Abstract:
The present invention describes a method of selective peptide isolation for the identification and quantitative analysis of proteins in complex mixture. The method comprises the selective isolation from every protein of those peptides that neither contain arginine nor histidine (NHNR peptides), and the determination of the relative concentrations of one or several proteins in different samples from the ratio between the areas of the estimated theoretical spectra for the NHNR peptides labeled with different isotopes in each sample. The determination of the relative concentration of proteins is valid for any type of isotopic label of the NHNR peptides. The method avoids the separation and purification of the proteins present in a complex mixture, and the analysis of all peptides generated from the enzymatic digest of the samples. The method is applicable to the identification of proteins with vacunal, therapeutic and diagnostic aims.

Description:
This application asserts priority to Cuban application number CU2003/0269 filed on Nov. 19, 2003. The specification of Cuban application number CU2003/0269 is hereby incorporated by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     The present invention relates to the biotechnology field, in particular with proteomics. Proteomics is defined as a group of tools, techniques and methods very close related to the proteomes studies. The term proteome is used to define the protein complement of the genome. 
     Nowadays the combination of separation technologies with mass spectrometry and automatic database search has made possible the high-trough put identification of proteins in complex mixtures. 
     Most of the emerging techniques analyze the peptides generated by hydrolysis of the proteins by the combination of liquid chromatography and mass spectrometry. 
     In 1999 Link et al. (Link, A. J. et al. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676-682, 1999), developed a method based in two dimensional liquid chromatography and mass spectrometry (LC-MS/MS). For this purpose they packed a microcapillary column with ion exchange and reverse phase media. By this way, all proteolytic peptides are initially absorbed by the ion exchanger. Then, a single fraction of peptides is transferred to the reverse phase using a discontinuing salt gradient. Finally, the peptides are directly eluted from the reverse phase to the mass spectrometer using an increased gradient of acetonitrile. The described procedure is repeated several times using increased salts concentration in order to released additional fractions of peptides from the ion exchanger. This method is better known as MudPiT (Multidimensional Protein Identification Technology). Using the MudPiT, Washburn M. P. et al. identified 1484 proteins from the yeast  Saccharomyces cerevisiae  (Washburn M. P. et al. Large-scale analysis of the yeast proteome by multidimensional protein identification technology,  Nature Biotechnology  19, 242-247, 2001). Although the MudPiT dramatically accelerated the proteins identification procedure, the relative quantitation of proteins could not be easily achieved. In this sense, Washburn M P et al., (Analysis of quantitative proteomic data generated via multidimensional protein identification technology. Analytical Chemistry. 74:1650-1656, 2002), quantified the proteins by the metabolic labeling of two  Saccharomyces cerevisiae  cultures. Cells were grown in enriched nitrogen-14/15 ( 14 N/ 15 N) media in a similar way to the previous studies of Oda et. al. (Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591-6596, 1999). These authors identified the proteins using two dimensional electrophoresis while Washburn M P et al. used MudPiT. For both cases the relative quantitation of proteins was done taking into account the relative intensities of the mass spectrum signals of a given labeled peptide according to the conditions cultures of its original sample. 
     Due to the high cost of reagents and media needed, the metabolic labeling is mainly applied to organisms such as bacteria and yeast. Moreover, in this kind of labeling all the nitrogen atoms of the proteins are labeled: the nitrogens atoms of the peptide bond as well as those of the amino acids side chains, which make impossible to predict the mass difference of homolog labeled peptides without the knowledge of the amino acid sequence. 
     An important step was taken by a group of authors using a different kind of metabolic labeling. In this case labeled essential amino acids are introduced to the cells cultures and hence they are incorporated to every expressed protein. This strategy was named SILAC (stable isotope labeling by amino acids in cell culture) and its potentialities have been proved using labeled amino such as leucine (1H/2D) and lysine ( 12 C/ 13 C) (S. E. Ong, B. Blagoev, I. Kratchmarova, D. B. Kristensen, H. Steen, A. Pandey, M. Mann, Mol. Cell Proteomics 1, 2002, 376-386); (Berger S J, Lee S W, Anderson G A, Lijiana P T, Tolić N, Shen Y, Zhao R, Smith R D, 2002, High-throughput Global Peptide Proteomic Analysis by Combining Stable Isotope Amino Acid Labeling and Data-Dependent Multiplexed-MS/MS. Analytical Chemistry 74:4994-5000); and (Precise Peptide Sequencing and Protein Quantification in the Human Proteome Through In Vivo Lysine-Specific Mass Tagging, J Am Soc Mass Spectrom 2003, 14, 1-7). 
     The enzymatic labeling of peptides has also been suggested and employed during the proteolytic digestion of the protein mixtures in the presence of water and oxygen-18 enriched water (H 2   18 O). In the latest case, one or two atoms of oxygen-18 are incorporated to the carboxy terminus of generated peptides. The comparative proteomic study is conducted mixing the samples of labeled and non-labeled peptides and analyzing by mass spectrometry, the pairs of homolog peptides. The ratio between the areas of the signals of a given peptide is proportional to the concentration ratio of the corresponding protein in the analyzed samples. 
     With this labeling technique there is not enough separation between the mass signals to avoid the overlapping of the isotopic envelops. Additionally, the incorporation of one or two  18 O atoms produces a complex pattern which makes difficult the analysis. In this sense Yao et al. (Yao X, Afonso C, Fenselau C. Dissection of proteolytic  18 O labeling: endoprotease-catalyzed  16 O-to- 18 O exchange of truncated peptide substrates. J Proteome Res. 2003, 2,147-52) proposed a procedure to catalyze the complete incorporation of two  18 O atoms to the C-terminus of peptides. 
     Besides the additional steps and experimental procedures, these methods are not saved from the possible appearance of other peptide(s) sharing part of the mass range covered by the isotopic envelope. 
     The inverse labeling methodology proposes a scheme to accentuate and emphasizes those ion signals that reflect the differential expression of proteins, by means of two experiments executed in parallel where each one use an inverse labeled respect to the other. The subtraction of the two mass spectra allows focusing the attention on those mass-changing-signals in one experiment respect to the other. The inverse labeling offers several advantages such as the identification of proteins which extreme expression changes and the detection of post-translational modifications. Furthermore, this methodology significantly reduces the time and efforts dedicated to the analysis of peptides MS/MS as well as protein identification and quantification. However, subtle changes in protein expression may no be detected by visual examination, especially when the differences do not exceed the noise of the mass spectra. In addition, the filtering and/or smoothing of the spectra results in lost of resolution and of relevant information in region of low signal-to-noise ratio. Another disadvantage of the inverse labeling method is the need of two experiments which certainly reduce the sensitivity. 
     Taking in to account the current resolution power of liquid chromatography and mass spectrometry systems, the analysis of all peptides generated during the hydrolysis of a complex mixture of protein results impracticable. For this reason, other alternatives of quantitative analysis of proteomes have emerged. In the new methods, the identification and quantification of protein is achieved by the selective isolation and analysis of a reduced group of peptides per protein present in the mixture. 
     An example of the emerging alternatives is the ICAT (isotope-code affinity tags) methodology (Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R.  Nat. Biotechnol.  17, 994-999, 1999). With ICAT, only cysteine containing peptides are isolated and analyzed through the use of reagent of three functional elements: an specific chemical reactivity, an isotopically coded linker and an affinity tag. In this method the free thiol of cysteines of a protein sample representative of a given cellular stage are modified with a light isotopically version of the ICAT reagent, while the sample representative of a second cellular stage is modified with the heavy version of the ICAT reagent. The two samples are combined, enzymatically digested and the cysteine contained peptides are isolated by affinity chromatography and analyzed by μLC-MS/MS. 
     The differential protein expression is quantify by measuring the relative intensities of the mass signals of the paired of peptides with identical sequences, but labeled with light and heavy isotopic version of the ICAT reagent. 
     An algorithm which reconstructs the spectra by means of a smoothing filter is used to determine the ratio of intensities. By this way the location and intensity of the local maxims (peaks) are identified. Finally, the average of the intensities of all identified peptides is determined to each protein. 
     The following limitations have been ascribed on the ICAT methodology:
         proteins not containing cysteines residues are excluded from the analysis.   the size of the ICAT reagent causes interferences during the ionization and the mass spectra interpretation.   peptides labeled with the light and heavy versions of the ICAT reagent (H 8 /D 8 ) could eluted significantly separated on RP-HPLC systems, a fact that could mislead the quantification process.   the quantification process could not be applied to labeling techniques that do not provide enough mass separation to avoid the overlapping of the isotopic distributions.   the quantification process could not offer reliable results if there is an overlapping of the isotopic distributions of a given peptide with the isotopic distributions of any of the pair (light/heavy) of homolog labeled peptides.       

     In spite of the limitations described for this method, it continues being necessary to identify and determine relative levels of proteins expressions in complex mixtures, through the selective and specific isolation of a small group of peptide per protein, i.e. the previous simplification of the mixture of proteolytic peptides before the mass spectrometry analysis. The reduction of sample complexity would allow the identification of proteins poorly represented in the mixture, and would avoid the sequencing of many peptides from the same protein. Additionally, it is also necessary the development of methods to analyze and process the spectra of overlapped mass signals without complicating the experimental procedures. 
     DETAILED DESCRIPTION OF THE INVENTION 
     When a complex mixture of proteins is enzymatically digested, it generates a number of peptides, which is beyond the resolving power of liquid chromatography and mass spectrometry systems, making the analysis of all proteolytic peptides impracticable. Nevertheless, the sequence of a peptide of at least 7 residues, obtained by the cleavage of a protein with a highly specific protease, is sufficient for the identification of the protein. 
     The analysis of a small number of peptides per protein is often accomplished by the specific isolation of peptides containing low abundance amino acid. However, it is also possible to achieve such a goal taking into consideration physical features of the peptides, and excluding from the analysis those peptides containing well-distributed as well as low abundance amino acid within the protein sequence, for example peptides that contain histidine and arginine residues. 
     In this sense, the present invention provides a method for the selective isolation of peptides that do not contain histidine neither arginine residues (NHNR peptides) based on ion exchange chromatography of proteolytic peptides previously modified by covalent derivatization of α-amino terminal groups and ε-amino groups of lysines residues. The present method of invention can be used efficiently in the identification of proteins in a complex mixture, and in the determination of the ratio of expression levels of one or more proteins in two different samples since all the proteins contain NHNR peptides. 
     In a specific embodiment of the methods herein, a polypeptide mixture, which may be generated from a variety of natural or synthetic sources, is subjected to the steps illustrated in  FIG. 1  and are explain as follows:
     (1) Alkylation of cysteines with any known alkylating reagent i.e. iodoacetamide, iodoacetic acid or acrylamide. This is particularly useful for tightly folded protein(s) due to disulfide bridges formation or for protein containing free cysteines residues. For the fist case the cysteine alkylation assists in the enzymatic digestion process and for the second case it would avoid the formation of dimers and other adducts through disulfide/dithiol exchange reactions.   (2) Hydrolysis of the proteins. This goal is achieved by the enzymatic digest with endoproteinase Glu-C, endoproteinase Asp-N, endoproteinase Lys-C, trypsin, quimotrypsin, termolisin, pepsin, papain, pronase or any other protease. The chemical hydrolisis of protein with cyanogen bromide or with organic or anorganic acids could also be employed. In addition, peptide mixtures subjected to the method of this invention may be the results of the combination of enzymatic and/or chemical procedures such as those mentioned above. Peptides thus generated, preferably range in size from about 10 to 50 amino acids in length and are more preferable to facilitate peptide sequencing using tandem mass spectrometric methods. Those of ordinary skill in the art can select a protein digestion protocol suitable for use in the protein sample(s) of interest.   (3) Covalent modification of α-amino terminal groups and ε-amino groups of lysine side chains. A variety of useful amine protective groups are known in the art and readily available for application in this method. The protective group selected must no contains or generate basic groups nor possible sites for protonation. The list of reagents that could be used includes: acetic anhydride, N-hidroxysuccinimide, N-acetoxysuccinamide, citraconic anhydride, maleic anhydride, succinic anhydride, phtalic anhydride, tetrahidroftalic anhydride and 9-fluorenylmethyl chloroformate. Some other suitable N-terminal amino protecting groups are: (a) aromatic urethane-type protecting groups which include benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, isonicotinyloxycarbonyl and 4-methoxybenzyloxycarbonyl; (b) aliphatic urethane-type protecting groups which include t-butoxycarbonyl, t-amyloxycarbonyl, isopropyloxycarbonyl, 2-(4-biphenyl)-2-propyloxycarbonyl, allyloxycarbonyl and methylsulfonylethoxycarbonyl; (c) cycloalkyl urethane-type protecting groups which include adamantyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl and isobornyloxycarbonyl; (d) acyl protecting groups or sulfonyl protecting groups. Preferred protecting groups include benzyloxycarbonyl, t-butoxycarbonyl, acetyl, 2-propylpentanoyl, 4-methylpentanoyl, t-butylacetyl, 3-cyclohexylpropionyl, n-butanesulfonyl, benzylsulfonyl, 4-methylbenzenesulfonyl, 2-naphthalenesulfonyl, 3-naphthalenesulfonyl and 1-camphorsulfonyl; (e) photosensitive protective groups which include carbamates derivatives from m-nitrophenyl, 3,5-dimetoxybenzyl, 1-methyl-1(3,5-dimetoxyphenyl)etyl, α-methylnitropiperonyl, o-nitrobenzyl, 3,4-dimetoxy-6-nitrobenzyl, phenyl(o-nitrophenyl)methyl, 2-(2-nitrophenyl)etyl, 6-nitroveratryl, 4-metoxyfenacyl and 3′,5′-dimetoxybenzoine.   

     Any method and/or reagents that achieve the function of selective derivatization of amino groups are intended to be encompassed by this invention. Examples of reagents and protocols for making such modification are easily find in the literature (Protective groups in organic synthesis, Teodora W. Greene and Peter G. M. Wuts, pag. 494-654, Ed. John Wiley &amp; Sons, Inc. (1990) and Peptide Chemistry, Bodanszky, N., pag. 74-103, Springer-Verlag, N.Y. (1988)).
     (4) Ion exchange chromatography of the mixture of modified peptides. The selection of peptides is accomplished by a strong cation exchange chromatography at pH 2-4. The acidic conditions include the use of formic acid, trifluoroacetic acid or any other buffer system, which gives the desired pH of work. This chromatographic step readily separates non-charged from single and double charged peptides. The charged peptides are retained by the exchanger while NHNR peptides, are collected in the flow through.   5) Regeneration of α-amino terminal groups and ε-amino groups of lysines. This is an optional step which greatly depends on the amino group modifier initially employed. If maleic or citraconic anhydride were used as modifying agents, the free amino groups could be restored by timed incubation in the same acid conditions employed during the cation exchange chromatography. For the case of reagents used in the peptide synthesis for the transitory protection of amino groups, the protective groups could generally be removed using basic conditions. If the modifier agent has a photosensitive properties, it could be eliminated by light irradiation of modified peptides.   

     The method of this invention as specifically exemplified employs steps of washing peptides on reverse phase columns to remove undesired materials from the peptide sample. 
     The determination of differences in concentration of one or more proteins in different samples is achieved through the mass spectra analysis of isolated NHNR peptides isotopically labeled in three different ways:
     a) The proteins are extracted from tissues or cell cultures grown in media with two isotopic version of certain nutrient. Among isotopically labeled nutrients could be used the fundamental source of nitrogen labeled with  14 N/ 15 N, and essential amino acids labeled with isotopes of hydrogen ( 1 H/ 2 H), nitrogen ( 14 N/ 15 N), carbon ( 12 C/ 13 C), oxygen ( 16 O/ 18 O), etc. After the protein extraction, at least a portion of the samples are mixed to yield a combined sample which is treated according to the step 1-5 of the method for the selective isolation of NHNR peptides explained above.   b) The protein samples are separately hydrolyzed as in the step 2 of the method in buffer solutions prepared one of them with normal water while the other one contains H 2   18 O. After that, at least a portion of the samples are mixed to yield a combined sample which is treated according to the steps 3-5 of the method for the selective isolation of NHNR peptides explained above.   c) The protein samples are separately hydrolyzed as in the step 2 of the method, and the generated peptides are modified with different isotopic versions, of the same amino modifier reagent, according to the step 3 of the method. The mixture of both samples consists of peptides of the same chemical nature but isotopically different due to the isotopic version of the amino modifier reagent used, for instance H/D,  12 C/ 13 C,  14 N/ 15 C  16 O/ 18 O, etc. Next, NHNR peptides are isolated by cation exchange chromatography as explained above.   

     In any of the three exposed labeling techniques, the isolated NHNR peptides are analyzed by mass spectrometry. The concentration ratio between the analyzed proteins is determined from the areas ratios of the estimated theoretical spectra for differentially labeled NHNR peptides. 
     The theoretical spectrum is estimated from the lineal combination of the isotopic distributions of the NHNR peptides which better fit to the observed spectrum. This combination indicates the peptide contribution to the envelope of the observed spectrum. The ratio of areas is equivalent to the ratio of contributions if the isotopic distributions areas are normalized. 
     The compounds present in the analyzed m/z range can be sorted out in three groups: 1) the NHNR peptide species, coming from the first sample, 2) the NHNR peptide species, coming from the second sample, 3) a compound without interest for the present analysis. 
     The ratio of the total contribution of the NHNR peptide species of interest, coming from each sample, represents the ratio of concentration of the proteins in each sample. Any interference caused by the overlapping of other uninteresting compounds signals, can be equally considered by the theoretical spectra estimation, providing robustness and generality to this quantification method. 
     The isotopic distribution calculation considers the elemental composition of the NHNR peptides and any particular isotopic enrichment. This, along with the analysis of the signal overlapping, decouples the method from a particular isotopic labeling. 
     During labeling in the presence of H 2   18 O, peptides prompted to deamidation such as those containing asparagine, glutamine or carbamidomethylcystein residues, might incorporate an additional  18 O. This produces a wider isotopic distribution for the NHNR peptides coming from the  18 O labeled sample, misguiding any method which estimate the light/heavy ratio based only on the first two peaks from the isotopic distribution. The estimation of the theoretical spectrum allows extracting the information provided by the composition of isotopic distributions of the NHNR peptides which better fit to the observed spectrum. The information contained in the isotopic distribution is highly restrictive, allowing estimating the noise with high precision. 
     Mass spectrometers based on Electrospray (ESI-MS) or Matrix Assisted Laser Desorption Ionization (MALDI-MS) as ionization sources, could be employed in the analysis. The information thus provided is useful in determining the peptides amino acid sequences and their corresponding proteins through database search. 
     The following references relate to the application of mass spectrometric techniques to protein identification, particularly those related to proteome analysis: Ideker T, Thorsson V, Ranish J A, Christmas R, Buhler J, Eng J K, Bungarner R, Goodlett D R, Aebersold R, Hood L “Integrated genomic and proteomic analyses of a systematically perturbed metabolic network.” Science. May 4, 2001;292(5518):929-34; Gygi S P, Aebersold R. “Mass spectrometry and proteomics.” Curr Opin Chem Biol. October 2000;4(5):489-94.; Gygi S P, Rist B, Aebersold R “Measuring gene expression by quantitative proteome analysis” Curr Opin Biotechnol.” August 2000;1 1(4):396-401; Goodlett D R, Bruce J E, Anderson G A, Rist B, Pasa-Tolic L, Fiehn O, Smith R D, Aebersold R. “Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching.” Anal Chem. Mar. 15, 2000;72(6):1112-8.; and Goodlett D R, Aebersold R, Watts J D. “Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry.” Rapid Commun Mass Spectrom. 2000;14(5):344-8; Zhou, H. et al (April 2001) Nature Biotechnol. 19:375-378. 
     Obtaining peptide structural information could be explained as follows. In a first stage of a tandem mass spectrometer, any given NHNR peptide is selected and subjected to a collision-induced dissociation (CID) experiment. The resulting fragment ions spectrum is recorded in a second stage of the mass spectrometer, as a so-called CID or MS/MS spectrum. Because the CID process usually causes fragmentation at peptide bonds along the peptide chain, the CID spectrum alone often provides enough information to determine a peptide sequence. 
     The sequence of the isolated peptides and the identification of proteins can be determined by a combination of tandem mass spectrometry and computer-assisted database search programs, such as MASCOT (Matrix Science Ltd, UK) (Perkins, D N, et al. (1999) “Probability-based protein identification by searching sequence databases using mass spectrometry data”  Electrophoresis  20, 3551-3567) or SEQUEST (Trademark, University of Washington, Seattle Wash.) (McCormack, A. L. et al. (1996) “Direct Analysis and Identification of Proteins in Mixtures by LC/MS/MS and Database Searching at the Low-Femtomole Level”, Anal. Chem. 69, 767-776; Eng, J. K. et al. (1994) “An Approach to Correlate Tandem Mass Spectral Data of Peptides with Amino Acid Sequences in a Protein Database” J. Amer. Soc. Mass. Spectrom., 5, 976-989; U.S. Pat. No. 5,538,897 (Jul. 23, 1996) Yates, III et al.). Both, MASCOT and SEQUEST takes all known genomic sequence, computes all possible theoretical CID spectra and compares them to experimental CID spectra for matches and sequence identification. 
     The protein identification process using the method of this invention could be greatly assisted with the use of genomic database of NHNR peptide sequences. Such databases would allow a fast and more reliable search using the MASCOT and/or SEQUEST programs, minimizing the possibilities of false positive identifications. 
     Once the NHNR peptides have been identified it is possible to determine the relative concentrations of the proteins, from the MS/MS spectra. This could be done by analyzing the isotopic distributions of the fragment ions containing the light and heavy versions of the labeling. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 . Scheme depicting the selective isolation of peptides by the method of this invention. 
         FIG. 2 . Results of the selective isolation of NHNR peptides from the protein recombinant streptokinase. The LEP digest of the protein was analyzed by ESI-MS before (A) and after (B) the application of the method of this invention. 
         FIG. 3  Mass spectra obtained after the application of the method of this invention to the mixture of proteins A and B which were digested in the presence of H 2   16 O y H 2   18 O respectively. The mixtures were composed by the proteins recombinant streptokinase, horse myoglobin and human alpha-2b interferon. The concentration ratios of the proteins recombinant streptokinase, horse myoglobin and human alpha-2b interferon in the mixture A respect to the mixture B were of 1:1, 1:2 and 1:4, respectively.
     A) Mass spectra corresponding to the selective isolation of NHNR peptides from the mixture of both samples.   (B), (C) y (D) Expanded mass ranges for the m/z signals 725.83, 908.46 y 1081.60 corresponding to the proteins human alpha-2b interferon, horse myoglobin and recombinant streptokinase, respectively. These signals contain the typical isotopic distributions of the inclusion of  16 O/ 18 O at the C-termini of every peptide. It could also be observed the contribution to the estimated theoretical spectrum of the present peptide species according to the following representation:
       IIII Observed Spectrum   
       
       Contribution of each present peptide specie to the total estimated spectrum:
           16 O labeled   labeled with one  18 O atom   labeled with two  18 O atoms     16 O labeled and one desamidation     16 O labeled and two desamidation   labeled with one  18 O atom and one desamidation   labeled with one  18 O atom and two desamidations   labeled with two  18 O atoms and one desamidation   labeled with three  18 O atoms and one desamidation   Theoretical total estimated spectrum which includes each peptide specie       

         FIG. 4 . MS/MS spectrum of the NHNR peptide of m/z 725.83 automatically assigned by the MASCOT program to the sequence DSSAAWDETLLDK corresponding to the protein human alpha 2b interferon. The N- and C-terminal ions are denoted in the spectrum according to the nomenclature proposed by Roepstorff P. and Fohlman J. (Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 11, 984, p.601) 
     
    
    
     EXAMPLES 
     Example 1  
     NHNR Peptide Isolation from Recombinant Streptokinase 
     Recombinant streptokinase was subjected to the method shown in  FIG. 1  according to the following steps. 1) Hydrolysis for 6 hours at 37° C. with trypsin in 0.1 M HEPES buffer,pH 8.6, urea 4M. 2) Citraconic anhydride was added in a 50 molar excess over amino groups. The reactions proceed for 15 min at 4° C. 3) Excess of reagent was removed by desalting of peptides by HPLC using a RP-C4 column (20×2.1 mm), and a solvent system consisting of solvent A: 0.05% TFA in water and solvent B: 0.05% TFA in acetonitrile. 4) Modified peptides were loaded onto a strong cation exchanger and the flow-through fraction was immediately collected. 5) Isolated peptide fraction was incubated for 12 hours at 37° C. in order to remove the citraconyl groups and further analyzed by mass spectrometry. 
     The ESI-MS of the entire digest of the protein is shown in the  FIG. 2A . Subjecting the streptokinase digest to the NHNR peptide isolation procedure, considerably reduced sample complexity, yielding only 4 peptides ( FIG. 2B ). Table 1 exhibits the m/z values of isolated peptides and its assignment to the protein sequence (sequence #1 at the end of the document). 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Observed and calculated m/z values for the isolated peptides 
               
               
                 from the protein recombinant streptokinase according to the 
               
               
                 method of this invention. 
               
             
          
           
               
                 (M + H) obs 
                 (M + H) cal 
                 Sequence 
               
               
                   
               
               
                 1307.71 
                 1307.68 
                 300-310 
               
               
                 1775.89 
                 1775.87 
                 259-274 
               
               
                 2162.15 
                 2162.08 
                 336-354 
               
               
                 2428.38 
                 2428.33 
                 188-210 
               
               
                   
               
             
          
         
       
     
     Example 2  
     Identification of proteins present in a membrane extract from the  Neisseria meningitidis  bacteria using the selective isolation of NHNR peptides. 
     The  Neisseria meningitidis  bacteria culture was grown during 16 hours and after that it was centrifuged at 5000 g for 15 min. The biomass was extracted with a buffer solution consisting in 0.1 M Tris-HCl pH 8.5, 10 mM EDTA and 0.5% sodium deoxicolate during 30 min at room temperature with constant agitation. Then, it was centrifuged at 20000 for 30 min at 4° C. and the supernatant was collected. Another extraction step was conducted over the pellet with the same buffer solution as above. This sample and the previously collected supernatant were mixed and further ultracentrifuged at 125000 for 2 hours at 4° C. The pellet was dissolved in 50 mM Tris-HCl, 2 mM EDTA, 1.2% sodium deoxicolate and 20% sucrose and the solution was subjected to another ultracentrifugation step. Finally, the protein pellet was homogenized in solution containing 3% sucrose and 0.01% tiomersal. 
     The extract of proteins (300 μg) were dissolved in 0.1 M HEPES buffer (pH 8.8), guanidinium chloride 6M, and the mixture was reduced and alkylated by addition of DTT (10 mM, 2 hours) followed by 1 hour of incubation with iodoacetamide. The sample were then diluted prior to digestion for 16 hours with trypsin at 37° C. The resulting peptide mixture was treated essentially as in example 1. 
     Isolated peptides by this method were analyzed by LC-MS/MS and the MS/MS spectra were recorded for database search identification of proteins. The table 2 shows the list of identified peptides and their corresponding proteins or genes. Nearly 40% of the identified proteins (58 in total) are localized in the bacteria membrane. Half of these proteins could not be detected in a previous study using two dimensional gel electrophoresis (results not shown). This perfectly agrees with the fact that membrane proteins are poorly represents when current two dimensional electrophoresis procedures are used. 
                                                         TABLE 2                   Proteins and peptides identified in the membrane extract from the  Neisseria           meningitidis  bacteria using the method of this invention.                        Identified Peptide           Access number to       (initial amino acid-final       Protein name   TREMBL or SWISSPROT   Protein #   amino acid)                    outer membrane protein class 1   Q9S4V0   2   67-77                   78-87                   225-235                   336-355       outer membrane protein class 3   P30690   3   64-73                   129-136                   118-128                   20-28                   185-197                   60-73       outer membrane protein class 5c   Q9AE79   4   171-179                   186-199                   201-214       outer membrane protein P64k   Q51225   5   87-99                   44-57                   367-381       outer membrane protein class 4   P38367   6   103-109                    56-102       outer membrane protein Omp85   Q9K1H0   7   146-153                   772-779                   687-701       Fe-regulated protein B precursor -   Q9JXL3   8   130-148         Neisseria meningitidis             298-304                   633-643                   305-314                   29-37                   656-671       Major ferric iron binding protein   P17940   9   142-150       precursor (FBP)           324-331                   266-274       transferrin-binding protein 1   Q09056   10   738-746                   800-810       lactoferrin-binding protein A   O87343   11   483-493                   837-847       hemoglobin receptor   Q9JYA8   12   600-605                   626-635                   636-644                   700-707                   711-717       ATP synthase F1, beta subunit   Q9JXQ2   13   144-154       ATP synthase F1, alpha subunit   Q9JXQ0   14   141-155       ABC transporter, ATP-binding   Q9K112   15   36-46       protein       Opacity protein   O30756   16   159-166                   156-173       putative pilus assembly protein   Q9JY02   17   72-88                    95-115       Pilus secretin   Q9JVW4   18   426-441       organic solvent tolerance   Q9K187   19   359-369       protein, putative           152-170       surface protein A   Q9RP17   20   80-98       pyruvate dehydrogenase, E2   Q9JZ11   21   364-372       component       dihydrolipoamide acetyltransferase       Elongation factor Tu   Q9JRI5   22   26-38       Elongation factor G (EF-G)   Q9K1I8   23   190-198       glyceraldehyde 3-phosphate   Q9JX95   24   216-227       dehydrogenase       alcohol dehydrogenase, propanol-   Q9K0P0   25    3-10       preferring       proline dehydrogenase   Q9K0Z9   26    996-1004       6-phosphogluconate dehydrogenase,   Q9K1Q5   27   38-47       decarboxylating       peroxiredoxin 2 family   Q9JQS4   28   220-230       protein/glutaredoxin       malate: quinone oxidoreductase   Q9JXD7   29   186-194       aspartyl-tRNA synthetase   Q9JT23   30   376-383       phosphoenolpyruvate synthase   Q9K012   31   527-533       citrate synthase   Q9JQX0   32   275-293       recA protein   P56988   33   287-297                   199-216       5-   Q9JZQ2   34   298-308       methyltetrahydropteroyltriglutamate-       homocysteine methyltransferase       phospholipase A1, putative   Q9K0U7   35   181-187       adenylosuccinate lyase   Q9K183   36   146-152       oxoacyl-(acyl-carrier-protein)   Q9K1D8   37   131-152       synthase III       aconitate hydratase, Putative   Q9X5I6   38   615-622       Succinyl-CoA synthetase   Q9JZP4   39   281-296       beta chain       homoserine dehydrogenase   Q9JR84   40   172-180       stringent starvation protein A   Q9JXN8   41   23-30       orotate phosphoribosyltransferase   Q9JR25   42   20-26       Glutamate dehydrogenase, NADP-   Q9JY71   43   105-113       specific.       IgA1 protease   Q51169   44   1376-1386                   1503-1515                   1490-1502       30S ribosomal protein S2   Q9JRG7   45   118-131                   65-73                   226-266       30S ribosomal protein S3   Q9JQX2   46   26-34                   80-88       30s ribosomal protein S5   Q9JQP3   47    96-102       30S ribosomal protein S10   Q9JR21   48   38-45       30S ribosomal protein S11   Q9JQR2   49   60-74       30S ribosomal protein S17   Q9JQL7   50   74-83       50S ribosomal protein L14   Q9JQY4   51   79-90       50S ribosomal protein L1   Q9JRJ1   52   142-154       50S ribosomal protein L6   Q9K1I3   53   19-27       50S ribosomal protein L16   Q9JR26   54   27-35       50S ribosomal protein L25   Q9JZW3   55   165-175       50S ribosomal protein L15   Q9K1I2   56    97-109       50S ribosomal protein L22   Q9JRD8   57   28-41       50S ribosomal protein L9   Q9JZ31   58   72-82       50S ribosomal protein L11   Q9K1J3   59   73-81                    
From table 2 it could be observed that out of the 95 positively identified peptides only one contains an arginine residue. These results probed the high degree of selectivity that could be achieved with the present method. On the other hand, 20 identified proteins (34%) do not contain cysteine, which means that could have not been detected using methods based on the selective isolation of cysteine residues. (Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R.  Nat. Biotechnol.  17, 994-999, 1999; Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides. Spahr C S, Susin S A, Bures E J, Robinson J H, Davis M T, McGinley M D, Kroemer G, Patterson S D. Electrophoresis, 21(9), 1635-1650, 2000). This fact affords for a broader application of the present invention.
 
     Example 3  
     Identification and relative quantification of the proteins components of the two mixtures (A and B) by the method of this invention. 
     The samples A and B where prepared with the proteins recombinant streptokinase, horse myoglobin and human alpha-2b interferon. The three proteins were mixed in a molar ratio of A respected to B of 1:1 for streptokinase, 1:2 for myoglobin and 1:4 for the alpha interferon. The isolation of NHNR peptides was achieved essentially as in the example 1, but for this case the enzymatic digestion with trypsin was carried out in the presence of normal water for the mixture A while the mixture B was hydrolyzed in presence of H 2   18 O. Both samples were mixed and the NHNR peptides were isolated according to the method of this invention. 
     The enzymatic labeling with  16 O/ 18 O was chosen due to the increasing level of complexity that this labeling procedure may introduce. For instance, it does not produce enough separation between the mass signals to avoid the overlapping of the peptides isotopic envelops. Additionally, the incorporation of one or two  18 O atoms creates a complex pattern which makes more difficult the quantification analysis. 
     The isolated NHNR peptides were sequenced and quantified in a single LC-MS/MS experiment. This allows an unambiguous identification of the three proteins present in the mixture as well as their precise quantification with less than 14% of error (table 3). 
     
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Identification and quantitative analysis of the proteins in the mixtures A and B 
               
               
                 by selective isolation of NHNR peptides labeled with  16 O and  18 O, respectively. 
               
             
          
           
               
                   
                   
                 Identified Peptide 
                   
                 Observed 
                   
                 Expected 
                   
                   
               
               
                   
                 Protein 
                 (initial amino acid - 
                   
                   16 O/ 18 O 
                 Mean Value 
                   16 O/ 18 O 
                   
                 % 
               
               
                 Protein 
                 No. 
                 final amino acid) 
                 m/z (charge) 
                 relation 
                   16 O/ 18 O 
                 Relation 
                 Desviation 
                 error 
               
               
                   
               
             
          
           
               
                 Streptokinase 
                 1 
                 300-310 
                  654.35 (+2) 
                 0.933 
                 1.04 
                 1 
                 0.09 
                 4 
               
               
                   
                   
                 2549-274  
                  888.43 (+2) 
                 1.010 
               
               
                   
                   
                 336-354 
                 1081.55 (+2) 
                 1.22 
               
               
                   
                   
                   336-354 (a)   
                 1082.04 (+2) 
                 0.93 
               
               
                   
                   
                   336-354 (b)   
                 1082.54 (+2) 
                 1.39 
               
               
                   
                   
                 188-210 
                 1214.69 (+2) 
                 1.126 
               
               
                   
                   
                 188-210 
                  810.11 (+3) 
                 0.952 
               
               
                 Myoglobin 
                 60 
                  1-16 
                  908.46 (+2) 
                 1.872 
                 1.87 
                 2 
                 0.13 
                 6.5 
               
               
                 Alpha 
                 61 
                 71-83 
                  725.83 (+2) 
                 3.46 
                 3.46 
                 4 
                 0.54 
                 13.5 
               
               
                 interferon 
                   
                   
                   
               
               
                   
               
               
                   (a) Peptide with one desamidated asparagine. 
               
               
                   (b) Peptide with two desamidated asparagines 
               
             
          
         
       
     
     The  FIG. 3A  shows the mass spectrum corresponding to the isolation of NHNR peptides from the mixture of both samples. The  FIGS. 3B ,  3 C and  3 D shows the expanded ranges of the spectrum for the m/z signals 725.83, 908.46 and 1081.55 respectively, containing typical isotopic distributions of  16 O/ 18 O at the C-terminus of each peptide. Furthermore, it can be observed the contribution of each peptide specie and isotopic variants to the total theoretical spectrum. In the case of the peptide of m/z 1081.55 it could be identified and quantified the presence of asparagine deamidations which where also considered for the calculations of the relative concentrations of peptides labeled with  16 O/ 18 O. 
     The quantitation procedure takes into account the possible deamidation of NHNR peptides according to their composition of Asn and Gln residues. The deamidation process occurring during the H 2   18 O exposition may introduces an additional atom of  18 O in the OH group that substitutes the NH 2  group of the Asn to form Asp. Hence, in addition to the  18 O atoms that are incorporated at the C-terminus of the peptides during the enzymatic hydrolysis, the NHNR peptides coming from the sample hydrolyzed in the presence of H 2   18 O might incorporate an additional  18 O in every Asn o Gin of the peptides. 
     For the spectra analysis, the theoretical isotopic distribution of each NHNR peptide are calculated and normalized including the isotopic variants and possible deamidations. The combination of the isotopic distributions that better matches to the observed spectra is estimated. 
     For each peptide it is calculated the ratio between the sum of areas of the isotopic envelops of the natural isotopic variants i.e.  16 O labeled species, and the sum of areas of the isotopic envelops of the  18 O enriched species. Due to the possible differences in the ionization efficiency of the peptide with Asn and the deamidated peptide containing Asp, the concentration ratio of their isotopic variant is separately calculated. For instance the peptide of m/z 1081.55 contains two Asn, and the calculations were made taking into account the areas of the isotopic envelopes of the three species of the peptides detected in the spectrum: 1) the peptide without deamidation, 2) with one deamidation and 3) with two de deamidation. 
     The application of the method of this invention to these samples resulted in the isolation and identification of 6 peptides, out of the 59 possible peptides that could be generated during the LEP hydrolysis of the mixture. This result shows the substantive simplification of the mixture composition obtained by this way. The mass spectrometry analysis is reduced to about 90% of the peptides, without detriment in the capacity to identify the proteins presents. For instance, the identification of the protein alpha interferon was feasible with the isolation of a single peptide with sequence DSSAAWDETLLDK and m/z 725.83 ( FIG. 4 ). 
     LC-MS/MS and database search. 
     Mass spectrometric measurements were done in a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTof-2™ (Micromass, Manchester, UK). The mass spectrometer was connected online with a liquid chromatographer AKTA Basic (Amersham Pharmacia Biotech, Sweden) by using a RP-C18, 200×1 mm column (Vydac, USA). Peptides were eluted using a lineal gradient of solvent B (CH 3 CN, 0.2% formic acid) from 5 to 45% in 120 min. 
     The capillary and cone voltages were set at 3000 and 35, respectively. Doubly and triply charged precursor ions to be fragmented were selected automatically once their intensity rose above a defined threshold (7 conts sec −1 ). The MS/MS was switched to MS mode once the TIC decreased below 2 count sec −1  or when MS/MS mode was achieved during 4 sec. Data acquisition and processing were performed using a MassLynx system (version 3.5) from Micromass. 
     Protein identification based on MS/MS spectra was made using the Internet available search engine Mascot. Search parameters included fixed (carbamidomethylcystein) and variable modifications, which included deamidation of asparagines and/or glutamines residues and oxidation of methionines.