unlabeled fragments by their ion/mass pattern in the resulting mass spectrum.
  • the label does not suppress the ionization efficiency of the peptide.
  • the label remains soluble in an MS buffer system of choice.
  • Suitable mass-altering labels include stable isotopes, including but not limited to isotopes of hydrogen, nitrogen, oxygen, carbon, or sulfur, such as, 2 H, 13 C, 15 N, 17 O, 18 O, or 34 S, and combinations thereof.
  • pairs of stable isotopes are used which provide heavy and light mass labels. Such pairs include, but are not limited to H and D, 16 O and 18 O, 16 O and 17 0, 12 C and 13 C, 14 N and 15 N, 32 S and
  • the mass-altering label is part of a peptide comprising a modification, i.e., peptides comprising the modification and peptides lacking the modification are distinguishable by mass.
  • the modification may comprise a phosphorylated residue, a glycosylated residue, an acetylated residue, a ubiquitinated residue, a ribosylated residue, methylated residue, a sulfated residue, a prenylated residue, a hydroxylated residue, or a farnesylated residue.
  • the mass of a labeled peptide is determined and correlated with the identity and/or activity of a protein (e.g., the presence of a particular modified form of a protein which is known to be active in the proteome being evaluated).
  • a mass-to-charge ratio is determined, e.g., by mass spectrometry.
  • a quantitative measure of the amount of protein in the sample may be obtained.
  • the site of a modification may be determined by reacting sample proteins or peptides with a label comprising a reactive site which reacts with a modified residue on the protein.
  • the relative amount of a modified protein compared to unmodified protein also may be determined.
  • samples are combined (generally, equal amounts of samples are combined) and contacted with Pt-substrate compositions according to the invention.
  • proteins bound to the Pt-substrate are exposed to a cleaving agent (e.g., such as trypsin) to generate peptide-Pt-substrate complexes.
  • peptides are eluted from the Pt-substrate under eluting conditions (e.g., such as exposure to a solution comprising molecules comprising cyanates, beta mercaptoethanol, sodium bisulfite, sodium sulfite, potassium thiocyanate, guanidinethiocyanate, sodium thiosulphate, sodium metabisulphite, aromatic or aryl cyanides and the like).
  • eluting conditions e.g., such as exposure to a solution comprising molecules comprising cyanates, beta mercaptoethanol, sodium bisulfite, sodium sulfite, potassium thiocyanate, guanidinethiocyanate, sodium thiosulphate, sodium metabisulphite, aromatic or aryl cyanides and the like).
  • Eluted peptides may be subjected to one or more separation steps. Such separations may be based on size, charge, hydrophobicity or a combination thereof to obtain purified peptides whose mass may be determined (e.g., after fragmentation in a mass spectrometer). Suitable separation techniques include, but are not limited to: High Pressure Liquid Chromatography (HPLC), Low Pressure Liquid Chromatography, Reverse Phase-High Pressure Liquid Chromatography (RP-HPLC), gel electrophoresis (including capillary gel electrophoresis or any other electrophorctic modes), cation or anion exchange chromatography, or any of a number of peptide purification methods as are known in the art. In some aspects, separation is performed using a device which may be interfaced to a mass spectrometer. For example, separations may be performed using microcapillary liquid chromatography.
  • the mass of a peptide selected using Pt-substrates according to the invention may be determined and correlated with the identity and/or activity of a protein (e.g., the presence of a particular modified form of a protein which is known to be active). Preferably, a mass-to-charge ratio is determined, e.g., by multistage mass spectrometric analysis. In addition to determining the identity of a protein, a quantitative measure of the amount of protein in the sample may be obtained. The method may also be used to determine the site of a modification of a protein in one or more samples, by reacting sample proteins with a tag molecule comprising a reactive site that reacts with a modified residue on the protein. In another aspect, the amount of a modified protein in a sample is also determined.
  • Peptide sequence information may be automatically generated by selecting peptide ions of a particular mass-to-charge (m/z) ratio for collision- induced dissociation (CID) or other means for generating peptide ions known in the art.
  • CID collision- induced dissociation
  • the resulting ionization spectra may then be correlated with sequences in sequence databases to identify the protein from which the sequenced peptide originated, e.g., using computer searching algorithms known in the art.
  • Peptides may be quantified by measuring the relative signal intensities for pairs of peptide ions of identical sequence that are tagged using different mass- altering labels, e.g., such as light or heavy forms of isotope, or which comprise label and unlabeled peptide pairs (which differ in mass by the mass of the label).
  • a peptide or mass-altering portion of a peptide may comprise a detectable label such as a radioactive label, spin label, chemiluminescent label, and the like.
  • mass spectrometry analysis may be used to determine both the quantity and identity of proteins from which labeled peptides are derived, for example, by using an automated multistage mass spectrometer and alternating scans which measure quantities of peptides eluting from a separation column and record sequence information from selected peptides.
  • agents which can be evaluated include, but are not limited to: drugs; toxins; proteins; proteins; peptides; amino acids; antigens; cells, cell nuclei, organelles, portions of cell membranes; viruses; receptors; modulators of receptors (e.g., agonists, antagonists, and the like); enzymes; enzyme modulators (e.g., such as inhibitors, cofactors, and the like); enzyme substrates; hormones; nucleic acids (e.g., such as oligonucleotides; polynucleotides; genes, cDNAs; RNA; antisense molecules, ribozymes, aptamers), and combinations thereof. Agents also can be obtained from synthetic libraries which are commercially available or generated through combinatorial synthesis using methods well known in the art.
  • Agents associated with a desired cell state or the transition from an undesired cell state (e.g., a pathology) to a desired cell state (e.g., absence of the pathology or reduction in symptoms or biomarkers diagnostic of the pathology) may be identified as candidate compounds for treating the undesired cell state, for example, in a patient from whom the sample of cells was derived.
  • Such compounds may be formulated as pharmaceutical compositions using methods known in the art.
  • expression of a protein or set or proteins, and/or modified and/or cleaved forms thereof, associated with a particular cell state may be used to generate diagnostic probes to detect or screen for the cell states.
  • Such proteins (or modified or cleaved forms) may be detected directly, e.g., using mass spectrometry techniques or indirectly, e.g., using antibody probes.
  • the invention further provides a system which interfaces Pt-substrate compositions with a protein analysis system such as a mass spectrometer.
  • the system comprises a Pt-substrate composition directly or indirectly coupled to a separation device such as a column, capillary, or a channel (e.g., a microchannel or nanochannel) in a substrate.
  • the platinum-substrate may comprise a Pt-resin which is itself disposed in a column, capillary, or channel and which communicates, directly or indirectly with the separation device.
  • the substrate may be part of or contained within a chamber and peptides eluted from the substrate may be collected via a tube, capillary, spray, or injection device for delivery to the separation device.
  • the separation device may be an HPLC device, RP-HPLC device, an LC- microcapillary device, a gel matrix, an ion exchange matrix, and the like. The separation device may function both to separate peptides, purify individual peptides, and concentrate purified peptides. Both the Pt-substrate and separation device may be contained within a micro-scale or nano-scale device.
  • Pt- substrates and separation devices are separated by channels or reservoirs which may be used to add or exchange buffers or elution solutions used for affinity purification for buffers or other solutions suitable for separation.
  • the invention provides a computer program product comprising a computer readable medium comprising instructions for controlling functions of a system described above.
  • the invention additionally provides computer program products comprising computer readable medium providing instructions to a processor in communication with a system described above to control one or more system functions, e.g., exposure of Pt-substrate compositions to elution conditions, contacting proteins with a cleaving agent, ionization or peptide fragments, delivery of peptides to a mass spectrometer, and analysis of mass spectra.
  • An embodiment of the invention also includes forwarding, communicating, or receiving data produced from any method of the invention.
  • Another embodiment can additionally include forwarding a sample to a remote location and receiving data communicated from the remote location using that sample in a method of the present invention.
  • the system comprises a computer readable medium comprising a memory, wherein the memory comprises mass spectral data relating to a plurality of peptides, wherein each peptide comprises at least one cysteine, methionine, and/or histidine residue.
  • the memory may additionally comprise data relating to the mass of a peptide in a sample, the type of sample, data relating to agents to which the sample has been exposed, data relating to an organism (e.g., such as a human patient) from which a sample has been derived (e.g., such as medical history, drug exposure, and the like).
  • the separation module and or affinity purification composition may be in communication with a detector.
  • peptides or their mass- altering tags when such are used may be labeled with a label detectable by the detector (e.g., such as a radioactive label, spin label, chemiluminescent label, and the like).
  • throughput may be increased by dividing eluted peptides into a plurality of sets and separating the sets using a plurality of separation devices operating in parallel and/or in series.
  • the separation module interfaces with an mass spectrometer device through an interfacing module (such as an electrospray device, such as an electrospray capillary or nozzle) which delivers substantially purified peptides comprising, methionines, histidines, reduced cysteines, or other sulfur or nitrogen groups (e.g., from derivatizing agents) comprising electron groups which may bind to coordination sites of Pt compounds (e.g., generated by derivatizing the peptides with one or more chemical moieties) to the mass spectrometer.
  • phosphorylated residues may be tagged with a derivatizing agent which comprises or generates a sulfur or nitrogen group suitable for binding to Pt coordination sites.
  • an automated spotter may be used to connect a separation capillary to a MALDI device (see, e.g., Figeys et al., 1998, Electrophoresis 19: 2338-2347).
  • Throughput of the delivery process may be increased using arrays of electrospray or nanospray needles.
  • arrays of electrospray or nanospray needles See, e.g., Zubritsky et al., 2000, Anal. Chem. 72: 22A; Licklider et al., Anal. Chem. 72: 367-375; Scherer et al., 1999, Electrophoresis 20: 1508-1517).
  • Fluids may be moved through the system using mechanisms known in the art such as pressure or electro-osmotic pumping.
  • the system comprises one or more detectors for detecting movements of fluids, proteins, and/or peptides through the system.
  • the mass spectrometer device of the system comprises an ionizer, an ion analyzer and a detector.
  • Any ionizer that is capable of producing ionized peptides in the gas phase can be used, such as an ion spray mass spectrometer (Bruins et al., 1987, Anal Chem. 59: 2642-2647), an electrospray mass spectrometer (Fenn et al., 1989, Science 246: 64- 71), and laser desorption device (including matrix-assisted desorption ionization and surfaced enhanced desorption ionization devices).
  • Any appropriate ion analyzer can be used as well, including, but not limited to, quadropole mass filters, ion-traps, magnetic sectors, time-of-flight, and Fourier Transform Ion Cyclotron Resonance (FTICR).
  • tandem MS instrument such as a triple quadropole, ion-trap, quadropole-time-of flight, ion-trap-time of flight, or an FTICR is used to provide ion spectra.
  • a FAB ionizer may also be used.
  • molecular ions generated by ionization of peptides delivered, for example, from an electrospray or nanospray are accelerated through an ion analyzer as charged molecules. Ions generated may be detected using any suitable detector. In one aspect, ions are isolated and fragmented to generate daughter ions which when detected may provide a unique signature for the peptide.
  • peptides typically fragment at the amide bond between amino acid residues and peaks correspond to particular amino acids or combinations of amino acids. While there may be additional peaks (ions) present in the product ion spectra, many of these other peaks can be predicted and their presence explained by comparison with spectral data of known compounds (e.g., standards). Many different processes can be used to fragment the parent ion to form product ions, including, but not limited to, collision-induced dissociation (CID), electron capture dissociation, and post-source decay.
  • CID collision-induced dissociation
  • electron capture dissociation electron capture dissociation
  • post-source decay post-source decay
  • the system further comprises a system processor which can convert signals obtained from different components of the system (e.g., such as electrical signals) into data and can provide instructions for controlling one or more system functions.
  • data includes, but is not limited to, data relating to an identifier on a Pt-substrate, data relating to binding conditions and/or elution conditions during affinity purification using the Pt-substrate, data relating to separation, concentration, and/or purification of peptides eluted from the Pt-substrate using the separation device, data relating to fluid movement in the system (e.g., the operation of pressure or electroosmotic pumps), as well as data relating to peptide fragmentation, ionization, peptide quantity and amino acid sequence.
  • the processor compares mass spectral data to sequences in a protein and/or nucleic acid sequence database which the processor may access remotely.
  • the system further comprises a memory for storing data relating to peptide masses, and/or amino acid sequence.
  • the system additionally comprises an information management system for searching and comparing data in the memory and obtained from mass spectrometry analysis.
  • the processor obtains sequence information directly from mass spectral data provided to it from the mass spectrometer. The type of protein or peptide analysis performed by the system processor will relate to the type of mass spectrometer or other protein analysis device used in the system.
  • the processor in response to data from various system components, alters one or more functions of the system.
  • the processor is programmed e.g., by a user of the system and/or remotely to provide particular system instructions.
  • the invention additionally provides computer program products comprising computer readable medium providing instructions to a processor in communication with a system described above to control one or more system functions, e.g., exposure of Pt-substrate compositions to elution conditions, contacting proteins to a cleaving agent, ionization or peptide fragments, delivery of peptides to a mass spectrometer, and analysis of mass spectra.
  • system functions e.g., exposure of Pt-substrate compositions to elution conditions, contacting proteins to a cleaving agent, ionization or peptide fragments, delivery of peptides to a mass spectrometer, and analysis of mass spectra.
  • the invention provides kits for facilitating methods according to embodiments of the invention.
  • the kit comprises a Pt coordination compound and a solid substrate, along with suitable reagents for stably associating the compound with the substrate.
  • the kit comprises Pt coordination compounds stably associated with the substrate.
  • the kit comprises a suitable sample contacting solution for promoting binding between sample proteins and/or peptides to a Pt substrate.
  • the solution promotes binding between proteins/peptides comprising sulfur or nitrogren groups, such as proteins comprising methionines, histidines, and/or reduced cysteines, or nitrogen or sulfur groups comprising electrons which may bind to Pt coordination agents, e.g., such as found on certain derivatized proteins.
  • the kit further comprises derivatizing agents.
  • the kit may additionally include a reagent for reducing di-sulfide bonded proteins and/or an alkylating agent.
  • the kit comprises an elution solution for removing a biomolecule bound to the Pt-coordination compound from the Pt-coordination compound.
  • Such a solution may include, beta mercaptoethanol, sodium sulfite, sodium bisulfite, potassium thiocyanate, guanidinethiocyanate, sodium thiosulphate, sodium metabisulphite, aryl and aromatic cyanides, cyanates, molecules which may compete with biomolecules bound to the coordination sites of the Pt compound and the like.
  • the kit comprises a cleaving agent such as trypsin.
  • Additional kit reagents may include labels, such as mass-altering labels, label pairs (such as heavy and light isotopes), reference molecules such as peptide standards or lock mass molecules, buffers and reagents for use in peptide separations such as HPLC, RP-HPLC, cation or anion exchange, electrophoresis, and/or buffers suitable for use in biomolecule analysis systems such as mass spectrometers.
  • labels such as mass-altering labels, label pairs (such as heavy and light isotopes), reference molecules such as peptide standards or lock mass molecules, buffers and reagents for use in peptide separations such as HPLC, RP-HPLC, cation or anion exchange, electrophoresis, and/or buffers suitable for use in biomolecule analysis systems such as mass spectrometers.
  • the platinum/linker complex is attached to the substrate by reacting a platinum/linker that is terminated by a reactive group with an activated substrate.
  • the substrate comprises a reactive moiety while the platinum/linker comprises a reactive group.
  • the reactive moiety is any organic or inorganic group which, under appropriate conditions or by the addition of suitable reagents, will react with the platinum/linker reactive group to form a bond so that a Pt-substrate composition is formed.
  • the reactive moiety can include any reactive species, including a cyanato group (— O-C ⁇ N), a sulfonic acid ester group (-CH 2 -OSO 2 -alkyl), or a primary amine group (-NH 2 ) (see Figure 3B).
  • the polyfunctional chemical moiety is attached to the solid support and is also attached to the active site.
  • the polyfunctional chemical moiety may be aliphatic, aromatic, alkyl, alkenyl, heteroaliphatic, heteroaromatic, any suitably reactive inorganic compound, or any combination thereof.
  • the reactive group on the linker is any organic or inorganic group that will react with the reactive moiety to form a bond so that a Pt-substrate composition is formed.
  • the reactive moiety on the substrate is a cyanato group
  • the reactive group on the platinum/linker complex is a primary amine group or a primary thiol group.
  • Such a reaction is performed under conditions that are well known in the art. The reaction conditions are selected such that the isourea linkage is formed without significantly degrading other parts of the molecule.
  • the attachment of the platinum/linker complex to the substrate is alternatively accomplished by forming an alkyl linkage.
  • the reactive moiety on the substrate is sulfonic acid ester or -CH 2 -OSO 2 -R
  • the reactive group on the platinum/linker complex is a primary amine group or a primary thiol group.
  • the organic group on the ester that is bonded to the sulfur is selected so that the sulfonic acid ester is a good leaving group to form an alkyl linkage between the platinum/linker complex and the solid substrate.
  • the attachment of the platinum/linker complex to the activated substrate is alternatively accomplished by forming an amide linkage.
  • the reactive moiety on the substrate is a primary amine or -NH 2
  • the reactive group on the platinum-linker complex is a carboxylic acid group.
  • Reaction of the amine with the carboxylic acid to form an amide bond may be undertaken directly at any suitable condition. In cases where the reaction between the acid and the primary amine does not occur readily, it may be necessary to elevate the reaction temperature for the formation of the amide bond to occur. Alternatively, the reaction may proceed in good yield at room temperature by the use of coupling agents, such as dicyclohexylcarodiimide. Other exemplary agents include N,N'- carbonyldiimidazole, POCl 3 , TiCl 4 , sulfuryl chloride fluoride, chlorosulfonyl isocyanate, pyridinium salts-Bu 3 N, or a mixture Of Bu 3 P and PhCNO.
  • coupling agents such as dicyclohexylcarodiimide.
  • Other exemplary agents include N,N'- carbonyldiimidazole, POCl 3 , TiCl 4 , sulfuryl chloride fluoride, chlorosulfonyl isocyan
  • the amide linkage may be formed by selecting a carboxylic acid succinimide ester as the reactive group on the platinum/linker complex.
  • Samples containing a multiplicity of proteins can be obtained from any source. However, typically the proteins will be procured from tissue or cells or from body fluid such as plasma, serum, CSF (cerebrospinal fluid) and urine. Tissue is homogenized and/or into smaller groups of cells in order to facilitate lysis.
  • tissue or cells or from body fluid such as plasma, serum, CSF (cerebrospinal fluid) and urine.
  • body fluid such as plasma, serum, CSF (cerebrospinal fluid) and urine.
  • Tissue is homogenized and/or into smaller groups of cells in order to facilitate lysis.
  • Cells are lysed via any of a number of protocols known in the art including physical disruption of cells, lysis in hypotonic solution, or lysis via ionic or non-ionic detergents. Following lysis, cell debris is removed via centrifugation and the supernatant containing the protein sample is collected. The concentration of protein is determined and the proteins are concentrated or diluted to a concentration in the range of about 0.1 -20.0 mg/ml.
  • the invention provides for selection of peptides or proteins comprising methionine alone; methionine and cysteine; methionine and histidine; and methionine, cysteine, and histidine.
  • peptides comprising cysteine residues not bind to the platinum.
  • the protein sample is treated with methyl methanethiosulfonate (MSSM) in order to form a disulfide bond which will prevent interaction of the sulfur groups with the platinum.
  • MSSM methyl methanethiosulfonate
  • the reaction mixture is loaded on a SephadexTM. (PD-10 G25 column with 5 mM MES and 2 mM CaCl 2 , pH 6.5).
  • the protein fraction is then dialyzed against 1 mM CaCl 2 and the dialysate is lyophilized or suitable buffer.
  • cysteine residues of peptides protected in order to prevent the formation of disulfide bonds
  • the protein sample is treated with iodoacetic acid or vinyl pyridine which will bond to the sulfur of the cysteine thus ensuring its availability to interact with the platinum.
  • the protein sample is prepared in 100 mM Tris pH 8.5. 10 ⁇ L IM dithiothreitol is added and the reduction reaction proceeds for 2hrs at ambient temperature. 20 ⁇ L IM iodoacetic acid is added to the mixture and incubated for 30 min at ambient temperature in the dark. 40 ⁇ L IM dithiothreitol is then added to quench the iodoacetic acid. The protein is then purified using dialysis, spin columns, or reverse phase chromatography or used "as is" for the nest step in the experiment. Alternatively, the Cys sulfhydryl group is alkylated by reaction with 4-vinyl pyridine using the method originally devised by Friedman and coworkers (1970 J. Biol. Chem. 245, p. 3868-3871).
  • Example 5 Digestion of Samples to Generate Peptides
  • sample proteins are cleaved into smaller fragments (e.g., peptides), before, during, or after contacting the proteins with the Pt-substrate composition.
  • proteins are contacted with one or more cleaving agents such as trypsin. Trypsin digestion is performed in buffer comprising 10OmM Tris-HCl (pH 8.5). The digestion is typically performed overnight at 37°C in the dark.
  • each protein sample comprises a different label or one sample is labeled while the other is unlabeled.
  • Relative quantitation between two proteomic samples is performed by derivatizing a specific side chain of a residue in the peptides or derivatizing the C- or N- terminal of peptides after an enzymatic digest.
  • derealizations include acylation of the N-terminus (Ji, J., Chakraborty, A., Geng, M. et al.
  • the labeled peptides from Example 6 are incubated in a spin tube with a 0.22 ⁇ m filter with the conjugated Pt-substrate of Example 1 for 2 hours at 6O 0 C. If peptides that contain histidine are to be bound to the Pt-substrate, the incubation is carried out at about pH 8.0. If peptides that contain histidine are not to be bound to the Pt-substrate, the incubation is carried out at about pH 3.0. The peptides not bound to the Pt-substrate are collected through the filter by centrifugation. The peptides of the flow-through can be saved and analyzed separately from the peptides bound to the platinum.
  • the Pt-substrate is washed and again the flow-through can be collected.
  • the bound peptides are eluted using any of a variety of elution reagents which include but are not limited to guanidine thiocyanate (GuSCN), Na 2 S 2 O 5 , Na 2 S 2 O 3 and beta-mercaptoethanol.
  • the particles are treated with an equal volume of 4M GuSCN, resulting in a final volume of 2M GuSCN.
  • This solution is incubated at 6O 0 C for 1 hour.
  • the Pt-substrate is once again separated by centrifugation and the flow-through collected.
  • This flow-through solution containing the targeted peptides can then be desalted by dialysis or reverse phase (RP).
  • RP reverse phase

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Abstract

L'invention concerne un procédé de séparation de protéines et de peptides d'échantillon : contact entre échantillon/composé de coordination Pt en association stable avec un substrat, ce composé se liant aux groupes soufre et/ou azote et donc utile pour la purification de biomolécules qui renferment des acides aminés de cystéine, méthionine, histidine ou des acides aminés/résidus dérivatisés à groupes soufre ou azote se liant aux sites de coordination sur les composé de coordination Pt, y compris leurs formes modifiées, non modifiées, traitées et non traitées. On décrit aussi des procédés, systèmes et kits pour l'utilisation du composés de coordination Pt. Selon certaines variantes, les protéines purifiées et/ou les peptides purifiés peuvent être analysés, par exemple en spectrométrie de masse pour l'identification et la quantification des protéines et/ou peptides dans un échantillon, du type protéome ou peptidome.
PCT/US2005/030380 2004-08-25 2005-08-25 Compositions, procedes, systemes, et kits pour purification d'affinite WO2006026416A1 (fr)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FICARRO SB ET AL: "Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.", RAPID COMMUNICATION IN MASS SPECTROMETRY., vol. 19, 2005, pages 57 - 71, XP008059397 *
GARCIA AA ET AL: "Immobilization of Silver and Platinium Ions for Metal Affinity Chromatography.", REACTIVE POLYMERS., vol. 23, no. 2-3, October 1994 (1994-10-01), pages 249 - 259, XP002994397 *
LESNEY MARCK.: "Sticking with Affinity Chromatography.", MODERN DRUG DISCOVERY., December 2002 (2002-12-01), pages 27 - 29, XP002994398 *

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