WO2003102022A1

WO2003102022A1 - Use of reagent homologs for comparative proteomics

Info

Publication number: WO2003102022A1
Application number: PCT/US2003/017021
Authority: WO
Inventors: James P. Reilly; Richard L. Beardsley
Original assignee: Advanced Research And Technology Institute, Inc.
Priority date: 2002-05-31
Filing date: 2003-05-30
Publication date: 2003-12-11
Also published as: AU2003232447A1

Abstract

A method for differentially labeling peptide and/or protein samples with at least two members of a set of reagent series wherein each member of the set differs by at least one carbon atom to determine the relative quantities of one or more proteins in two or more proteins samples is provided. Mass spectral analysis of the labeled samples permits determination of the ratios of one or more proteins in the different samples without isolating the proteins of interest.

Description

USE OF REAGENT HOMOLOGS FOR COMPARATIVE PROTEOMICS

FIELD OF THE INVENTION

This invention relates generally to labeling methods for comparative proteomics and more specifically, to differential non-isotopic labeling methods for determining the ratios of peptides and proteins in different samples by mass spectral analysis.

BACKGROUND OF THE INVENTION

The common methods employed for comparative proteomics involve reagents in which a mass differential is introduced by incorporation of costly, low- abundance heavy isotopes into one of the reagents. A common isotope approach, isotope coded affinity tagging (ICAT), utilizes affinity tags and capture molecules, such as the well known biotin-avidin interaction, to affinity-capture peptides that contain cysteine. One of the limitations of this approach is that the biotin-avidin interaction is very strong, and as a consequence, some of the sample may not be recovered from avidin. Therefore, since the most popular approach is to use non- cleavable biotin affinity tags, some proteins are missed. Additionally, only peptides containing cysteine are captured and analyzed.

Another isotope approach is global internal standard technology (GIST) in which the proteins in the sample are subjected to proteolytic digestion, and then the amino- or carboxy-termini are labeled with a heavy isotope-containing moiety. However, the acetyl tags commonly used in GIST tend to reduce MALDI ion yields. Chakrabarty, A. et al, J Chromatogr. A 949:173 (2002) Furthermore, the introduction of a heavy isotope yields a mass spectral shift difference of only about 3 Daltons. This small difference limits the analysis to smaller peptides where such a difference can be readily observed; and it did not allow analysis of intact proteins. Thus it would be desirable to have methods and reagents to label proteins and peptides for comparative proteomic analysis that are not dependent on heavy isotopes. It would also be desirable if such reagents increased ion yield and had a large mass spectral shift difference. SUMMARY OF THE INVENTION

The present invention provides a method for quantification of proteins or peptides in complex mixtures. The method includes differentially labeling protein/peptide mixtures with at least two members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differs from other members of the set by at least one carbon atom. The method is particularly applicable in comparative proteomics, where the difference in concentration of a peptide or protein in at least two different samples is detected. The method of the present invention includes labeling the proteins/peptides in a first sample by covalently bonding them to a first chemical entity through predetermined functional groups, and covalently labeling the proteins/peptides in a second sample with a second chemical entity wherein the first and second chemical entities are members of a homologous series. The first and second samples may then be combined in a predetermined mass ratio and subjected to mass spectral analysis to determine the ratios of the common peptides/proteins in the first sample and the second sample. The nature of the reagents to be used in the method of the present invention depend on the specific protein/peptide functional groups to be labeled. Therefore, if cysteine residues in the protein/peptide components are to be labeled, the reagent will be specific for covalently linking the homologous chemical entities tlirough a thiol group. Alternatively, if the amino termini and/or lysine residues are to be modified, homologous reagents which specifically react with amino groups are used. At least two different protein/peptide samples are each reacted with a different labelling reagent for covalently linking the respective homologous chemical entities to the proteins and/or peptides in the samples. The homologous reagents are typically selected to, differ by at least one carbon atom, typically as part of a methylene group. Thus, for example, a first sample can be reacted with a reagent to covalently link an acetamidino to the amino groups of the proteins/peptides in the sample; a second sample is reacted to covalently link propionamidino groups to the amino groups on the protein/peptide components. The samples may then be combined in a predetermined ratio and subjected to mass spectral analysis to determine the ratios of the peptides/proteins common to the first sample and to the second sample. Additional objects, advantages, and features of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and by referencing the following drawings in which:

Figure 1 is a schematic illustrating the acetamidination and propionamidination of peptide amino groups; Figure 2 is a graph showing the experimental signal intensity ratio of acetamidinated to propionamidinated hemoglobin tryptic peptides;

Figure 3 is a graph showing the experimental intensity ratio vs. actual concentration ratio of acetamidinated to propionamidinated cytochrome C tryptic peptides; Figure 4 is a MALDI mass spectra comparing acetamidinated to propionamidinated undigested yeast ribosomal proteins; and

Figure 5 is a bar graph showing the relative mass spectral intensities between the unamidinated and acetamidinated peptides from a cytochrome C digest.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for protein identification and quantification of proteins or peptides in complex mixtures. The method includes labeling protein/peptide mixtures with at least two members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differ from other members of the set by at least one carbon atom. The method is particularly applicable in comparative proteomics, where the difference in concentration of peptides/proteins common to at least two different samples is detected. The method of the present invention includes labeling the proteins/peptides in a first sample by covalently bonding them to a first chemical entity through predetermined functional groups and covalently labeling the proteins/peptides a second sample with a second chemical entity wherein the first and second chemical entities are members of a homologous series. The first and second samples may then be combined in a predetermined mass ratio and subjected to mass spectral analysis to determine the ratios of the common peptides/proteins in the first sample and the second sample.

It should be understood that the term "protein," as used herein, refers to a polymer of amino acids and does not connote a specific length of a polymer of amino acids. Thus, for example, the terms oligopeptide, polypeptide, and enzyme are included within the definition of "protein," whether produced using recombinant techniques, chemical or enzymatic synthesis, or naturally occurring. This term also includes polypeptides that have been modified or derivatized, such as by glycosylation, acetylation, phosphorylation, and the like. The term "peptide" as used herein, usually refers to shorter polymers of amino acids. Peptides may be naturally formed within a sample of the present invention or may be formed by fragmentation of larger proteins by methods known to the skilled artisan. It will be appreciated that the method of the present invention can be applied to peptides, proteins or mixtures thereof. Therefore, reference to protein or peptide mixtures is not intended to be limiting.

It has been discovered that by differentially labeling proteins/peptides in at least two different samples with at least two members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differs from other members of the set by at least one carbon atom, the ratios of the common proteins/peptides in the first sample to the second sample can be determined by mass spectral analysis. The peptides/proteins of a first sample are labeled by covalently bonding them to a first chemical entity through predetermined functional groups, and covalently labeling the proteins/peptides in a second sample with a second chemical entity wherein the first and second chemical entities are members of a homologous series. It was previously not known that peptides labeled with the chemical entities of the present invention would have similar spectral ionization yields allowing for such determinations. However, not only are the ionization yields within an acceptable range when the chemical entities differ by at least one carbon, but the mass difference, or shift, is much greater than the shift obtained with heavy isotope labeling. For example, the addition of one methylene group leads to a mass difference of 14 for each labelled site on a protein or peptide. In contrast, the substitution of three deuterium atoms for hydrogen in a methyl group produces a mass difference of only 3 and O¹⁸ for O¹⁶ produces a shift of only 2 per labeled site. The advantage of the larger mass difference in the present invention is that larger peptides and proteins can be analyzed as compared to the smaller masses that can be analyzed with heavy isotope labeling. The ability to analyze larger peptides and proteins will decrease the complexity of the samples being analyzed. Therefore a sample having larger peptides can be analyzed without further proteolysis. The advantage is that longer peptides produce more distinctive spectra. Alternatively, fragmentation of a sample can be done such that larger peptides are produced. For example, instead of treating a protein sample with a protein such as trypsin that cleaves at both lysine and arginine, the sample may be treated with the arg-C proteinase, which cleaves only at arginine producing fewer peptides in the sample and decreasing the complexity of the analysis. The method of the present invention includes reagents for differentially labeling at least two different samples. The reagents are at least two members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differs from other members of the set by at least one carbon atom. Preferably, the reagents will differ from at least one to at least ten carbon atoms and more preferably, from about one to about five carbon atoms. Typically the carbon atoms are in the form of methylene groups. A non-limiting example of a homologous reagent series is a series of reagents wherein each reagent has a chemical entity defined by a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl group. For example, a homologous reagent series for the amidation of peptide/protein amino groups may include, but not be limited to S-methyl thioamidinates that have a methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl or isopropyl group attached to a central carbon. Another example of a series of compounds would be acetylating reagents such as, but not limited to acetic anl ydride, propionic anhydride, butanoic anhydride and pentoic anhydride.

If only two reagents are used, the reagents are chosen from a homologous series to give the desired mass shift. Examples of pairs of reagents are ones that contain chemical entities defined by, but not limited to, ethyl and methyl, propyl and methyl, propyl and ethyl, butyl and methyl, butyl and ethyl, butyl and propyl, isopropyl and methyl or isopropyl and ethyl. The reagents may also contain heteroatoms such as, but not limited to, nitrogen, oxygen, sulfur, iodine, phosphorus, silicon, chlorine or mixtures thereof. The organic reagents of the present invention are reacted with a protein/peptide sample to covalently link chemical entities to peptide/protein functional groups wherein the chemical entities are part of a homologous series differing by at least one carbon atom, typically as part of a methylene. The nature of the reagents to be used in the method of the present invention depend on the specific protein/peptide functional groups to be labeled.

Non-limiting examples of protein and/or peptide functional groups or amino acid side chains that can be covalently modified are thiol (cysteine), carboxyl (C-terminus, aspartic acid, glutamic acid), amino (N-terminus, lysine, arginine), hydroxyl (serine, threonine, tyrosine), and/or histidine. The homologous reagents include, but are not limited to, alkylation, acetylation or amidination reagents. By way of non-limiting example, the reagents may be acetylating reagents that attach acetyl and propionyl moieties to the peptides. Alternatively, the amino terminus may be amidinated, with one sample being acetoamidinated and the other propionamidinated (Fig. 1). Alternatively, the chemical entities covalently linked to the proteins/peptides can be charge tags, which have the advantage of increasing the ion yield of the sample during mass spectral analysis. The covalent modification of proteins and/or peptides is well known in the art and reagents available to use with the method of the present invention will be known to the skilled artisan.

The method of the invention is designed for use in complex samples containing a number of different proteins. The sample may contain a single protein or it may include all the proteins found in a total cell lysate. A sample can therefore include total cellular protein or some fraction thereof. For example, a sample can be obtained from a particular cellular compartment or organelle, using methods such as centrifugal fractionation. The sample can be derived from any type of cell, organism, tissue, organ, or bodily fluid, without limitation. The method of the present invention can be applied to determine the relative quantities of one or more proteins in two or more protein samples. For example, cellular protein expression in two different samples may be analyzed. One sample may be treated to induce expression, or different cellular growth conditions may be used to manipulate protein expression. The methods of the present invention allow for analysis of protein expression without having to actually isolate the protein of interest.

The present invention further provides a post-synthetic differential labeling method useful for detecting differences in the concentration of metabolites between two samples. Application of the differential labeling method of the invention is not limited to proteins, but can be used to identify or quantitate other metabolites as well such as carbohydrates, lipids, nucleic acids, polysaccharides, glycopeptides, glycoproteins, and the like. The samples are preferably complex mixtures, and the metabolite is preferably a protein or a peptide. Advantageously, the method can be utilized with complex mixtures from various biological environments. For example, the method of the invention can be used to detect a protein or family of proteins that are in regulatory flux in response to the application of a stimulus. Or, samples can be obtained from different organisms, cells, organs, tissues or bodily fluids, in which case the method permits determination of the differences in concentration of at least one protein in the organisms, cells, organs, tissues or bodily fluids from which the samples were obtained.

To aid in the mass spectral analysis, proteins and/or larger peptides in the samples to be compared may be fragmented, either before or after the differential labeling of the present invention. In one aspect of the invention, the protein/peptide samples are differentially labeled and then subsequently fragmented. The differentially labeled samples may then be combined before fragmentation or they may be fragmented separately. Preferably they are combined before fragmentation and subsequent analysis. Fragmentation in solution can be achieved using any desired method, such as by using chemical, enzymatic, or physical means. It should be understood that as used herein, the terms "cleavage", "proteolytic cleavage",

"proteolysis", "fragmentation" and the like are used interchangeably and refer to scission of a chemical bond within peptides or-proteins in solution to produce peptide or protein "fragments" or "cleavage fragments." No particular method of bond scission is intended or implied by the use of these terms. Fragmentation and the formation of peptide cleavage fragments in solution are to be differentiated from similar processes in the gas phase within a mass spectrometer. These terms are context specific and relate to whether bond scission is occurring in solution or the gas phase in a mass spectrometer.

Fragmentation of proteins can be achieved by chemical, enzymatic or physical means, including, for example, sonication or shearing. Preferably, a protease enzyme is used, such as, but not limited to, trypsin, chymotrypsin, papain, arg-C, glu- C, endo lys-C, proteinase K, carboxypeptidase, calpain, subtilisin, staph V8 protease and pepsin; more preferably, a trypsin digest is performed. Alternatively, chemical agents such as cyanogen bromide can be used to effect fragmentation. The proteolytic agent can be immobilized in or on a support, or can be free in solution. Proteolytic enzymes and agents are well known in the art and the skilled artisan may chose the enzyme and agent based on the sample and the number of peptides desired.

Alternatively, the samples may be fragmented before differential labeling. The samples are subjected to fragmentation and then differentially labeled individually. At least a portion of each sample is then typically mixed together to yield a combined sample, which is subjected to mass spectrometric analysis. Control and experimental samples are mixed after labeling, fractions containing the desired components are selected from the mixture, and a concentration ratio is determined to identify analytes that have changed in concentration between the two samples.

In a further alternate aspect of the present invention, the samples are differentially labeled but not subjected to fragmentation before analysis. The method of the present invention provides differential labeling that results in a larger mass difference between the samples than previously reported in the prior art. This larger mass difference allows for the analysis of large peptides and small proteins that would not be possible with the isotope labeling methods of the prior art. Therefore although the specification describes the differential labeling and analysis of peptide samples, the method of the present invention may also be used for protein samples as well. By way of non-limiting example, differentially amidinated proteins typically differ by hundreds of Daltons depending on the number of lysines in their sequence. Thus amidination facilitates the challenging task of resolving labeled whole protein signals by MALDI-TOF MS. Comparing MALDI-TOF protein signals via an isotope-coded labeling method would be of limited utility since this imparts much smaller mass-shifts. Figure 5 shows mass spectra of six differentially amidinated yeast ribosomal proteins. The first set of labels denote acetamidinated proteins while the corresponding propionamidinated proteins are the second set of labels. The fact that differentially amidinated proteins were present in equal quantity is reflected by the equal intensity of their signals. The method of the present invention may also be used to quantify undigested proteins. In a further alternate aspect of the present invention, the samples are differentially labeled but not subjected to fragmentation before analysis. The method of the present invention provides differential labeling that results in a larger mass difference between the samples than previously reported in the prior art. This larger mass difference combined with labeling of multiple functional groups, allows for the analysis of large peptides and small proteins that would not be possible with the heavy isotope labeling methods of the prior art. Therefore although the specification describes the differential labeling and analysis of peptide samples, the method of the present invention may also be used for protein samples as well. Mass spectrometric analysis can be used to determine peak intensities and quantitate ratios in the combined sample, and determine whether there has been a change in the concentration of a protein between two samples. Preferably, changes in peptide concentration between the control and experimental samples are determined by their ratios using MALDI-mass spectrometry because MALDI-MS allows the analysis of more complex peptide mixtures, but ESI-MS may also be used when the peptide mixture is not as complex. Alternatively, LC-MS may also be used, but the increased mass difference will result in a slightly different elution profile for differentially labeled proteins/peptides. This difference would need to be taken into account when doing the analysis. In a complex combined mixture, there may be hundreds to thousands of peptides, and many of them will not change in concentration between the control and experimental samples. These peptides whose levels are unchanged are used to establish the normalized ratio for peptides that were neither up nor down regulated. All peptides in which the ratio exceeds this value are up regulated. In contrast, those in which the ratio decreases are down regulated. A difference in relative ratio of a peptide pair, compared to peptide pairs derived from proteins that did not change in concentration, thus signals a protein whose expression level did change between the control and experimental samples. The peptide used to determine the concentration change can be used according to the method of the invention to identify the protein from which it was derived.

The method of the present invention may be used in the global internal standard technology (GIST) method, which involves labeling all peptide fragments. The method is described in U.S. Patent Application Publication No. 2002/0037532, hereby expressly incorporated by reference. Components from control samples function as standards against which the concentration of components in experimental samples are compared. When the differential labeling process is directed at primary amine, carboxyl groups, or both in peptides produced during proteolysis of the proteome, an internal standard is created for essentially every peptide in the mixture. Because virtually all peptide fragments in the sample are labeled, the method is referred to as a global labeling strategy. This global internal standard technology (GIST) for labeling may be used to quantify the relative concentration of all components in complex mixtures. It is also contemplated that the methods of the present invention can be used with affinity capture methods for isolating specific peptides before analysis. In affinity capture methods the differentially labeled peptides are sorted to produce a specific sub-population of peptides. For example, these may be cysteine or histidine containing peptides, glycosylated peptides, lipid modified peptides or peptides with which an exogenous affinity tag such as, but not limited to, biotin has been attached. Examples of affinity capture methods are described in U.S. Patent Application Publication No. 2002/0037532 and PCT Application WO 00/11208 and are well known to the skilled artisan.

An affinity tag used for selection can be endogenous to the protein, or it can be added by chemical or enzymatic processes. The term "affinity tag," as used herein, refers to a chemical moiety that functions as, or contains, an affinity ligand that is capable of binding (preferably noncovalently, but covalent linkages are contemplated also) to a second, "capture" chemical moiety, such that a protein or peptide that naturally contains or is derivatized to include the affinity tag can be selected (or "captured") from a pool of proteins or peptides by contacting the pool with the capture moiety. The capture moiety is preferably bound to a support surface, preferably a porous support surface, as a stationary phase. Examples of suitable supports include porous silica, porous titania, porous zirconia, porous organic polymers, porous polysaccharides, or any of these supports in non-porous form.

Preferably the interactions between the affinity tag and the capture moiety are specific and reversible (e.g., noncovalent binding or hydrolyzable covalent linkage), but they can, if desired, initially be, or subsequently be made, irreversible (e.g., a nonhydrolyzable covalent linkage between the affinity tag and the capture moiety). It is important to understand that the invention is not limited to the use of any particular affinity ligand.

Examples of endogenous affinity ligands include naturally occurring amino acids such as cysteine (selected with, for example, an acylating reagent) and histidine, as well as carbohydrate and phosphate moieties. A portion of the protein or peptide amino acid sequence that defines an antigen can also serve as an endogenous affinity ligand, which is particularly useful if the endogenous amino acid sequence is common to more than one protein in the original mixture. In that case, a polyclonal or monoclonal antibody that selects for families of polypeptides that contain the endogenous antigenic sequence can be used as the capture moiety. An antigen is a substance that reacts with products of an immune response stimulated by a specific immunogen, including antibodies and/or T lymphocytes. As is known in the art, an antibody molecule or a T lymphocyte may bind to various substances, for example, sugars, lipids, intermediary metabolites, autocoids, hormones, complex carbohydrates, phospholipids, nucleic acids, and proteins. As used herein, the term "antigen" means any substance present in a peptide that may be captured by binding to an antibody, a T lymphocyte, the binding portion of an antibody or the binding portion of T lymphocyte. A non-endogenous (i.e., exogenous) affinity tag can be added to a protein or peptide by, for example, first covalently linking the affinity ligand to a derivatizing agent to form an affinity tag, then using the affinity tag to derivatize at least one functional group on the protein or peptide. Alternatively, the protein or peptide can be first derivatized with the derivatizing agent, then the affinity ligand can be covalently linked to the derivatized protein or peptide at a site on the derivatizing agent. An example of an affinity ligand that can be covalently linked to a protein or peptide by way chemical or enzymatic derivatization is a peptide, preferably a peptide antigen or polyhistidine. A peptide antigen can itself be derivatized with, for example, a 2,4-dinitrophenyl or fluorescein moiety, which renders the peptide more antigenic. A peptide antigen can be conveniently captured by an immunosorbant that contains a bound monoclonal or polyclonal antibody specific for the peptide antigen. A polyhistidine tag, on the other hand, is typically captured by an IMAC column containing a metal chelating agent loaded with nickel or copper. Biotin, preferably ethylenediamine terminated biotin, which can be captured by the natural receptor avidin, represents another affinity ligand. Other natural receptors can also be used as capture moieties in embodiments wherein their ligands serve as affinity ligands. Other affinity ligands include dinitrophenol (which is typically captured using an antibody or a molecularly imprinted polymer), short oligonucleotides, and polypeptide nucleic acids (PNA) (which are typically captured by nucleic acid hybridization). Molecularly imprinted polymers can also be used to capture. The affinity ligand is typically linked to a chemical moiety that is capable of derivatizing a selected functional group on a peptide or protein, to form an affinity tag. An affinity ligand can, for example, be covalently linked to maleimide (a protein or peptide derivatizing agent) to yield an affinity tag, which is then used to derivatize the free sulfhydryl groups in cysteine.

Alternatively, the desired proteins/peptides can be captured by covalently linking the proteins/peptides to a solid surface. For example, cysteine containing peptides can be linked to a said surface by reacting with another thiol to form a disulfide bond. The isolated cysteine containing peptides can then be released by reduction of the disulfide bond. In one aspect of the method of the present invention, affinity tags can be attached to the peptides in the sample in addition to differentially labeling the same peptide sample. As an example, an investigator can differentially label all peptides (by labeling the free amino group or the free carboxyl group that characterizes nearly every peptide), then independently affinity label the differentially labeled peptides at other sites, either in parallel or in series. Perhaps tyrosines in an aliquot of a differentially labeled peptide pool could be affinity labeled (either before or after protein fragmentation), after which peptides containing tyrosines could be selected. Then, another aliquot of the same peptide pool could be selected for histidine-containing peptides. Alternatively, the selected tyrosine- containing peptide subpopulation could be further selected for histidine, depending on the interests of the investigator. In a preferred aspect of the invention, the affinity tags may be cleavable. For example, the affinity tag may be a cleavable biotin affinity tag. The affinity tagged peptides are then isolated by binding to immobilized avidin and subsequently released from the immobilized avidin by cleaving the biotin tag from the peptide. The result is a higher recovery of tagged peptides than if the peptide were released from the immobilized avidin by competition with free biotin. The ratios for any of these selected peptides could be determined using mass spectrometry.

The present invention also provides a kit for differential labeling of protein/peptide samples according to the method of the present invention. The kit comprises at least two members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differ from the other members of the set by at least one carbon atom. The reagents in the kits covalently at least a first and a second chemical entity to at least a first and a second protein/peptide sample, wherein the chemical entities are members of homologous series wherein each member of the series differs from other members of the series by at least one carbon atom, typically as part of a methylene. The kit may also comprise any other reagents necessary to differentially label the peptide samples. The kit may fiirther contain at least one protease and any required buffers and reagents for carrying out fragmentation of the protein samples. Finally, the kit may also contain an affinity tag and capture moiety for isolating subpopulations of the peptides in the samples.

It is also contemplated that the method of the present invention be used to identify and quantify proteins/peptides in more than two samples. Unlike isotope labeling, wherein there are only two labels, the method of the present invention provides more than two labels. Therefore, a first protein/peptide sample may be labeled with one reagent of a set of reagents in a homologous reagent series, a second sample labeled with a second reagent from the same series having a difference of one carbon atom from the first reagent, and a third sample can be labeled with a third reagent from the series, differing from the first reagent by two carbon atoms and differentiating from the second reagent by one carbon atom. It will be appreciated that the number of samples that can be labeled and analyzed are only limited by the number of members in the set of reagents in a homologous series. Therefore, it will be appreciated by one skilled in the art that any description of the invention can apply to multiple samples greater than two.

The foregoing and other aspects of the invention may be better understood in connection with the following examples, which are presented for purposes of illustration and not by way of limitation.

EXAMPLE Preparation of Amidination Reagents S-methyl thioacetimidate was prepared from thioacetamide and iodomethane in anhydrous diethyl ether. 1 lg of thioacetamide were dissolved in 1L diethyl ether at room temperature. To this solution 8.8 ml of iodomethane was added. The mixture was briefly stirred and allowed to stand for 14 h. After the reaction a light yellow precipitate was collected by vacuum filtration. The powder was not further purified.

S-methyl thiopropionimidate was prepared from thiopropionamide and iodomethane in acetone. 1.8 g of thiopropionamide were dissolved in 100 ml of 99.5% pure acetone. This solution was placed in a 60°C water bath and 3.8 ml of iodomethane were added. The reaction was performed for 60 min. at the bath temperature. The reaction product was collected after drying the solution in a room temperature vacuum chamber. The dark yellow crystals were not further purified. Amidination of Peptides

The amidination of the lysines and N-termini of peptides was accomplished using very similar procedures for both reagents. For both acetamidination and propionamidination reactions the reagent was dissolved to a concentration of approximately 100 mM in a 250 mM trizma buffered solvent. This stock solution was mixed in equal volume with the peptide solution. The reaction pH was about 8.0 and the mixture was incubated at ambient temperature for lh. The reaction was terminated by addition of 10% v/v trifluoroacetic acid in sufficient quantity to result in a pH of about 1.0. See Thumm, M. et al., Biochim. Biophys. Ada 923:263-267 (1987) for methods for preparing reagents and modifying proteins and peptides.

Mass Spectometry of Amidinated Peptides

Amidinated peptides were mixed with an excess of MALDI matrix, typically α-cyano-4-hydroxycinnamic or 2,5-dihydroxybenzoic acid. The amidinated mixture does not need to be purified prior to analysis. A MALDI spot was prepared by depositing 0.7 μl of the matrix/analyte mixture onto a 2 mm diameter stainless steel target and allowing it to air dry. MALDI-TOF mass spectra were acquired in positive-ion mode with a Bruker Reflex III mass spectrometer.

RESULTS

Comparison of Differentially Amidinated Tryptic Peptides

MALDI ion-yields of lysine-containing peptides are dramatically increased by amidination. The plot in Figure 5 convincingly demonstrates this signal enhancement effect. The average relative intensities displayed in this plot were derived from 5 MALDI-TOF MS spectra of samples containing equal quantities of an underivatized and an acetamidinated cytochrome c tryptic digest. Ionization enhancement appears to be greater for small peptides and similar enhancements were observed with propionamidination. Another unique characteristic of amidination for MALDI analyses is that sample purification is not necessary following the reaction. We have routinely analyzed amidinated tryptic digests using both 2,5-DHB and CHCA matrices without removal of amidination reagents. The lack of need for a purification step is an uncommon feature of peptide modifications and is advantageous because minimal sample handling leads to improved peptide recovery and a shorter procedure.

The tryptic digest of human hemoglobin was separated into 2 equal aliquots. One aliquot was acetamidinated while the other was propionamidinated. Following the reactions, the mixtures were recombined. Since the quantities of hemoglobin that were amidinated and combined were equal, the MALDI signal intensities of the differentially amidinated peptides should also have been equal. Prior to this analysis it was unknown whether separate samples could be amidinated in this way and yield mass spectrometric signals that reflected their relative abundances. If the reactivity of propionamidination and acetamidination reagents were significantly different, then the MALDI-MS signal intensities of equal quantities of peptides could be unequal due to the varied levels of amidination. Another possible source of error could come from unequal ionization between propamidinated and acetamidinated peptides. If one derivative possessed greater gas-phase basicity than the other, then its MALDI ion yield would be greater. The data in Figure 2 demonstrate that these problems are not observed. This figure is a plot of the ratio of relative intensities of acetamidinated to propionamidinated tryptic peptides derived from hemoglobin. Fourteen differentially amidinated peptides were analyzed and the masses of the two types of derivatives are displayed on the x-axis. The mass difference between the two types of derivatives is 14 Daltons per amidination since the two reagents differ by only one methylene group. The 1:1 ratio acetamidinated to propionamidinated samples resulted in an average intensity ratio of 0.995±0.153. This average was derived from the intensity ratios of 50-100 laser shot mass spectra, taken form 50 separate MALDI spots. Since 14 derivatized peptide pairs were analyzed, this average is from 700 individual ratios. It is worth mentioning that MALDI-MS signals are known to vary significantly form one laser shot to another, as well as from one MALDI spot to another. Since 50 MALDI spots and thousands of laser shots were used in this experiment, this source of error is accounted for. Also worth mentioning is the fact that typical applications requiring a comparative approach such as differential amidination are intended to measure relatively large differences in protein abundances (i.e. 2:1 or greater). With a relative error of only 15%, differential amidination is well suited for these applications. In addition to comparing equal quantities of amidinated samples, similar experiments have been performed using a 1 :2 ratio of acetamidinated to propamidinated hemoglobin tryptic digests. The average intensity ratio attained form this experiment was 0.57±0.12. Once again, this results reflects the relative abundances of the differentially amidinated samples.

It is essential that a range of actual relative protein concentrations be accurately reflected in MALDI spectra. To test this capability, spectra were acquired of samples mixed in varying concentration ratios. Five different samples were prepared by mixing acetamidinated and propionamidinated cytochrome c tryptic digests. Data are displayed in Figure 3. The theoretical intensity ratio curve is displayed as a solid line, while the measured ratios are displayed as a dashed line.

Those skilled in the art will now appreciate from the foregoing description that the broad teachings of the present invention can be implemented in a variety of forms. Therefore, while this invention has been described in connection with particular examples thereof, the true scope of the invention should not be so limited since other modifications will become apparent to the skilled practitioner upon a study of the drawings, specification and claims.

All patents and other publications cited herein are expressly incorporated by reference.

Claims

1. A method for comparative analysis of two or more samples to determine ratios of the amounts of common components of said samples, said method comprising the steps of reacting components of a first sample with a first reagent to covalently label components of said first sample with said first reagent, reacting components of a second sample with a second reagent to covalently label components of said second sample with said second reagent, said first and second reagents being members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differs from other members of the set by at least one carbon atom, combining portions of the first and second reagent-reacted samples in a predetermined ratio and subjecting the resulting mixture to mass spectral analysis to determine the ratios of components common to the first and second samples.

2. The method of claim 1 further comprising the steps of reacting the components of a third sample with a third reagent member of the set of homologous reagents to covalently label the components of the third sample with said third reagent, combining portions of the three reagent-reacted samples in a predetermined ratio and subjecting the resulting mixture to mass spectra analysis.

3. The method of claim 1 wherein the reagents are selected to be reactive with protein and peptide components of the respective samples.

4. The method of claim 1 wherein the first and second reagents are reactive with amine groups on components of the respective samples.

5. The method of claim 1 further comprising the step of subjecting the respective samples to conditions to fragment protein and peptide components in each sample before reaction with the reagent.

6. The method of claim 1 wherein the reagent-reacted samples are subjected to conditions selected to promote fragmentation of peptide and protein components prior to combining portions of the respective reagent-reacted samples for mass spectral analysis.

7. The method of claim 1 wherein the first and second samples are biological samples obtained from a biological environment before application of a stimulus and after application of a stimulus, respectively.

8. The method of claim 1 wherein the first and second samples are obtained from different organisms, cells, organs, tissues or bodily fluids.

9. A kit for facilitating comparative mass spectral analysis of biological samples, said kit comprising at least two members of a set of reagents in a homologous reagent series wherein each member of the set of reagents differ from other members of the set by at least one carbon atom, said reagents capable of reacting with and labeling one or more components of the biological sample.

10. The kit of claim 9 further comprising at least one protease.

11. The kit of claim 9 further comprising an affinity tag and capture moiety for isolating subpopulations of peptides or proteins in a biological sample.