WO2003031982A1

WO2003031982A1 - Method for analysing protein/peptide expression

Info

Publication number: WO2003031982A1
Application number: PCT/EP2002/011099
Authority: WO
Inventors: Peter James
Original assignee: Peter James
Priority date: 2001-10-05
Filing date: 2002-10-03
Publication date: 2003-04-17
Also published as: GB0123858D0

Abstract

The present invention relates to a method of analysing protein and/or peptide expression, which method comprises the steps of (a) providing a sample comprised of disintegrated cells; (b) treating the sample with a protecting agent so as to block epsilon amino groups present on lysine residues of proteins present therein; (c) cleaving the proteins into a peptide mixture; (e) treating at least partially separated peptides with a labelled agent which binds directly to N-terminal amino acids of peptides; and (d) at least partially separating the labelled peptides.

Description

METHOD FOR ANALYSING PROTEIN/PEPTIDE EXPRESSION

Technical Field

The present invention relates to methods for quantitating, identifying and characterising proteins or peptides. The invention is especially advantageous in the context of membrane or membrane-associated protein analysis.

Background

In the spring of year 2000, the results of the Human Genome Project (HGP) were first presented and the sequence of the DNA in the human genome became publicly accessible, including more than 30,000 human genes.

However, even though protein structure and function can be predicted from the DNA sequences, genomics alone does not provide enough information for an understanding of how protein function is regulated and the mechanisms that govern e.g. whether and when gene products are translated, the relative concentration of gene products, the extent of post- translational modifications of the gene products, and the effects of under- or over- expression. Thus, within the field of proteomics, one now seeks to define the function and expression profiles of all proteins encoded within a given genome. Accordingly, large numbers of proteins expressed are now separated, identified, and characterised in routine experiments. From this follows a growing need of high-throughput methods for separation, quantitation, and identification of hundreds of proteins from biological samples. Conventionally, separated proteins are identified by using peptide mass spectrometry coupled to genome database searching. Since the quality of the sample entered into a detection instrument is crucial for the result achieved, the effectiveness of proteome analysis will however always be dependent upon the quality of the electrophoretic separation technology utilised.

More specifically, it is estimated that 15-30% of proteins expressed by pro- and eukaryotes are either membrane spanning or covalently bound to membranes. A considerable number of other proteins are tightly associated with the membrane. The major difficulty encountered with the analysis of these proteins lies in their physicochemical properties, since on one hand they are extremely hydrophobic (to allow membrane penetration) and on the other, highly hydrophilic in the extra-membranar regions. These properties give rise to extreme difficulties in formulating a general separation scheme for this class of proteins. At present, the primary tool for protein separation and quantitation is two-dimensional gel electrophoresis. However, the denaturing conditions used therein result in the hydrophobic regions becoming very prone to self-aggregation. As is easily realised, this is an essential drawback and a major limiting factor in the conventional analysis of membrane proteins e.g. by 2D PAGE.

To overcome these difficulties, Schagger et al. (Schagger H, Cramer WA, von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem. 1994 Mar; 217(2):220-30) suggested in 1994 to use mild detergents that are non-denaturing to allow a partial separation by either non-denaturing PAGE or by column chromatography. However, the above discussed dual nature of the proteins often leads to multiple elution points during chromatography depending on which part of the protein surface has bound to the separation phase.

Further modifications of the two-dimensional separation methods resulted in the BAC/SDS diagonal gel method disclosed by Hartinger in 1996 (Hartinger J, Stenius K, Hogemann D, Jahn R. 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal Biochem. 1996 Aug 15; 240(1): 126-33), which currently is the only viable two-dimensional separation method available. However, this method has still a rather low resolution and has therefore not proven satisfactory in practice for an efficient protein analysis.

Simpson et al. (Simpson RJ, Connolly LM, Eddes JS, Pereira JJ, Moritz RL, Reid GE. Pro- teomic analysis of the human colon carcinoma cell line (LIM 1215): development of a membrane protein database. Electrophoresis. 2000 May; 21(9): 1707-32) have demonstrated that virtually all membrane proteins can be separated by one-dimensional SDS-PAGE and identi- fied by mass spectrometry. However, the disadvantage of this method is that the quantitative aspect is lost and comparative analyses of protein expression by gel densitometry cannot be carried out.

WO 02/08767 (Procter & Gamble) discloses methods and kits that enable polypeptide sequencing, wherein a novel group of acidic labelling agents is used. More specifically, the method suggested therein comprises to provide a sample comprising peptides, to protect the epsilon amino group of lysine on the lysine containing peptides, to label the peptides at the N-te-πtnini thereof with the above-mentioned novel acidic reagent and to analyse the thereby derivatised analytes using MS.

WO 01/74842 (Proteome Systems) discloses a method of quantitating, identifying and characterising proteins and peptide fragments wherein proteins are labelled to allow relative protein quantitation in 1 and 2D gel separations.

Further, WO 00/57183 (Biovation) discloses a method of analysing protein mixtures comprising the steps of digestion or cleavage of the protein mixture followed by fractionation of the resultant peptides. More specifically, the fractionation step uses a library of protein affinity reagents, such as libraries of recombinant antibody fragments. Since generation of antibodies to all peptides in a large peptide mixture is difficult, the disclosed method isolates N and/or C terminal peptides by preabsorption of the protein to a solid phase via its N and/or C te-rminus prior to cleavage for subsequent isolation after cleavage. The subsequent analysis is a mass analysis, especially mass spectrometry.

In addition, Mϋnchbach et al (Mϋnchbach M, Quadroni M, Miotto G, James P. Quantitation and Facilitated de Novo Sequencing of Proteins by Isotopic N-Te-rminal Labeling of Peptides with a Fragmentation-Directing Moiety. Anal Chem. 2000 Sep l;72(17):4047-57) describe a method for comparative quantitation and de novo sequencing of proteins, which method was specifically developed to enable correlation of data from DNA sequencing with changes in membrane protein expression and the state of posttranslational modification. More specifi- cally, the Mϋnchbach method starts with a sample obtained from gels obtained -from 2D electrophoresis of sulphate-starved E. coli bacteria. Thus, peptides obtained from E. coli digests and isolated by electrophoresis are treated with an agent to protect epsilon amino groups present on lysines and the treated peptides are then N-terminally modified with isotopic reagents.

However, there is a need within this field of an alternative and preferably a general method for protein analysis and quantification.

Summary of the present invention

One object of the present invention is to provide a method for protein analysis, which avoids the above-discussed problem of self-aggregation of hydrophobic regions of protein during protein separation in a gel. This can according to the present invention be achieved by a method wherein a sample comprising proteins is first digested into peptides, which peptides are subsequently separated. Thus, the method according to the invention does not include any separation of protein, as appears from the appended claims and the detailed description below. Accordingly, the present method will provide a sample wherein all possible peptides are easily accessible.

Another object of the present invention is to provide a method for protein analysis, which is useful for quantitative analysis. A further object of the invention is to provide a method for protein analysis wherein the resolution obtained in the detection step is improved as compared to previously known methods. Further objects and advantages of the invention will appear from the detailed description and the experimental part below.

Brief description of the drawings

Figure 1 shows an embodiment where the present invention is used for isotopic analysis of membrane protein expression. The spectra show realative intensity on the Y-axis and Mass/Charge m/z on the X-axis. Figure 2 outlines how an alternative method developed to allow the quantitation of multiple proteins in a single spot from a two-dimensional was recently described. The spectra are as explained above.

Detailed description of the invention

More specifically, a first aspect of the present invention is a method of analysing protein expression, which method comprises the steps of

(a) providing at least one sample comprised of one or more disintegrated cells;

(b) treating the sample(s) with at least one protecting agent to block epsilon amino groups present on lysine residues of protein(s) present therein;

(c) cleaving the so treated protein(s) into a peptide mixture;

(d) treating the peptide(s) with labelled reagent, which binds directly to N-terminal amino acids of peptides; and

(e) at least partially separating the peptides resulting from step (d).

The present invention can also be viewed as a novel method of labelling peptides, wherein the peptides have been obtained from disintegrated cells wherein proteins have been treated with protecting agent as described above and which proteins have subsequently been cleaved into peptides.

The crucial difference between the present invention and WO 02/0867, WO 01/74842 and Mϋnchbach et al is that the analysis takes place without the use of 1 or 2D gels. Furthermore in contrast to WO 00/57183 and WO 02/0867 is that the method allow direct a direct comparison of protein amounts in multiple samples to be carried out. Furthermore, the approach can be multiplexed, using a label with different numbers of isotopic substitutions. As an example, sample one may be labelled with an H6 nicotinic acid derivative, sample 2 with a D2H4 derivative, sample three with D4H2, sample four with D6 etc. Thus, contrary to the above-discussed WO 02/08767, the method according to the present invention uses a sample comprising disintegrated cells, wherein epsilon amino groups of proteins are protected in a first step. After completed labelling, such protected proteins are cleaved into peptides. As will be discussed in more detail below, the present method is also suitable for use with isotopic labelling reagents, which forms can be distinguished on the basis of mass. Hence, the present method is suitable for comparative studies of two samples, such as proteins expresses under different conditions, which is not possible using the acidic labelling reagents presented in WO 02/08767.

Further, as compared to the above-discussed WO 00/57183, there is no need for use of any affinity tags in the present method. In addition, the present method can handle a large mixture of peptides without any need of absorbing proteins to a solid phase as required by the WO 00/57183 method.

In one embodiment, the sample provided in step (a) has been obtained by mechanical or chemical cell disintegration and centrifugation. In another embodiment, the sample provided in step (a) comprises membrane or membrane-associated protein(s). This embodiment is especially advantageous, since such proteins have shown to be quite problematic to label in the prior art. As mentioned above, the dual function of both hydrophilicity and hydrophobicity of such proteins often results in self-aggregation thereof, which in turn makes them inaccessible for any further analysis. In a specific embodiment of the present invention, the first digest of choice is carried out in formic acid, which dissolves virtually all proteins. After the digest, the acid is removed and the smaller peptides are all soluble in chaotrope solutions like urea where they can easily and efficiently be digested with enzymes into small peptides, most of which do not show the tendency of the intact protein to aggregate. Thus, according to the present invention, even though a few peptides from a protein may indeed aggregate, there will still be at least 50% minimum who do not. Accordingly, the present method has shown to be more advantageous than the prior art methods in the context of membrane and/or membrane associated proteins. The labelling agent used in step (d) above will label the N-te-rminal amino acids by virtue of its reaction with free amino groups. Accordingly, it is necessary to pre-treat the proteins to block binding of the labelling agent to amino groups present on internal amino acid residues, especially lysine. If the epsilon groups on lysine were not blocked, then the labelling agent would also bind to all free amino groups on the lysines making it difficult to interpret the amino acid sequence of the labelled peptide. Thus, in one embodiment, step (b) of the above- described method is a succinylation of protein(s). In a specific embodiment, the protecting agent used in step (b) is succinic anhydride, but the protecting agent could be any suitable protecting agent that fulfils the above-described function. For example, N- hydroxysuccinimide can be added, e.g. at a pH of about 8. However, this protecting agent adds not only to lysine residues but also to tyrosine and serine/threonine. Thus, for such agents, a further step will be required, wherein these side-reactions are removed. This further step should be accomplished after the derivatisation of step (d), but before the peptide separation of step (e), and can for example be an addition of hydroxylamine (0.2 M), pH 8, for about 30 minutes. The skilled in this field is familiar with the art of protection and deprotec- tion of amino acids and will be capable of selecting the appropriate conditions for each situation.

The cleaving in step (c) can be an enzymatic digestion, such as with an enzyme, such as a protease (e.g. trypsin, V8 protease, LysC, AspN etc) or a glycosidase, or a chemical digestion, such as with cyanogen bromide. However, as regards membrane and/or membrane associated proteins, due to their compact structure and tendency to aggregate when denatured, enzyme digestions can be found to be inefficient. In one embodiment which is especially advantageous for membrane and/or membrane proteins, the cleaving in step (c) is an enzymatic digestion preceded by addition of a digestive chemical, such as cyanogen bromide. More specifically, the present inventors have used a scheme wherein the proteins are first digested with cyanogen bromide in a powerful solvent, such as 70% formic or trifluoroacetic acid, with or without hexafluoropropanol. This generates medium sized fragments which can be readily solubilised by a conventional method, e.g. in 1% SDS, before dilution to about 0.01% and digestion with LysC protease. In an alternative embodiment, acid-based cleavages are used, as reported by the group of Tsugita (Kamo et al. 1998 and Kawakami et al. 1997). Thus, in one embodiment, the cleaving in step (c) is a serme/threonine cleavage with a fluorinated acid. In a detailed embodiment, site specific cleavage at serine and threonine is carried out in peptides and proteins with S-ethyltrifluorothioacetate vapour as well as at as- partic acid residues by exposure to 0.2% heptafluorobutyric acid vapour at 90°C. Such a serme/threonine cleavage method is advantageous, since Ser and especially Thr are found often in transmembrane segments. In summary, the skilled in this field can select the most appropriate method to cleave the proteins in the sample depending on factors such as the source of the sample, the purpose of the labelling etc. The digested proteins obtained according to the present invention are much easier to handle since physicochemically they are much simpler. Thus, an essential advantage with the present invention is that the separation of peptides obtained according to the invention can be selected to pick out virtually any one or ones of those present in the original sample as proteins, since the present digestion will be essentially total. Accordingly, in the step of separation and the subsequent labelling, any one of all possible peptides (fragments of proteins) can be treated, even cysteine-containing peptides, as will be discussed in more detail below. This should be compared to the prior art methods, wherein proteins can be hidden or concealed due e.g. to self-aggregation. Prior methods required the separation of intact proteins and could not deal with peptide digests without losing the quantitative aspect. The present method of cleavage provides homogenous peptides, which can be separated without the problems associated with proteins have multiple domains (hydrophobic and hydrophilic) which cause them to run at multiple positions. The present digestion method also allows the analysis of proteins that are otherwise completely insoluble or are parts of large complexes which cannot be easily separated, especially cytoskeletal aggregates or proteoglycans.

The labelling reagent can be any moiety, which reacts with an N-terminal amino group. Advantageously, the present invention utilises labels that can be produced in two or more forms which confer the ability to distinguish the different forms of labelled reagents and the peptides to which they are linked by mass, but which importantly do not affect the ionisation efficiency of the peptides to which they are linked when subject to mass spectrometry. In one embodiment of the present method, step (d) is treating with a reagent available in different forms that can be distinguished on the basis of mass, such as mass. Thus, the reagent is selected from the group that consists of C12/C14; H/D; C135/37; positively charged aromatic amines; positively charged tertiary quaternary amines; and phosphorous-based compounds. In an illustrative embodiment, two samples are provided in step (a), one of which is treated with the reagent 1-(H4 nicotinoyloxy) succinimide (H4 Nic-NHS) ester and the other one with 1-(D4 nicotinoyloxy) succinimide (D4 Nic-NHS) ester. (For the synthesis of reagents, see Mϋnchbach et al. 2000 Anal Chem, below). However, as the skilled in this field easily realises, virtually any other pair of heavy/light isotopes can be used to this end.

To illustrate this, reference is made to the above-mentioned Mϋnchbach et al, wherein the development of an isotopic labelling approach for the quantitation of proteins isolated by 2D SDS-PAGE (essentially the process is described in Figure 1) is reported. The membranes of interest are isolated, washed and then the proteins are denatured with 1% SDS before being diluted fifty-fold. The proteins are then succinylated and passed through a synthetic hydro- phobic membrane such as PVDF or an Empore membrane. The membrane is washed to remove any detergent and the membrane is inserted into the chemical modification apparatus for digestion. After this the distinct cell digests are either labelled with the D4 or H4 reagent prior to HPLC-MS analysis as outlined in this specification. Those peptides showing a change in expression or modification level are then chosen for MS/MS analysis, either in a second HPLC run or dynamically in the first. In very complex systems, this method could be combined with prefractionation by BAC/SDS-PAGE orlD-SDS PAGE as a first dimension separation (which functions for greater than 99% of membrane proteins). The advantage of the isotopic labelling approach is that it allows individual proteins to be quantitated even if multiple proteins are found in the same spot or band. Thus, in this context, it is stressed that even though the present invention in an advantageous embodiment utilises a labelling scheme as described by M nchbach et al, the present method utilises a principle which was not suggested by Mϋnchbach et al, namely to protect e.g. by succinylation a sample of proteins before cleavage of said protected proteins into peptides and subsequently N-terminal labelling thereof for the purpose of MS. Contrary, M nchbach et al do not start from such a complex sample but suggest to succinylate already isolated peptides obtained from a gel spot.

In one embodiment of the present method, the separation according to step (e) is by multidimensional chromatography. In another embodiment of the present method, standard reverse phase HPLC is used to separate the majority of the peptides. In a specific embodiment which is efficient if it is desired to get the most hydrophobic peptides, a hydrophilic interaction chromatography (HILIC) approach is used. Alternatively, a first dimension separation can be carried out by ion exchange in the presence of a detergent such as octylglucoside as demonstrated previously (James P, Inui M, Tada M, Chiesi M, Carafoli E. The nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum. Nature. 1989 Nov 2;342 (6245):90-2). The detergent is then easily removed prior to RP-HPLC-MS analysis by a using normal phase precolumn.

Thus, contrary to the above-discussed WO 01/74842, according to the present invention, the proteins or peptide extract is not separated but treated with a protecting agent, e.g. by suc- cinylation, and subsequently digested and labelled with reagent. The resulting complex peptide is according to the invention then separated, preferably by multi-dimensional chromatography, before analysis which is most advantageously performed by mass spectrometry (MS).

In an additional embodiment, the present method comprises labelling peptides by the method above followed by the step of

(f) detecting or measuring the amount of label on the peptides. Accordingly, the present invention also embraces a quantitative method, which is advantageously based on isotopic labels as discussed above.

In one embodiment, the detection or measuring is by mass spectroscopy (MS). In a specific embodiment, said mass spectroscopy is matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In one embodiment, the detection is a dynamic MS/MS. In an alternative embodiment, the detection comprises dividing the sample into two portions, one of which is directed to MS and the other one of which is directed to a fraction collector.

In another embodiment of the present method, the detected label is present on a cysteine- containing peptide. Accordingly, contrary to the prior art, such as Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Related Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 1999 Oct; 17(10): 994-9, the present invention provides a method that is useful on any peptide or protein, regardless of its cysteine content. Since about 20-30% of the proteins of the human genome contains the amino acid cysteine, this is an essential advantage of the invention, which broadens its applicability and makes it a more general method than the ones previously disclosed.

In yet another embodiment, the present method comprises the method described above, which further comprises the step of

(g) identifying the amino acid sequence of at least one of the labelled peptides.

In one embodiment, step (g) is by mass spectral analysis using an ion trap spectrometer or a quadrupole time of flight (TOF) instrument. However, as is realised by the skilled in this field, any MS instrument capable of carrying out and measuring peptide fragmentation spectra can be used to this end.

A second aspect of the present invention is a method of comparing or determining the expression of a protein in a first cell and a second cell, which method comprises the steps of (i) providing samples comprised of a crude cell preparation from a first cell and a second cell;

(ii) labelling the protein from the first cell and the second cell by a method as described above, wherein a first labelling reagent is used to label peptide(s) originating from the first cell and a second labelling reagent is used to label peρtide(s) originating from the second cell and wherein the first and second labelling reagents can be distinguished; and (iii) detecting or measuring the amount of first and second labelling reagent on at least one peptide from each sample.

In one embodiment, the first and the second labelling agent can be distinguished on the basis of mass. There are then two alternative analysis methods depending on the number of peptides in the starting extract. In a first embodiment, the HPLC-MS is carried out with a flow splitter, directing half of the sample to the MS and half to a fraction collector. The MS spectra can be processed after the HPLC-MS run and the peptides showing an change in expression level as observed from the isotopic ratio can be scheduled for MS/MS after reloading the appropriate fraction. In an alternative embodiment, which is advantageous if the peptide mixture is not too complex, the isotope ratios can be determined on the fly and dynamic MS/MS can be done in one HPLC run.

An alternative method developed to allow the quantitation of multiple proteins in a single spot from a two-dimensional was recently described (Mϋnchbach M, Quadroni M, Miotto G, James P. Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labelling of peptides with a fragmentation-directing moiety. Anal Chem. 2000 Sep l;72(17):4047-57), which can be extended to direct labelling of peptides released from a digestion of a whole cell protein extract. The isotopic labelling allows a quantitative analysis of protein expression levels as well as facilitating the identification of the peptides generated (see Figure 2).

In another embodiment, step (iii) is MALDI-MS.

In an advantageous embodiment, the first labelling reagent comprises a light isotopic label and the second labelling reagent comprises a heavy isotopic label, or vice versa. Labelling reagents can be selected from the group discussed above in relation to the first aspect of the invention. Thus, in a specific embodiment, said first and second labelling reagents are 1-(H4 nicotinoyloxy) succinimide (H4 Nic-NHS) ester and 1-(D4 nicotinoyloxy) succhiimide (D4 Nic-NHS) ester. A further aspect of the invention is the method described above, which further comprises the step of

(iv) identifying the amino acid sequence of at least one of the labelled peptides.

In one embodiment, step (iv) is by mass spectral analysis using an MS/MS capable mass spectrometer, such as an ion trap mass spectrometer.

In a specific embodiment, the labelled peptide mixtures are combined prior to step (iii).

A last aspect of the invention is the use of two or more differentially isotopically labelled succinylating agents as labelling reagents for proteins in a complex mixture, such as a crude cell preparation. In a specific embodiment, said agents are 1-(H4 nicotinoyloxy) succinimide and 1-(D4 nicotinoyloxy) succinimide esters, as discussed above.

The present invention can be used in a wide variety of applications, such as for example to identify peptides presented by a major bistocompatibility complex (MHC) molecule.

Another application where the present method is useful is for the analysis of peptides being carried around or in solution in body fluids such as cerebro-spinal and synovial fluids as well as in urine and blood serum. Accordingly, the method according to the present invention can be used e.g. in diagnosis of diseases.

As discussed above, the use of two or more labelling agents with different labels allows a determination of relative amounts of proteins in two or more different samples. In particular, the labelling techniques of the present invention may be used to compare protein expression in two different cells. The two different cells may for example be cells of the same type but under different conditions (or states), or they may be cells of a different type (under the same or different conditions). Thus, by way of example, a first cell may be treated with an agonist and a second cell untreated, and the expression of one or more proteins in each cell compared.

The two conditions could also be cells resting versus cells induced or treated in some manner. Often, differential expression in cells under different conditions can provide useful information on the activity in the cells.

Detailed description of the drawings

Figure 1 shows an embodiment where the present invention is used for isotopic analysis of membrane protein expression. More specifically, in Figure 1, Cell state 1 and 2 are shown, wherein membranes are isolated, proteins are succinylated and chemically digested and peptide N-temiini are modified with H4 and D4 reagent, respectively. Digests 1 and 2 are combined and analysed by HPLC M8. In the first spectrum shown, relative protein levels are quantitated by H4D4 peak height ratios. In the spectrum below, sequence is determined from b ion series which appear as goal posts separated by 4 mass units.

Figure 2 outlines an alternative method developed to allow the quantitation of multiple proteins in a single spot from a two-dimensional gel. More specifically, in Figure 2, in Cell state 1 and Cell state 2, spots are excised, succinylated, and Asp/Glu-C protease digested. The peptides' N-termim are modified with H4 reagent and D4 reagent, respectively, and the digested are then combined and analysed by MALDI MS. In the first spectrum shown, relative protein levels are quantitated by H4D4 peak height ratios. In the spectrum below, sequence is determined from b ion series which appear as goal posts separated by 4 mass units.

EXPERIMENTAL PART The following examples are providing for illustrative purposes only and should not be construed as limiting the invention as defined by the appended claims. All references given below and elsewhere in the present specification are hereby included herein by reference. Example 1: Identification of proteins (comparative^{^})

The model system was set up as described in Dainese P, Staudenmann W, Quadroni M, Ko- rostensky C, Gonnet G, Kertesz M, James P. Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry. Electrophoresis. 1997 Mar- Apr; 18(3-4):432-42; and Quadroni M, Staudenmann W, Kertesz M, James P. Analysis of global responses by protein and peptide fingerprinting of proteins isolated by two- dimensional gel electrophoresis. Application to the sulphate-starvation response oϊEscher- ichia coli: A set of 8 proteins (SSI, sulphate starvation induced proteins) was observed by comparative 2D electrophoresis to be upregulated when Escherichia coli were grown using compounds other than sulphate or cysteine as the sole sulphur source.

In accordance with the prior art method, these proteins were isolated after two-dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The proteins were identified as: ssil = tauA, ssi2 = sulphate binding protein, ssi3 =tauD, ssi4 = ssuE, ssi5 = cysteine synthase A, ssi6 = suD, ssi7 cystine binding protein fliY and ssi8 is alkylhydroperoxide reductase. However, three proteins, which should have been found since they are expressed in the same operons (tau and ssu) were tauB, a membrane associated protein, tauC and ssuC both integral membrane proteins.

To test the efficiency of the method according to the present invention, the experiment was then repeated in accordance with the present invention using the sulphate starved and fed cultures. Thus, after cell disruption, the membranes were pelleted and extensively washed. The proteins were succinylated in SDS and then digested with cyanogen bromide in 70% formic acid and then the acid was evaporated and the pellet was taken up in 8M urea and digested with Glu(C) protease under slightly acidic conditions where it cuts at glutamate and aspartate. The resulting peptides were labelled with the isotopic reagents H4/D4 and the two culture conditions (sulphate fed labelled with the light, the sulphate starved with the heavy reagent) were mixed and analysed by HPLC-MS(/MS). A program, Autofrag, which automates the collection of CID fragmentation spectra from unknown samples, was written in Finnigan MAT instrument control language. The program was subsequently modified to incorporate features of a similar program presented by Dr. T. Lee at the 1993 ASMS (American Society for Mass Spectrometry) meeting. The program monitors the masses of the peptides eluting from the column, using the first quadrupole Ql to scan the mass range from 500 to 2000 amu every 4 seconds (with 2 mTorr argon in Q2 but with the collision offset voltage set to zero). The program switches to MS/MS mode every time a signal lasting more than two scans is detected with a signal/noise ratio >5. Quadrupole Ql filters out the selected ion that undergoes fragmentation in the second quadrupole, which is filled with a collision gas (argon) to a pressure of 2 mTorr. The program returns to scanning Ql after 5 MS/MS scans so that less intense, co-eluting peptides can also be analysed. The collision offset voltage (the voltage for accelerating the ions in the collision chamber, Q2) is automatically adjusted to a value determined by the mass of the ion selected. The resulting fragments are analysed with the third quadrupole Q3, scanning the mass range from 50 to 2000 amu in 3.0 s. This procedure allows both parent ion mass measurement (for protein mass fingerprinting) and sequence analysis by fragmentation (for peptide mass fingerprinting) to be carried out during the same HPLC run. All those peptides showing a greater than four fold increase in the D4/H4 isotope ratio were chosen for MS/MS.

Using the method according to the present invention, the three membrane bound (associated) proteins which had been missed in the two-dimensional gel based approach, namely tauB, tauC and ssuC, could be identified.

Example 2: Alternative application of the present isotopic labelling method

Peptide presentation proteomics: The ability to distinguish between 'self and 'non-self is the basis for one of the most vital defences that the multi-cellular organism possesses and ensures that defence responses are limited to invading pathogens with mhiimum self damage. The fundamental features are highly polymorphic cell-surface recognition structures and mechanisms for the destruction of non-sel The major histocompatibility complex (MHC) forms the basis of recognition structures which present processed protein fragments to the T- cell receptor (TCR) stimulating cytotoxic and helper T-cells. There are two main classes, I and II, which are themselves highly polymorphic. The MHC molecules are almost ubiquitously expressed in human tissues with the exception of the central nervous system and a few specialised tissues such as the corneal epithelium. The diverse set of MHC molecules serve to present a large number of peptides, with each MHC species carrying up to around 2,000 different peptides. Thus it has been estimated that there are on the order of 10-30,000 different peptides being presented on the surface of each cell.

Peptide extraction and derivatisation: As mentioned above, it has been shown that specific MHC molecules can carry up to 2,000 different peptides and that there are, depending on the specific tissue, up to 1 x 10⁶ MHC molecules per cell. In order to release the peptides, two approaches can be taken. Either a specific MHC molecule can be isolated by antibody affinity purification after detergent solubilisation of the cells and then the peptides released from the MHC by acidification. Alternatively a broader approach can be taken and the cell membrane purified by centrifugation after cell lysis. The entire set of peptides bound to the membrane can be released by adding trichloroacetic acid to a final concentration of 12% which precipitates the proteins and membrane but leaves the peptides in solution. Size-exclusion chromatography or ultra-filtration through a specific molecular weight cut-off membrane is carried out to remove any traces of protein that would interfere with the subsequent chromatography.

Claims

1. A method of analysing protein expression, which method comprises the steps of

(a) providing at least one sample comprised of one or more disintegrated cells;

(c) cleaving the so treated protein(s) into a peptide mixture;

(e) at least partially separating the peptides resulting from step (d).

2. A method according to claim 1, wherein the sample(s) provided in step (a) are obtained by mechanical or chemical cell disintegration and centrifugation.

3. A method according to claim 1 or 2, wherein the sample(s) provided in step (a) comprise membrane and/or membrane associated protein(s).

4. A method according to any one of the preceding claims, wherein step (b) is a succinyla- tion of protein(s).

5. A method according to any one of the preceding claims, which comprises a further step of removing protecting agent bound to tyrosine, serine and/ or threonine residues before the separation according to step (e).

6. A method according to any one of the preceding claims, wherein the cleaving in step (c) is an enzymatic digestion preceded by addition of a digestive chemical, such as cyanogen bromide.

7. A method according to any one of the preceding claims, wherein step (d) is treating with a reagent available in different forms that can be distinguished on the basis of mass, which reagent is selected from the group that consists of C12/C14; H/D; C135/37; positively charged aromatic amines; positively charged tertiary quaternary amines; and phosphorous- based compounds.

8. A method according to claim 7, wherein two samples are provided in step (a), one of which is treated with the reagent 1-(H4 nicotinoyloxy) succinimide (H4 Nic-NHS) ester and the other one with 1-(D4 nicotinoyloxy) succinimide (D4 Nic-NHS) ester.

9. A method according to any one of the preceding claims, wherein the separation according to step (e) is by multi-dimensional chromatography.

10. A method according to step 9, wherein the separation according to step (e) is by strong anion exchange followed by reverse phase HPLC.

11. A method of identifying and/or characterising a protein, which method comprises the method of any one of claims 1-10 followed by the step of

(f) detecting or measuring the amount of label on the peptides.

12. A method according to claim 11, wherein the detection or measuring is by mass spectroscopy (MS), such as matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS).

13. A method according to claim 11 or 12, wherein the detection is a dynamic MS/MS.

14. A method according to claim 11 or 12, wherein the detection comprises dividing the sample into two portions, one of which is directed to MS and the other one of which is directed to a fraction collector.

15. A method according to any one of claims 11-14, wherein the detected label is present on a cysteine-containing peptide.

16. A method according to any one of claims 11-15, which further comprises the step of (g) identifying the amino acid sequence of at least one of the labelled peptides.

17. A method according to claim 16, wherein step (g) is by mass spectral analysis using an ion trap spectrometer.

18. A method of comparing or determining the expression of a protein in a first cell and a second cell, which method comprises the steps of

(i) providing samples comprised of a disintegrated first cell and a disintegrated second cell; (ii) labelling the protein from the first cell and the second cell by the method of any one of claims 1-10 wherein a first labelling agent is used to label peptide(s) originating from the first cell and a second labelling agent is used to label peptide(s) originating from the second cell and wherein the first and second labelling agent can be distinguished; (iii) detecting or measuring the amount of first and second labelling agent on at least one peptide from each sample.

19. A method according to claim 18, wherein step (iii) is by mass spectroscopy (MS), such as MALDI-MS.

20. A method according to claim 18, wherein the detection is a dynamic MS/MS.

21. A method according to claim 18, wherein the detection by dividing the sample into two portions, one of which is directed to MS and the other one of which is directed to a fraction collector.

22. A method according to any one of claims 18-21, wherein the detected label is present on a cysteine-containing peptide.

23. A method according to any one of claims 18-22, which further comprises the step of (iv) identifying the amino acid sequence of at least one of the labelled peptides.

24. A method according to claim 23, wherein step (iv) is by mass spectral analysis using an ion trap mass spectrometer.

25. A method according to any one of claims 18-24, wherein the labelled peptide mixtures are combined prior to step (iii).