WO2005052182A2

WO2005052182A2 - A method of analyzing plasma membrane protein content of cells

Info

Publication number: WO2005052182A2
Application number: PCT/IL2004/001085
Authority: WO
Inventors: Michal Linial; Alex Inberg; Yaniv Bledi
Original assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Current assignee: Yissum Research Development Co of Hebrew University of Jerusalem
Priority date: 2003-11-26
Filing date: 2004-11-25
Publication date: 2005-06-09
Anticipated expiration: 2006-05-26
Also published as: WO2005052182A3

Abstract

The present invention is of a method of characterizing proteins present on the plasma membrane of live cells which can be used in the identification of diagnostic markers and potentials drugs. Specifically, the present invention can be used to identify drug targets useful in treating disorders, such as cancer, which are associated with abnormal representation of cell surface proteins.

Description

A METHOD OF ANALYZING PLASMA MEMBRANE PROTEIN CONTENT OF CELLS

FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a method of identifying proteins present on the plasma membrane of intact cells and, more particularly, to the identification of potential drugs useful treating disorders, such as cancer, which are associated with abnormal representation of cell surface proteins on living cells. The plasma membrane (PM) consists of proteins and polar lipids and provides a strict separation between the internal and external environments of the cell. PM proteins exhibit a fundamental role in defining the physiological state of the cell by performing functions such as signal transduction, cell-cell contact, the selective transport of molecules and other essential functions. Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Thus, about two-thirds of all drug targets are directed against PM proteins (Hopkins and Groom, 2002). However, despite their crucial role in cell function, PM proteins are disproportionately under-characterized from the biochemical, topographical and structural perspectives. PM proteins include proteins which are genuine integral membranous proteins, i.e., transmembrane proteins which cross the membrane (e.g., single pass or multipass proteins), proteins which are associated on the extracellular face of the cell via ionic interactions with the lipid layer or with other proteins (e.g., β2-microglobulin which is attached to the heavy chain of MHC class I protein), proteins which are associated via a lipid moiety (e.g., alkaline phosphatase and acetylcholine esterase) and secreted extracellular proteins which are not membrane bound. Most protein identification methods rely on dissolving the proteins in aqueous solutions prior to their separation and identification. Thus, proteins which are associated via ionic interactions with the PM can be readily identified using standard protocols. However, due to their lipophilic domains and low-abundance, the integral PM proteins which cross the membrane at least once, are not readily dissolved in such solutions. In addition, the heterogeneous characteristics of PM proteins, i.e., the variations in the number of membrane spanning helixes (from 1 to 15), ratio of membrane hydrophobic imbedded domains to soluble intra - and extracellular portions, and the amount of modifications such as glycosylations may lead to differential, and often unpredicted, solubilization propensities. Furthermore, the lipid bilayer of the PM is far from being homogeneous as regions of high cholesterol, caveolae rich domains and lipid rafts, drastically affect protein concentrations and compositions (Foster et al., 2003). Membrane proteins can be dissolved using ionic detergents such as sodium dodecyl sulfate (SDS), urea-based solutions with non-ionic detergents such as triton X-100 and CHAPS and chaotropic agents, detergents, and organic solvents (Molloy, 2000; Santoni et al., 2000; Taylor et al., 2002; Ferro et al., 2002; Ferro et al., 2000). However, due to the low ratio of membranous to cytosolic proteins the detection of low-abundant integral membrane proteins is limited. The fraction of integral membrane proteins in the protein mixture can be increased using several approaches. For example, a biochemical enrichment of membranes (Chang PS et al., Anal Biochem. 2004; 325: 175-84) may remove some of the contaminating cytosolic proteins. However, intracellular proteins such as, cytoskeletal proteins, often remain peripherally attached to the membrane via weak interactions. To increase the representation of integral membrane proteins in the sample, undesirable proteins may be discarded by rinsing the membranes with strong ionic solution, by alteration the pH (i.e., a sodium carbonate wash) and or by applying cytoskeletal depolymerizing conditions. Once obtained, the protein mixture can be separated using two-dimensional gel electrophoresis (2DE) and/or liquid chromatography. Two-dimensional gel electrophoresis (2DE) can separate a large number of proteins in a single experiment. Depending on sample complexity, size ofthe gel and,. pH range, a few hundred to a few thousand of individual spots can be identified. The introduction of immobilized pH gradient gels to the 2DE (Celis and Gromov, 1999; Fey and Larsen, 2001) resulted in highly reproducible results with high-resolution separation on narrow pH ranges (Walsh and Herbert, 1999). The method is ideal for resolving large numbers of proteins and detecting posttranslational modifications (Lognonne, 1994). Many modifications such as phosphorylation result in a shift of the isoelectric point (pi), giving rise to a set of protein spots. Sophisticated comparative gel imaging programs coupled with automated spot picking and digestion procedures has turned 2DE technology into a robust, high throughput method (Spandidos and Rabbitts, 2002). However, due to inherent physical limitations such as protein size, extreme pi range, and the hydrophobic nature, the separation of plasma membrane integral proteins on 2DE is limited, resulting in an a priori biased under-representation ofthe plasma membrane extracellular proteome (PMEP). Multidimensional liquid chromatography (LC) is based on the separation of peptides/proteins using different chromatographic media, such as reversed phase, cation/anion exchange and hydrophobic interaction columns (Wagner et al., 2000). Combinations of such columns provide high-resolution, multidimensional separation of peptide and protein mixtures. In the on-line LC approach (Jalili PR and Dass C, Rapid Commun Mass Spectrom. 2004; 18: 1877-84), the protein mixture undergoes enzymatic treatment to produce a complex peptide mixture which is then separated using a multi-column chromatographic separation followed by tandem mass spectrometry (MS) analysis. The success of the 2DE and the LC techniques mainly depends on the initial membrane solubilization procedure. Thus, while the LC method is compatible with a wide variation of buffers, ions solvents and detergents, the 2DE application is limited to the use of mild non-ionic detergents and extremely low concentrations of salt ions. A critical difference between the LC and the 2DE methodologies is the step of protein fragmentation. In the LC methodology protein fragmentation is conducted prior to the separation stage, resulting in the representation of each protein by a number of fragments, all of which belong to the same protein. Such a fragmentation increases the probability that at least some fragments will be soluble under the experimental condition and will be available for the following MS/MS phase. Prior art studies describe the application of 2DE and LC separation techniques on integral membrane proteins in a high throughput fashion (Binz et al., 1999; Poutanen et al., 2001). However, while 2DE is successfully applied in many aspects of proteomics research, at present, satisfactory results are limited to secreted and , ,„,_,„ O 2005/052182

soluble proteins and to the study of bacterial proteomes (Celis and Gromov, 1999; Collins et al., 1994; Fey and Larsen, 2001). Following protein separation, whether performed by 2DE or by LC, the peptide mixture is analyzed using MS. Mass spectrometry (MS) analysis of peptides and proteins depends primarily on soft ionization techniques that create gas-phase ions from biomolecules, thus enabling accurate measurement of molecular weight. The two main MS methods are the electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). MALDI ionization is provided by a laser energy, which is applied to the peptides (or proteins) co-crystallized with small UV absorbing molecules. Ionization occurs due to proton transfer from the protein sample to the matrix. On the other hand, ESI produces charged solvent droplets, which release ions under drying conditions. This method uses a stream of solvent, and therefore can be easily coupled to a high-pressure liquid chromatography (HPLC) instrument. The main operational mode of ESI is a nano-electrospray (flow rate is approximately 20-100 nl/min) in which a very small sample amounts can be successfully analyzed. As an alternative, the off-line MS method is used, where the peptide mixture is first separated and then treated with proteolytic enzymes and analyzed by MS. Both variations on the techniques provide a convenient method for analyzing complex mixtures, containing low-abundance proteins, proteins with exceptionally high or low pi, high or low mass as well as highly hydrophobic proteins. The final step of most MS experiments is the database searching of the resulting mass spectra. Several search algorithms are available, allowing high throughput database searching techniques. Prior art proteomic studies adopted the chromatography platform exploiting the tactical advantage of digesting proteins prior to their isolation. In a benchmark study, Wu et al, 2003 (Nature Biotechnology, 21: 532-538), used a combination of high pH conditions and proteinase K (PK) treatment to fragment both soluble and membrane proteins. The source of the proteins for this large-scale test was either un-fractionated rat brain or enriched liver Golgi membranes. Under such experimental conditions 1610 rat brain proteins were identified, of which, 28.2 % were membrane proteins according to protein annotation and computational prediction algorithms. Washburn et al., 2001, demonstrated the use of enriched yeast membranous fractions in a proteome analysis. Yeast membranes were solubilized overnight using 90 % Formic acid in the presence of CNBr (cyanogen bromide), following which the pH was adjusted to 8.5 using Ammonium Bicarbonate and the samples were treated with LysC and Trypsin. Such conditions resulted in the detection of 1484 yeast proteins, of them, only 131 were authentic integral membrane proteins. An additional level of sensitivity was achieved by Han et al., 2001. In their experiments, the microsomal fraction of human myeloid leukemia (HL-60) cells was covalently labeled with isotope-coded affinity tag (ICAT) reagent and the labeled preparation was then trypsinized and subjected to MS identification scheme. Using this approach 491 proteins were identified, of them, 11.2 % were either known cell surface antigens, receptors, or proteins whose membranous nature could be inferred. Blonder et al., 2002, investigated the membrane proteome of the bacteria

Deinococcus radiodurans. The bacteria membrane fraction was treated in a sequential solubilization protocol including heating, high pH extraction, organic solvent-assisted solubilization and finally trypsinization. In this study, 503 proteins were isolated, of which 135 were predicted as genuine transmembrane proteins. However, although all of these studies exhibited an unprecedented success in identifying membranous proteins they all resulted in the identification of a mixed population of proteins, i.e., cytosolic proteins, membrane proteins which face the cytosol, as well as membrane proteins facing the outer surface ofthe cell. While reducing the present invention to practice and experimentation, the present inventors have devised a novel method of exclusively identifying membrane proteins present on the outer surface ofthe cell. In addition, since many cell surface proteins are modified in a disease state, the present inventors have uncovered that proteins identified using such a method can be used for diagnostic purposes and for the identification of drugs useful for treating disorders such as cancer which are associated with abnormal representation of proteins on the plasma membrane. SUMMARY OF THE INVENTION According to one aspect ofthe present invention there is provided a method of characterizing proteins present in a plasma membrane of a cell, comprising: (a) subjecting a cell having an intact plasma membrane to a protease treatment to thereby obtain peptide fragments derived from proteins present in the plasma membrane ofthe cell; (b) deterrniiiing a composition or sequence of the peptide fragments thereby characterizing the plasma membrane proteins ofthe cell. According to another aspect of the present invention there is provided a peptide composition comprising a plurality of peptide fragments each derived from an extracellular portion of a cell membrane protein According to further features in preferred embodiments of the invention described below, determining the composition of the peptide fragments is effected using mass spectrometry. According to still further features in the described preferred embodiments determining the sequence of the peptide fragments is effected using protein sequencing. According to still further features in the described preferred embodiments, the method further comprising a step of labeling the proteins present in the plasma membrane with a non-permeable in vivo label prior to step (a). According to still further features in the described preferred embodiments the non-permeable in vivo label is selected from the group consisting of biotin and ICAT. According to still further features in the described preferred embodiments the method further comprising a step of subjecting the cell to a glycosidase treatment prior to and/or concurrently with the protease treatment. According to still further features in the described preferred embodiments the glycosidase treatment is effected using a glycosidase selected from the group consisting of Ceramide Glycanase, Endo-b-galactosidase, Endo-a-N- acetylgalactosaminidase, b-Endo-chitinase, Endoglycoceramidase II ACT, Endoglycosidase D, Endoglycosidase FI, Endoglycosidase F2, Endoglycosidase F3, Endoglycosidase H, N-Glycosidase A, N-Glycosidase F, and N-Glycosidase F. According to still further features in the described preferred embodiments the method further comprising a step of denaturing and/or digesting the peptide fragments prior to step (b). According to still further features in the described preferred embodiments denaturing is effected using a urea-based denaturant and/or an ionic detergent. According to still further features in the described preferred embodiments the urea-based denaturant is 8 M UREA. According to still further features in the described preferred embodiments the ionic detergent is SDS. According to still further features in the described preferred embodiments the

SDS is provided at a concentration range of 0.01-0.5 %. According to still ftirther features in the described preferred embodiments digesting is effected using trypsin, Lys-C, chemotrypsin, and/or Asp-N. According to still further features in the described preferred embodiments the method further comprising subjecting the peptide fragments to a glycosidase treatment prior to and/or concurrently with the digesting. According to still further features in the described preferred embodiments the method further comprising a step of subjecting the peptides to a protease inhibitor following the protease treatment. According to still further features in the described preferred embodiments the protease inhibitor is selected from the group consisting of AEBSF, Antithrombin III, Aprotinin, Benzamidine, Bestatin, Calpeptin, Chymostatin, E-64 Protease Inhibitor, EST, Leupeptin, a2-Macroglobulin, Pepstatin A, Na-Tosyl-Lys Chloromethyl Ketone, Na-Tosyl-Phe Chloromethyl Ketone, and Trypsin Inhibitor. According to still further features in the described preferred embodiments the cell is selected from the group consisting of a mammalian cell, a plant cell, a cyanobacteria cell, a yeast cell and a gram negative bacterial cell. According to still further features in the described preferred embodiments the protease treatment is effected using a protease selected from the group consisting of proline-endopeptidase, Staphylococcus aureus V8 protease, low specificity chymotrypsin, high specificity chymotrypsin, trypsin, Proteinase K (PK), and Asp-N. O 2005/052182

According to still further features in the described preferred embodiments the mass spectrometry is LC-ESI-TOF-MS. According to still further features in the described preferred embodiments the peptide is labeled using a non-permeable in vivo label. j According to still further features in the described preferred embodiments the cell is embryonic carcinoma P19 cells and whereas the peptide composition is set forth by SEQ ID NOs:l-25 and 27-66. According to still further features in the described preferred embodiments the cell is a yeast cell and whereas the peptide composition is set forth by SEQ ID NOs:67-84. The present invention successfully addresses the shortcomings ofthe presently known configurations by providing a method of determining the composition of the plasma membrane proteins. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion ofthe preferred embodiments ofthe present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms ofthe invention may be embodied in practice. In the drawings: FIGs. la-k illustrate the PROCEED flowchart protocol. FIG. 2 is a schematic illustration depicting the release of extracellular exposed domains from plasma membrane (PM) proteins by a proteolytic treatment on live cells. The integral membrane proteins (proteins P2, P3 and P4) represented contain exposed domains. The illustration mimics the first stage in PROCEED in which a non-specific protease is supplemented and according to the accessibility of the cleavage sites, a set of peptides is obtained for further analysis (see Example 1 ofthe Examples section). Left, a soluble protein (protein Pl) is cleaved by the low- specificity protease. This protein is included in the analysis due to its direct interaction with an authentic integral membrane protein (P2). Middle, the exposed domain of the integral protein (P3) is heavily glycosylated and thus the potential protease cleavage sites are masked. Right, some of the extracellular loops in a mulipass integral membrane protein (P4) are susceptible for proteolytic cleavage. Note that only a loop that has more than one cleavage site is released to the medium. The resulting post-cleavage peptides are shown. The B symbol indicates an in vivo biotin label. An affinity purification of the biotin labeled peptides following solubilization of the PM membrane increases the coverage of the peptides analyzed and therefore the validity of protein membrane topology determination.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a method of characterizing proteins present on the plasma membrane of live cells which can be used in the identification of diagnostic markers and potential drugs. Specifically, the present invention can be used to identify drug targets useful in treating disorders, such as cancer, which axe associated with abnormal representation of cell surface proteins. The principles and operation of the method of identifying proteins present on the plasma membrane according to the present invention may be better understood with reference to the drawings and accompanying descriptions. O 2005/052182

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Before explaining at least one embodiment ofthe invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. However, despite their crucial role in cell function, plasma membrane (PM) proteins are disproportionately under-characterized from the biochemical, topographical and structural perspectives. Current methods of identifying PM proteins rely on dissolving the proteins in aqueous solutions prior to their separation and identification. However, due to their lipophillic domains and low-abundance, the integral PM proteins which cross the membrane at least once, are not readily dissolved in such solutions. To circumvent such limitations, prior art proteomic studies adopted the chromatography platform exploiting the tactical advantage of digesting proteins prior to their isolation. These include the combination of high pH conditions and proteinase K (PK) treatment [Wu, 2003 (Supra)], Formic acid in the presence of cyanogen bromide (CNBr) and LysC - Trypsin treatment [Washburn, 2001 (Supra)], isotope-coded affinity tag (ICAT) and trypsin [Han, 2001 (Supra)], and the combination of heating, high pH extraction, organic solvent-assisted solubilization and trypsinization [Blonder, 2002 (Supra)]. However, such methods resulted in the identification of a mixed population of proteins, of which, only 8-26 % were membrane proteins. In addition, using the prior art approaches (i.e., homogenization and digestion of all cell proteins) it is impossible to know which of the membrane proteins are extracellular proteins which face the outer surface ofthe cell. While reducing the present invention to practice the present inventors have devised a novel method of exclusively characterizing membrane proteins present on the outer surface of the cell. As is shown in Tables 1 and 3 and Examples 2 and 3 ofthe Examples section which follows, the present inventors were capable of identifying with high confidence 65 or 22 membrane proteins from the P19 or yeast cells, respectively. Thus, there is provided a method of characterizing proteins present in a plasma membrane of a cell. As used herein, the phrase "proteins present in a plasma membrane" refers to all proteins which transverse the plasma membrane, associate with the extracellular face of the plasma membrane, and/or secreted proteins which are present in the extracellular space but are not bound to the plasma membrane. Proteins which transverse the plasma membrane include single pass transmembrane proteins (type I and type II) membrane proteins, such as integrin beta, Serine/Threonine protein kinase and/or multipass transmembrane proteins (i.e., proteins which transverse the plasma membrane at least twice), such as uracil permease and AAA transporter. Proteins which are associated on the extracellular face of the plasma membrane include proteins which are associated via (i) ionic interactions with the phospholipid head groups of the lipid layer, (ii) ionic protein-protein interactions with other plasma membrane proteins (e.g., β2-microglobulin which are attached to the heavy chain of MHC class I protein) and/or (iii) a lipid moiety such as phosphatidyl-inositol (e.g., alkaline phosphatase and acetylcholine esterase). The cell of the present invention can be any cell having a plasma membrane.

Examples include, but are not limited to eukaryotic cells (e.g., a mammalian cell and a yeast cell) as well as prokaryotic cells (e.g., a gram negative bacterial cell). Preferably, the cell of the present invention is a mammalian cell. Non-limiting examples of mammalian cells include fibroblast cells, kidney cells, chondrocytes, chromaffin cells, neuronal cells, embryonal cells, and immunologically originated cells. It will be appreciated that the cell of the present invention can be part of a cell culture, e.g., cells grown in suspension such as jurkat cells or cells cultured on a tissue culture surface (e.g., P19 cells) and include both adherent and non-adherent cells. Alternatively, the cell of the present invention can be directly derived from an individual using cell and/or tissue biopsy using e.g., fine needle aspiration, surgery and the like. The method is effected by subjecting a cell having an intact plasma membrane to a protease treatment to thereby obtain peptide fragments derived from proteins present in the plasma membrane of the cell and determining a composition or sequence of the peptide fragments thereby characterizing the plasma membrane proteins ofthe cell. The phrase "an intact plasma membrane" refers to an un-ruptured, undamaged, integral and/or well-ftmctioning plasma membrane, i.e., a plasma membrane which involves in functions such as cell division, metabolism, diffusion, secretion, signal transduction, transport, cell-cell contact, selective transport of molecules, and the like. It will be appreciated that intact plasma membrane is found in live cells, i.e., cells undergoing proliferation and/or differentiation in vitro and/or ex vivo. The phrase "peptide fragments" refers to a portion of the proteins present in the plasma membrane which is dissociated from the plasma membrane following the protease treatment of the present invention. It will be appreciated that since such a treatment is employed on a cell having an intact plasma membrane, such peptides are derived from the extracellular portion ofthe proteins present in the plasma membrane i.e., the portion ofthe proteins which faces the outside ofthe cell. The length of such peptide fragments largely depends on the protease treatment employed. Preferably the protease treatment is effected such that resulting peptide fragments are at least 3 amino acids, more preferably, at least 7, more preferably, at least 10, more preferably, at least 12, more preferably, at least 14, more preferably, at least 16, more preferably, at least 18, most preferably, at least 20 amino acids. For example, digestion of a yeast proteome with Trypsin results in a mean peptide mass of 1229 Da (i.e., approximately 10-11 amino acids), digestion of a yeast proteome with Chymotrypsin, results in a mean peptide mass of 1496 Da (i.e., approximately 12-13 amino acids) and digestion of a yeast proteome with CNBr results in a mean peptide mass of 5554 Da (i.e., approximately 46-47 amino acids). The phrase "determining a composition or sequence" as used herein refers to determining the molecular mass, amino acid composition, amino acid sequence and post-translational modifications such as phosphorylation, glycosylation, carboxylation and sulfation of the peptide fragments. Methods of determining the composition or 2005/052182

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sequencing of peptides are well known in the art and include, for example, the use of mass spectrometry and protein sequencing as is further described hereinbelow. Thus, according to the method ofthe present invention live cells (i.e., cells in a cell culture, a cell line, a primary cell culture, cell and/or tissue biopsy) are subjected to a protease treatment and the resulting peptide fragments (i.e., which were present on the outer surface of the cells) which are released to the culture medium are subjected to composition or sequence analysis. The protease treatment of the present invention can be effected using any protease known in the art, including, but not limited to, prolyl endopeptidase (US Biological), Staphylococcus aureus V8 protease (Roche, #1420399), low specificity chymotrypsin (Roche), high specificity chymotrypsin (Roche, #1418467), trypsin (Promega # V5111), Asp-N (Sigma, # P3303) and Proteinase K (PK) (Sigma, #39450-01-6). For example, as is shown in Example 2 of the Examples section which follows, to dissociate the extracellular portion of the proteins present in the plasma membrane, 60-70 % confluent P19 cells cultured in 100-rnm cell culture dishes were overlayed with 3 ml of trypsin (10 μg/ml) for an incubation time of approx. 6 minutes at 37 °C until the cells were gently detached from the surface (as judged using an inverted microscope (Nikon TMS). Following protease treatment the culture medium (containing the released peptide fragments) was centrifuged for 5 minuets at 4 °C at a centrifugation speed of 500 g, following which the supernatant was centrifuged again for 5 minutes at 4 °C at 3000 g. It will be appreciated that the protease concentration, incubation time and temperature can be calibrated depending on the type of protease as well as the type of cell and the cell medium. As is mentioned hereinabove, peptide fragment length is largely dependent on the type of protease treatment used. For example, high concentrations of proteases and/or longer incubation periods result in efficient protein degradation and consequently, shorter peptides. Since the peptide fragments are further analyzed by mass spectrometry and/or protein sequencing methods and since the protease utilized to release such peptide fragments from the cells is a protein and can therefore interfere with such analysis, the protease used by the method of the present invention is preferably removed from the culture medium. Methods of removing the protease from the sample are known in the art and include for example, the use of protease inhibitors conjugated to sepharose or agarose beads via, for example, CNBr activation (Anagli J, et al., Eur. J. Biochem. 1996; 241: 948-54). Non-limiting examples of protease inhibitors which can be used along with the present invention include, but are not limited to, AEBSF (Hydrochloride, Calbiochem #101500), Antithrombin III (Human Plasma Calbiochem #169756), Aprotinin (Bovine Lung, Calbiochem #616398), Benzamidine (Hydrochloride, Calbiochem #199001), Bestatin (Calbiochem #200484), Calpeptin (Calbiochem #03- 34-0051), Chymostatin (Calbiochem #230790), E-64 Protease Inhibitor (Calbiochem #324890), EST (Calbiochem #330005), Leupeptin (Hemisulfate, Calbiochem #108975), a2-Macroglobulin (Human Plasma Calbiochem #441251), Pepstatin A (Calbiochem #516482), Na-Tosyl-Lys Chloromethyl Ketone (Hydrochloride, Calbiochem #616382), Na-Tosyl-Phe Chloromethyl Ketone (Calbiochem #16387), Trypsin Inhibitor (Soybean, Calbiochem #65035). For example, trypsin can be removed from the supernatant (which contains the released peptide fragments) using agarose beads conjugated to a trypsin inhibitor (e.g., Cat No. T0637, Sigma), essentially as described in Example 2 of the Example section which follows. Briefly, 50 μl of the agarose-conjugated trypsin inhibitor solution (Cat. No. T0637, Sigma) are added to the supernatant and incubated for 30 minutes at 4 °C while in a head over tail rotation, following which the tube is centrifuged for 5 minutes at 4 °C at a centrifugation speed of 500 g and the protease- free supernatant is transferred to a new tube. Since many extracellular membrane proteins include a polysaccharide modification (i.e., N-linked and/or O-linked glycosylation) the protease treatment employed on such proteins may result in incomplete digestion of the extracellular portion. To overcome such a limitation, a glycosidase treatment is preferably employed prior to or concurrently with the protease treatment. The glycosidase of the present invention can be any known glycosidase such as Ceramide Glycanase (Macrobdella decora, Calbiochem, EMD Biosciences, Inc, an Affiliate of Merck KGaA, Darmstadt, Germany #219484), Endo-b-galactosidase (Escherichia freundii, Calbiochem #324721), Endo-a-N-acetylgalactosaminidase (Streptococcus pneumoniae, Recombinant, E. coli, Calbiochem, #324716), b-Endo- chitinase (Recombinant, Calbiochem #220470), Endoglycoceramidase II ACT (Calbiochem #324722), Endoglycosidase D (Streptococcus pneumoniae, Calbiochem #324719), Endoglycosidase FI (Chryseobacterium meningosepticum, Recombinant, E. coli, Calbiochem #324725), Endoglycosidase F2 (Chryseobacterium meningosepticum, Recombinant, E. coli, Calbiochem #324726), Endoglycosidase F3 (Chryseobacterium meningosepticum, Recombinant, E. coli, Calbiochem #324727), Endoglycosidase H (Streptomyces plicatus, Recombinant, E. coli, Calbiochem #324717), N-Glycosidase A (Almond, Calbiochem #362180), N-Glycosidase F (Chryseobacterium meningosepticum, Calbiochem #362185), N-Glycosidase F (Chryseobacterium meningosepticum, Recombinant, E. coli, Calbiochem #362300). For example, as is shown in Example 2 of the Examples section which follows, the present inventors used the N-Glycosidase F (12500 units/ml) on P19 cells in a culture medium for a 45-rninutes incubation at 37 °C. It will be appreciated that the concentration of glycosidase used and the time and temperature of incubation depends on the type of cells, cell medium and the type of glycosidase used, and determination of optimal incubation conditions are well within the capabilities of anyone skilled with the art. Determination of the composition and sequence of the peptide fragments of the present invention requires that the preparation of the peptide fragments is amendable to common protein sequencing, chromatography and mass spectrometry analyses. However, while proteins exhibiting a short extracellular portion are readily dissolved in common detergents (e.g., Triton X-100), proteins having a long extracellular portion, with a complex three-dimensional structure (e.g., transporters, channels) often require the use of surfactants which can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry (Goshe MB, et al., 2003. J. Proteome Res. 2: 153-61). To circumvent the use of surfactants the method of the present invention preferably uses a non-permeable in vivo tagging of the cells prior to protease treatment thereof. In vivo tagging is well known in the art [see for example, (Goshe, 2003 (Supra), Blonder et al, J Proteome Res. 2002; l(4):351-60] and is based on the attachment of a molecule (i.e., the tag) to the protein. For example, a biotin molecule can be attached to cystein residues of a protein using the biotinyl-iodoacetamidyl-3,6- dioxaoctanediamine as a cysteinyl-alkylating reagent [(Goshe, 2003 (Supra)]. The use of a non-permeable in vivo tagging enables the labeling of the extracellular portion of the plasma membrane proteins and can facilitates the characterization of protein topology. Moreover, in vivo tagging can be used to retrieve the peptide fragments of the present invention using, for example, affinity binding columns or gels. According to preferred embodiments of the present invention in vivo tagging utilizes various molecules, including, but not limited to, biotin and ICAT (Smolka M, Zhou H, Aebersold R., Mol Cell Proteomics. 2002; l(l):19-29., Zhao Y, Zhang W, Kho Y, Zhao Y., Anal Chem.2004; 76(7): 1817.23). In order to analyze the peptide fragments present in the supernatant, the peptide mixture is preferably concentrated. Various approaches can be used to concentrate the peptide sample in the supernatant, including, but not limited to, protein precipitation [using e.g., acetone, trichloroacetic acid (TCA), TCA-DOC, Ethanol precipitation] followed by pellet resuspension; resin capturing [using e.g., zip-tips (C4, C-18, Cation exchange, Millipore) and/or Sep-Pack (Waters)] followed by peptide elution; and/or concentration via centrifugation [using e.g., Vivaspin (Vivascience, Germany)]. For example, as is shown in Example 2 of the Examples section which follows, the peptide - containing supernatant can be concentrated using four volumes of acetone or 12.5 % tricholoroacetic acid (TCA) for a one-hour incubation at 4 °C, following which the supernatant is centrifuged for 30 minutes at 4 °C at a centrifugation speed of 20, 000 g. It will be appreciated that prior to determination of peptide composition and/or sequencing the peptide fragments contained within the sample are preferably denatured and/or further digested using MS-grade proteases. Such a digestion can be effected using trypsin, Lys-C, chemotrypsin and/or Asp-N. For example, if the peptide fragments sample is analyzed by SDS-PAGE followed by band excision and mass spectrometry, the peptide fragments pellet (or the concentrated peptide fragments sample) is resuspended in 10-45 μl of sample buffer (Bio-Rad, #161-0737) depending of the protein concentration and approximately 10 μg of peptide sample is loaded on 1-D SDS-PAGE (gel concentration varied between 15-20 %). Bands of interest are excised from the gel and further subjected to mass spectrometry analysis using the Q-TOF mass spectrometer (Q-TOF Ultima, Micromass, United Kingdom) according to manufacturer's instructions. Alternatively, if the peptide fragments sample is analyzed by LC-MS/MS or offline LC-MALDI, the peptide fragments pellet (or the concentrated peptide fragments sample) is preferably denatured and/or further digested (using e.g., trypsin, Lys-C, and/or Chymotrypsin). According to preferred embodiments of the present invention, denaturation is achieved by resuspending the peptide pellet (or the concentrated peptide sample) in a denaturing solution containing protein denaturants such as 500 mM DTT, 350 mM β- mercapethanol, 8 M urea, 6M Guanidium Chloride, and/or 2 % SDS. For example, as is shown in Example 2 of the Examples section which follows, a pellet containing the peptide fragments sample is diluted in 1 :3 water and 1 μg/ml trypsin is added for a one-hour incubation. The trypsin is then removed using a agarose-conjugated trypsin inhibitor as described hereinabove, and the sample is denatured using 10 μl of 8 M urea and loaded on a LC C-18 column for LC-MS/MS or offline LC-MALDI analysis. It will be appreciated that in cases where SDS is used for denaturation of the peptide fragments the denatured fragments are preferably diluted prior to digestion and mass spectrometry analysis. For example, if SDS is used at a concentration range of 1 %, a 1 to 15 dilution is preferably employed. Mass spectrometry (MS) is based on measuring the molecular weight of gas- phase ions. There are currently two main MS methods which are known in the art: the matrix-assisted laser desorption/ionization (MALDI) and the electrospray ionization (ESI). MALDI is based on the incorporation of a peptide fragment to a matrix molecule followed by laser irradiation and the formation of molecular ions (M. Karas, U. Bahr, A. Ingendoh, F. Hillenkamp, Angew. Chem. 1989, 101, 805-806). Briefly, a biomolecule sample (e.g., the peptide fragments ofthe present invention) is dissolved in a solid, organic matrix (e.g., 2,5-dihydroxybenzoic acid or -cyano-4- hydroxycinnamic acid) and a laser light (e.g., a UV or IR) having a wavelength that is absorbed by the solid matrix but not by the biomolecule is used to excite the sample. Following the excitation of the matrix by the laser light, the matrix sublimes into the gas phase carrying with it the biomolecule sample. As a result, the biomolecules are ionized by a proton, electron, or a cation transfer from the matrix to the biomolecule sample. The MALDI process is typically used in conjunction with time-of-flight (TOF) analysis, known as MALDI-TOF MS and can be used to measure the molecular weights of proteins in excess of 100,000 daltons. The ESI method is based on the production of charged solvent droplets which release ions under drying conditions [J. B. Fenn, M. Mann, C. K. Meng, S. F. Wong, C. M. Whitehouse, Mass Spectrom.Rev. 9, 37 (1990)]. Briefly, a liquid sample ofthe peptide fragments of the present invention flows from a microcapillary tube into the orifice of a mass spectrometer. Such a flow is associated with the generation of a fine mist of charged droplets. Following the evaporation of the medium, charged ions are formed. Since the ESI method uses a stream of solvents it can be easily coupled to a high-pressure liquid chromatography (HPLC) instrument. The main operational mode of ESI is a nano-electrospray (flow rate is approximately 20-100 nl/min) in which a very small sample amounts can be successfully analyzed. It will be appreciated that the peptide fragments of the present invention can be also connected in line with liquid chromatography (LC) systems that automatically purify and deliver the sample to the mass spectrometer. Examples of this method are LC (Davis MT, and Lee TD, 1998. J. Am. Soc. Mass Spectrom. 9: 194-201), reverse-phase LC (RP-LC) (Gygi SP, et al., 2000. Proc. Natl. Acad. Sci. USA 97: 9390-9395) and reverse-phase microcapillary LC (RP-μLC; Deterding, LJ., et al., 1991. J. Chromatogr. 554:73-82). Multidimensional liquid chromatography (LC) is based on the separation of peptides/proteins using different chromatographic media, such as reversed phase, cation/anion exchange and hydrophobic interaction columns (Wagner et al., 2000). Combinations of such columns provide high-resolution, multidimensional separation of peptide and protein mixtures. In the on-line LC approach (Jalili PR and Dass C, Rapid Commun Mass Spectrom. 2004; 18: 1877-84), the protein mixture undergoes enzymatic treatment to produce a complex peptide mixture which is then separated using a multi-column chromatographic separation followed by tandem mass spectrometry (MS) analysis. For example, as is shown in Example 2 of the Examples section which follows, the present inventors successfully determined the presence of 65 proteins present on the outer surface ofthe membrane of P19 cells using the LC-ESI-TOF-MS. Although presently less preferred, the sequence of the peptide fragments of the present invention can be determined using protein sequencing. Protein sequencing is based on N-teiminal protein degradation (i.e., using Edman's degradation) (Edman, P. (1950) Method for determination ofthe amino acid sequence in peptides. Acta Chem. Scand. 4. 277-282 and 283-293). Since the success of N-terminal sequencing mainly depends on lack of N-terminal modifications (i.e., methionine, tryptophan), such modifications are preferably removed prior to subjecting the peptide fragments to Edman's degradation. For example, the methionine modification can be removed using cyanogen bromide (CNBr) and the tryptophan modification can be removed using skatole (Graves PR, et al., Microbiol Mol Biol Rev. 2002; 66: 39-63). Thus, to characterize the proteins present in the plasma membrane of a cell

(e.g., a mammalian cell), a cell culture containing 6 x 10⁶ cells per ml is first subjected to in vivo tagging using e.g., biotinyl-iodoacetamidyl-3,6- dioxaoctanediamine. For example, 10 μl of 100 mg/ml of biotinyl-iodoacetamidyl- 3,6-dioxaoctanediamine solution is applied on 6 x 10⁶ cells for an incubation of 15 minutes at room temperature. Following in vivo tagging, the cells are washed using e.g., PBS and are further subjected to a glycosidase treatment (N-Glycosidase F, O 2005/052182

20

Calbiochem #362300) followed by a protease treatment (Trypsin Promega, #V5111). Briefly, 12500 U/ml of the glycosidase and 1:100 enzyme-substrate ratio of trypsin are incubated with the cells at 37 °C for 45 minutes and 20 minutes, respectively. To remove the protease from the peptide fragments of the present invention, 50 μl the agarose-conjugated protease inhibitor (Sigma Cat. No. T0637) are added to the peptide fragments sample and incubated for 30 minutes at 4 °C, following which the peptide fragment sample is centrifuged for 1-5 minutes at 500 g and the protease-free peptide fragment supernatant is collected. Prior to mass spectrometry, the eluted peptide fragments are denatured with Urea (or Thiourea) at a final concentration of 8M. Following a 10-15 minutes of incubation in the presence of Urea, the denatured peptide fragments are subjected to an overnight incubation at 37 °C with 1:100 enzyme:substrate ratio of trypsin (Promega, #V5111). For LC-ESI-TOF-MS, 10-20 μl of the digested peptide fragments are subjected to HPLC separation followed by mass spectrometry analysis. Alternatively, if a tissue biopsy is to be analyzed using the teaching of the present invention, the tissue sample is first subjected to collagenase treatment (Roche, Liberase Blendzyme 3, #1814176) following which the cells are gently dissociated using e.g., a Pasteur pipette until single cell suspension are obtained. Single cell suspension can be then treated as described hereinabove. Thus, the method of the present invention can be used to characterize an extracellular membrane proteomic fraction, i.e., the overall expressed proteins present on the extracellular side ofthe plasma membrane of a cell. It will be appreciated that the present method can be used in diagnosing disorders associated with abnormal expression of proteins on the extracellular side of the membrane, such as the presence of pre-cancerous cells (e.g., mild and moderate dysplastic cells in a Pap smear specimen), cancerous cells (e.g., cervical cancer cells, colon cancer cells), cells harboring a genetic mutation (e.g., bronchial epithelial cells derived from a cystic fibrosis patient) and the like. It will be appreciated that in cases where a quantitative evaluation is needed, such as in the case of over- or under-expression of specific extracellular proteins in cancerous cells as compared with normal cells, the method of the present invention preferably uses various isotopes (e.g., ICAT) in the in vivo tagging step. Thus, peptide fragments which are attached to the in vivo tag can be compared between normal and cancerous cells using the isotope-specific mass in the MS analysis (see for example, Gygi SP, Nat Biotechnol. 1999; 17(10):994-9. Moreover, the extracellular membrane proteomics obtained using the method ofthe present invention can be used to identify drug targets which can be used in drug design for disorders associated with abnormal representation of proteins on the plasma membrane. It is expected that during the life of this patent many relevant mass spectrometry methods will be developed and the scope ofthe term mass spectrometry is intended to include all such new technologies a priori. As used herein the term "about" refers to + 10 %. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., Ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., Ed. (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (Eds.), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., Ed. (1984) "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985) "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984) "Animal Cell Culture" Freshney, R. L, Ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are beheved to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. EXAMPLE 1 DESIGN OF THE PROTEOME OF CELL EXPOSED EXTRACELLULAR DOMAINS PROCEED METHOD The PROCEED method of the present invention is based on the selective release and collection of extracellular domains of integral or peripheral membrane proteins from live cells. Experimental Methods Design ofthe PROCEED method - The principles of the PROCEED method are illustrated in Figures la-k. The method is based on subjecting live mammalian cells such as cell culture, tissue culture, tissue biopsy, yeast and/or bacteria cells to successive treatments in order to determine the topology of cell surface proteins within such cells. The most critical step in the PROCEED protocol is to achieve maximal representation ofthe plasma membrane (PM) exposed extracellular domains while avoiding damage to the cells treated. It is essential to maintain the integrity of the PM as intracellular contamination may override the selective collection of peptides that are authentic extracellular regions. Cell culture/tissue culture/yeast/bacteria (Figure la) - Live cells are cultured in tissue culture flasks using standard culturing conditions (see for example, Jennie P. Mather and Penelope E. Roberts, INTRODUCTION TO CELL AND TISSUE CULTURE: Theory and Technique, Plenum Press 1998)). In order to remove secreted and contaminating serum proteins the cells are rinsed using for example, PBS, Hank's medium, and/or Earl's Medium. Tag labeling (Figure lb) - In vivo tagging enables the labeling of the extracellular portion of the plasma membrane proteins and can facilitates the characterization of protein topology. For example, a biotin molecule can be attached to cystein residues of a protein using the biotinyl-iodoacetamidyl-3,6- dioxaoctanediamine as a cysteinyl-alkylating reagent [(Goshe, 2003 (Supra)]. In addition, in vivo tagging can be used to retrieve the released peptide fragments using, for example, affinity binding columns or gels. Glycosidase treatment (Figure lc) - Treatment with a mixture of glycosidases

(e.g., O-linked and/or N-linked glycosidase) prior to or in parallel with the "shaving" process (Figure Id) results in a limited enrichment in the amounts and composition of cleaved domains. However, the inaccessibility of glycosidases to their cleavage sites suggests that highly glycosylated domains may be under represented in the analysis. Low specificity protease treatment (Figure Id) - Proteases of choice [e.g., proline-endopeptidase, Staphylococcus aureus V8 protease, chymotrypsin (both, low and high specificity), trypsin, Proteinase K (PK)] are introduced to the medium to cleave exposed extracellular proteins in a process referred to as "shaving". The choice of proteases and the conditions used (i.e., incubation temperature, incubation time) should be calibrated to attain the desired size of peptide fragments. Interestingly, different cell types show a large variation in the conditions required for optimal "shaving", probably as a result of differences in the type and extent of glycosylation, and in the abundance of cell adhesion proteins on cell surface. Generally, "shaving" proteins from the cell membrane is performed using mild conditions, thus dissociating the exposed domains in their native conformation. In most instances, only limited cleavage by the protease occurs (Figure 2), reflecting the fact that many potential cleavage sites are masked by the high degree of glycosylation, the globular nature of extracellular domains and/or the potentially tight cell-cell and cell-matrix protein interactions. Protease inhibition and removal (Figure le) - The medium which contains peptides from extracellular domains of PM proteins is collected and the protease which was introduced during the "shaving" step (Figure Id) is *inactivated by the addition of specific inhibitors and further removed by inhibitor-immobilized affinity depletion. The resulting peptide mixture contained within the medium is a crude representation of the PM exposed domains. Due to the masking effect by associated secreted proteins many ofthe potential trypsin cleavage sites remain uncleaved. Denaturation of peptides (Figure If) — To achieve efficient cleavage, the mixture of peptides and protein fragments must be fully denatured in the presence of urea-based denaturants (8M) combined with a low level of ionic detergent (i.e., 0.1 % SDS) prior to the MS grade cleavage with trypsin. Glycosidase treatment (Figure Ig) —. A further glycosidase treatment is preferably performed as described hereinabove. Trypsin — Lys-C cleavage (Figure lh) — An MS grade cleavage using enzymes such as trypsin, Lys-C is performed following the dilution (by 3-5 fold) of the denatured peptide mixture. Chromatographic affinity separation (Figure li) - The enzyme-digested peptide sample is loaded onto a column (e.g., PepmaplOO, LC Packings) connected to an HPLC instrument (e.g., Ultimate Plus, LC Packings). Samples are then eluted by multi-step gradient elution using an acetonitrile: formic acid:ammonium acetate buffer. Mass spectrometry (Figure lj) - The eluted peptides are electrosprayed directly into an ESI-TOF-MS (Q-TOF Ultima, Micromass). Identification and Analysis (Figure Ik) - The MS/MS spectra are then analyzed using the SEQUEST and ProteinProspector software against a custom based membrane protein database. Such a database was constructed from experimentally established and predicted membrane proteins. This specialized database facilitates the protein identification and analysis by improving the match score ofthe peptides. The peptides identified as having posttranslational modifications, are then evaluated manually, including additional searches in order to intensify the confidence of the analysis. EXAMPLE! DETERMINATION OF MEMBRANE TOPOLOGY OF CELL SURFACE PROTEINS IN THE P19 CELLS To identify surface proteins on undifferentiated embryonal carcinoma P19 cells (Parnas and Linial, 1997), the PROCEED method was employed, as follows. Materials and Experimental Methods Extracellular proteolysis - P19 cells were cultured in 100-mnι cell culture dishes in the presence of DMEM tissue culture medium (Biological Industries, Israel) until 60-70 % confluence was achieved (approx. 2 days). Prior to protease and/or glycosidase treatment the culture medium was aspirated and the cells were rinsed five times with 10 ml of PBS or Hanks Medium. Glycosidase treatment - To remove protein glycosylation and to enable a wider protein coverage, the cells were treated for 45 minutes with 12500 units/ml of N-linked glycosidase (Calbiochem) and 25 mU/ml of O-linked glycosidase (Calbiochem). To remove cell debris and any protein remnant following glycosidase treatment the cell's medium was aspirated and the cells were rinsed 5 times with 10 ml of PBS. Protease treatment — To digest the extracellular membrane proteins the cells were gently overlayed with 3 ml of trypsin (10 μg/ml) and were incubated at 37 °C until all cells were detached (approx. 15 minutes). Cell detachment was evaluated using an inverted microscope Nikon TMS. The detached cells were then gently collected and further centrifuged for 5 minutes at 4 °C at a centrifugation speed of 500 g. Following centrifugation, the supernatant was gently transferred to a new tube and centrifuged again for 5 minutes at 4 °C at 3000 g following which the supernatant was transferred to a new tube. Protease inhibition - To stop the protease (i.e., trypsin) enzymatic reaction and to further remove the trypsin from the supernatant, 50 μl of the agarose- conjugated trypsin inhibitor solution (Cat. No. T0637, Sigma) were added to the supernatant and incubated for 30 minutes at 4 °C while in a head over tail rotation, following which the tube was centrifuged for 5 minutes at 4 °C at a centrifugation speed of 500 g and the protease-free supernatant was transferred to a new tube. Sample concentration - was alternatively performed using protein precipitation or resin capture. Protein concentration - Tricholoroacetic acid (TCA) to the final concentration of 12.5 % was added to the supernatant and incubated for at least 1 hour at 4 °C, following which the supernatant was centrifuged for 30 minutes at 4 °C at a centrifugation speed of 20, 000 g. Resin capture concentration - The protein sample was concentrated using zip-tips (C4, C-18, Cation exchange) or Sep-pak (Waters #WAT051910) according to the manufacturers specifications. Samples concentrated by resin were further analysed by LC-MS/MS or offline LC-MALDI as is further described hereinbelow. Peptide analysis - Two methods of peptide analysis were alternatively performed on the precipitated pellet: SDS-PAGE followed by band excision and analysis by mass spectrometry or peptide denaturation followed by trypsin treatment and analysis by LC-MS/MS or offline LC-MALDI. For SDS-PAGE the peptide pellet was resuspended in 10-45 μl of sample buffer (Bio-Rad, #161-0737) depending of the protein concentration and approximately 10 μg protein was loaded on 1-D SDS-PAGE (gel concentration varied between 15-20 %). Bands of interest were excised from the gel and were further subjected to mass spectrometry analysis (Q-Tof Ultima, Micromass, United Kingdom). For LC-MS/MS or offline LC-MALDI analysis, the pellet was resuspended in 10 μl of a denaturing solution containing 10 mM DTT in 25 mM Ammonium Bicarbonate. The sample was diluted 1:3 in 25 mM Ammonium Bicarbonate and 10 μg/ml trypsin was added for further cleavage. Following overnight incubation with trypsin, the protease was removed using the agarose-conjugated trypsin inhibitor as described hereinabove. The trypsin-free peptide solution was further subjected to LC- MS/MS or offline LC-MALDI (Ultimate Plus nano-LC system, LC Packings, Voyager DE-STR MALDI-TOF, Applied Biosystems, Q-TOF Ultima ESI-MS, Micromass). Experimental Results The PROCEED method successfully identified plasma membrane proteins from embryonial carcinoma P19 cells - Using the PROCEED method of the present invention on live embryonal carcinoma P19 cells, 61 proteins were identified with high confidence out of a total of almost 100 proteins (see Table 1, hereinbelow). In a repeating experiment utilizing the same P19 cells, the PROCEED method identified 46 out of the previously identified 61 proteins, (i.e., 70 % overlap), ensuring the reproducibility of the PROCEED method of the present invention. In the repeating experiment, 5 additional proteins were uncovered. 28

Table 1 Identification of surface proteins from live embryonal carcinoma P19 cells

29

Table 1: Proteins identified from P19 cells using the PROCEED method are presented along with their GenBank GI No. * = proteins identified in two independent PROCEED experiments.

Altogether, as is shown in Table 1 hereinabove, the PROCEED method identified with high confidence 65 proteins which are present on the plasma membrane of P19 cells. Uncovering the subcellular localization of tensin using the PROCEED method - One of the proteins identified in human cells (PC 12) using the PROCEED method (in two independent experiments) exhibits a clear membranous nature and is best matched with a putative transmembrane protein-tyrosine phosphatase (S wissprot: TPTE_HUMAN, GenBank Accession No. P56180, SEQ ID NO:26) annotated as a tensin protein (EC 3.1.3.48') and consists of 551 amino acids. Due to their phosphatase activity, tensin - related proteins are assumed to be involved in intracellular signaling, however, their subcellular localization has yet not been elucidated. The identified peptides were all associated with the most C-terminal third of the protein. A very close mouse homologues is known (gi|14787415, GenBank Accession No. CAC44243) consisting of 664 amino acids. While no information is available for the mouse homologue, the human protein is predicted to be a membrane protein, potentially function in mitosis by controlling cell division. The results obtained using the PROCEED method suggest that the human protein identified in the P19 cells is presented on the surface ofthe cell rather than on internal membranes as expected from the putative role in nuclear function. Conflicting protein topology were obtained using prior art prediction methods - When the prior art prediction methods HMMTOP [Tusnady GE, and Simon I. J Mol Biol. 1998; 283: 489-506; Tusnady GE and Simon I. Bioinformatics. 2001; 17: 849-50], SOSUI (TUAT; Tokyo Univ. of Agriculture & Technology), TMHMM (CBS; Denmark), TMAP/EMBOSS (Karolinska Institut; Sweden), TMpred (K. Hofmann & W. Stoffel, 1993; Biol. Chem. Hoppe-Seyler 374,166), TopPred 2 (Gunnar von Heijne, J. Mol. Biol. 1992; 225: 487-494) and PredictProtein (Columbia University) which are available through ExPASy Proteomics tools (http:/ /www.expasy.org/tools) were employed on the mouse TPTE homologue (GenBank Accession No. CAC44243) were employed conflicting results were obtained (Table 2, hereinbelow).

Table 2 Membrane topology predictions for the mouse tensin-related protein

Table 2: Membrane topology predictions for the mouse tensin-related protein (gi|14787415, GenBank Accession No. CAC44243) by several programs (available in http://www.expasy.org/tools ). transmembrane domain (TMD), ^bConsistency with peptides topology identified by the PROCEED protocol. Results from programs that only predict the TMD but not the membrane topology are noted by a question mark.

Since the PROCEED method of the present invention is based on an experimental set of data obtained from live cells, which thereby reflects the actual topology of cell surface proteins, such a method is far superior over prior art approaches. EXAMPLE 3 DETERMINATION OF PM PROTEINS IN YEAST SPHEROPLASTS USING THE PROCEED METHOD In order to test and tune the experimental conditions of the PROCEED method, the PROCEED protocol was applied to yeast spheroplasts. In the yeast model, the number of expected membrane proteins is limited and the topology for many of them had been verified. Annotation obtained from Gene Ontology database as well as from other yeast databases [i.e., YPD (Yeast Proteome Database] yielded a cellular compartment assignment for the majority (~76 %) of the yeast proteome (Hodges et al., 1998). Consequently, the composition for yeast membrane proteome and the subset of PMEP extracellular domains is fairly well defined. Materials and Experimental Methods Removal ofthe yeast cell wall - A yeast culture (50 ml in 500 ml) was incubated overnight at 30 °C while vigorously shaken up to an OD ₆₀ of -1.0. The cells were then centrifuged for 5 minutes at 4 °C using a centrifugation speed of ~3000 rpm, following which the cell pellet was washed with ice-cold water. The cells were washed using a cold solution of sorbitol (1 M, 50 ml), centrifuged again and resuspended in a sorbitol based solution (1 M sorbitol, 10 mM sodium phosphate, pH 7.5, 10 mM EDTA and 2 mercaptoethanol). Yeast lytic enzyme solution (ICN 152270) was added at a concentration of 50 mg/ml. The efficiency of spheroplast preparation was tested by cells diluted in SDS; efficient spheroplast formation is determined by formation of 80- 90 % "ghost" cells (i.e., cells in which the membrane is ruptured by SDS). Following removal of the cell wall, the PROCEED protocol was employed as described in Examples 1 and 2, hereinabove. Experimental Results Various proteases varying in their level of specificity were applied to yeast spheroplasts. The efficiency ofthe proline-endopeptidase, Staphylococcus aureus V8 protease, chymotrypsin (both, low and high specificity), trypsin, Proteinase K (PK) and their mutual combinations was tested in order to optimize the average size and the total amount of "shaved" fragments. Note that the yeast spheroplasts are single cells and thus lack any cell-cell or cell-matrix protection which occurs in mammalian cell adhesive cultures. Following calibration (using Immunostaining), the degree of cell disruption which was caused by the proteolytic treatment was negligible and comparable to the background level of untreated culture. Sampling the "shaved" peptides using MS/MS technology revealed many of the genuine membrane proteins (mostly transporters and ATPases). The topology of the peptides identified by the MS/MS was tested using the immunostaining and/or computational topology prediction methods and the results validated the capability of the PROCEED method to unveil not only the localization of proteins on the PM but also their expected membrane topology. The PROCEED protocol identified with high confidence 22 out of approximately 90 proteins of the yeast proteome - When the PROCEED protocol was employed on yeast cells 22 proteins were identified with high confidence. Of them, at least two proteins were confirmed in two independent experiments. Most of the yeast membrane proteins identified were combinations of a permerase (glucose), Na+/H+ antiporter and the STE6 YKL209c, an AAA representative that is related to the mating respond and uracil permease. Few of the identified proteins were not considered as membrane proteins by the prior arts. Four of such unknown proteins exhibited signals which suggest that the proteins are secreted from the cells or being released through vesicle translocation.

Table 3

Table 3: Proteins identified from yeast cells using the PROCEED method are presented along with their GenBank GI Numbers.

A full mass scan was recorded and the most prominent peptide profiles were selected for identification. In a routine scan a few hundreds well-separated peptide picks were detected and were further analyzed. Routinely, a reverse phase based was applied on hydrophobic separation, however, in instances that the initial protein mixture suggested a high complexity (as revealed by the HPLC profile), additional steps of cation and anion exchange chromatography were included. Such additional steps significantly increased the success of peptide identifications. Thus, these results demonstrate that the method ofthe present invention can be used to determine the composition ofthe plasma membrane proteins on a cell (e.g., a mammalian cell or a yeast cell). It is worth mentioning that a clear bias towards larger proteins is seen in proteins identified from both the P19 cells and the yeast cells. Analysis and Discussion The present inventors have devised a high throughput method of determining protein topology, named, the PROteome of Cell Exposed Extracellular Domains (PROCEED). The use of the PROCEED approach circumvents the need of isolating extracellular integral proteins in order to determine their presence. In addition, while during the preparation of plasma membranes (using prior art approaches) proteins which are bound by weak interactions are lost, the use of the PROCEED method retains such elusive peripheral extracellular proteins. Moreover, proteins or peptides involved in protein-protein interactions at the cell surface can be identified using the PROCEED method. In instances that peripheral proteins are undesirable, a more rigorous washing procedure can be adopted, prior to the protease 'shaving' step. Furthermore, while in most classical purification methods several tedious steps of purifications are required to reach a significant enrichment of PM relative to a crude membrane fraction, in the present method no cell disruption is involved, thus the risk of contamination is miriimized. In addition, using the PROCEED method unambiguous determination of membrane topology may be achieved even if a relatively small number of peptides are obtained from the MS/MS. This is also true for multipass membrane proteins, where an unambiguous topology can be determined based on an anchor for the 'in-out' orientation (van Geest and Lolkema, 2000). Lastly, since the outcome of the PROCEED protocol is a relatively restricted set of proteins for a specific genome, only a limited set of proteins are to be considered in the data analysis identification step. Consequently, the computerized search time and other problems in data analysis and retrieval are reduced. Altogether, in the PROCEED method live cells are exposed to an in vivo exhaustive biotin labeling using a non-permeable biotin reagent prior to protease treatment (Goshe et al., 2003). In such procedure, the biotin labels the PM proteins as well as the immediate extracellular interacting proteins. The use of PROCEED in combination with biotin labeled PM proteins increased the coverage and peptide representation of PM exposed proteins by 2-3 folds. Separation of the biotinylated proteins by one-DE (or two-DE) followed by MS/MS identification on all biotin labeled bands (or spots) was essential to confirm the membrane topology and to increase the coverage ofthe PM proteins having exposed domains. The pharmacological and clinical importance of identifying proteins that resides on cell membranes in mammalian cells led to the development of a large battery of techniques over the last 50 years. The present PROCEED method combines both classical biochemical approaches with the current advances in MS- based proteomic methodologies in order to gain knowledge on integral PM proteins. A byproduct of the PROCEED methodology is the determination of the membrane topology for proteins that are characterized by having exposed extracellular domains. Determining experimentally, the topology of an integral membrane protein is essential for developing drugs, toxins, antibodies and other pharmacological agents (Bichsel et al., 2001 ; Whitelegge and le Coutre, 2001). The potential in several areas of high throughput proteomic analysis has not been fully explored. Posttranslational modifications in particular glycosylations play an important role in functional analysis of membrane proteins (Scanlin and Glick, 1999). Although substantial difficulties are inherent, PROCEED is suitable to pursue such endeavors. Combining the elementary mode of PROCEED with in vivo tagging of the PM proteins (i.e., using biotin) turned out to be a powerful approach. Furthermore, quantitative differential expression of membrane proteins has not been attempted on a large scale. The use of membrane protein isotopic labeling technique combined with PROCEED will make this possible. The immediate motivation for such comparative study is to unveil the exposed domains of plasma membrane extracellular proteome (PMEP) in diseased versus healthy cell, in a fully differentiated versus embryonic cell, in transformed versus quiescent cell, in naϊve cells and cells exposed to an apoptotic signals or various pharmacological treatments.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. O 2005/052182

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Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

REFERENCES CITED (Additional references are cited in the text)

1. Bichsel, V.E., Liotta, L.A. and Petricoin, E.F., 3rd. (2001) Cancer proteomics: from biomarker discovery to signal pathway profiling. Cancer J, 7, 69-78.

2. Binz, P.A., Muller, M., Walther, D., Bienvenut, W.V., Gras, R., Hoogland, C, Bouchet, G., Gasteiger, E., Fabbretti, R., Gay, S., Palagi, P., Wilkins, M.R., Rouge, V., Tonella, L., Paesano, S., Rossellat, G., Karmime, A., Bairoch, A., Sanchez, J.C., Appel, R.D. and Hochsfrasser, D.F. (1999) A molecular scanner to automate proteomic research and to display proteome images. Anal Chem, 71, 4981-4988.

3. Blonder, J., Goshe, M.B., Moore, R.J., Pasa-Tolic, L., Masselon, CD., Lipton, M.S. and Smith, R.D. (2002) Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry. J Proteome Res, 1 , 351 -360.

4. Celis, J.E. and Gromov, P. (1999) 2D protein electrophoresis: can it be perfected? Curr Opin Biotechnol, 10, 16-21.

5. Collins, P.J., Juhl, C and Lognonne, J.L. (1994) Image analysis of 2D gels: considerations and insights. Cell Mol Biol (Noisy-le-grand), 40, 77-83. 6. Ferro, M., Salvi, D., Riviere-Rolland, H., Vermat, T., Seigneurin-Berny, D., Grunwald, D., Garin, J., Joyard, J. and Rolland, N. (2002) Integral membrane proteins ofthe chloroplast envelope: identification and subcellular localization of new transporters. Proc Natl Acad Sci U S A, 99, 11487-11492.

7. Ferro, M., Seigneurin-Berny, D., Rolland, N., Chapel, A., Salvi, D., Garin, J. and Joyard, J. (2000) Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis, 21, 3517-3526.

8. Fey, S.J. and Larsen, P.M. (2001) 2D or not 2D. Two-dimensional gel electrophoresis. Curr Opin Chem Biol, 5, 26-33. 9. Foster, L.J., De Hoog, CL. and Mann, M. (2003) Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A, 100, 5813-5818. /052182

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10. Goshe, M.B., Blonder, J. and Smith, R.D. (2003) Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis. J Proteome Res, 2, 153-161.

11. Han, D.K., Eng, J., Zhou, H. and Aebersold, R. (2001) Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat Biotechnol, 19, 946-951.

12. Hodges, P.E., Payne, W.E. and Garrels, J.I. (1998) The Yeast Protein Database (YPD): a curated proteome database for Saccharomyces cerevisiae. Nucleic Acids Res, 26, 68-72. 13. Hopkins, A.L. and Groom, CR. (2002) The draggable genome. Nat Rev Drug Discov, 1, 727-730.

14. Lin, D., Tabb, D.L. and Yates, J.R. (2003) Large-scale protein identification using mass spectrometry. Biochim Biophys Acta, 1646, 1-10.

15. Lognonne, J.L. (1994) 2D-page analysis: a practical guide to principle critical parameters. Cell Mol Biol (Noisy-le-grand), 40, 41-55.

16. Molloy, M.P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal Biochem, 280, 1-10.

17. Parnas, D. and Linial, M.(1997) Acceleration of neuronal maturation of P19 cells by increasing culture density. Brain Res Dev Brain Res, 101, 115-124. 18. Poutanen, M., Salusjarvi, L., Ruohonen, L., Penttila, M. and Kalkkinen, N. (2001) Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis. Rapid Commun Mass Spectrom, 15, 1685-1692.

19. Santoni, V., Molloy, M. and Rabilloud, T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis, 21, 1054-1070.

20. Scanlin, T.F. and Glick, M.C (1999) Terminal glycosylation in cystic fibrosis. Biochim Biophys Acta, 1455, 241-253. 21. Spandidos, A. and Rabbitts, T.H. (2002) Sub-proteome differential display: single gel comparison by 2D electrophoresis and mass spectrometry. J Mol Biol, 318, 21-31. 22. Taylor, S.W., Warnock, D.E., Glenn, G.M., Zhang, B., Fahy, E., Gaucher, S.P., Capaldi, R.A., Gibson, B.W. and Ghosh, S.S. (2002) An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and ID PAGE on highly purified human heart mitochondria. J Proteome Res, 1, 451-458.

23. van Geest, M. and Lolkema, J.S. (2000) Membrane topology and insertion of membrane proteins: search for topogenic signals. Microbiol Mol Biol Rev, 64, 13-33.

24. Wagner, K., Racaityte, K., Unger, K.K., Miliotis, T., Edholm, L.E., Bischoff, R. and Marko-Varga, G. (2000) Protein mapping by two-dimensional high performance liquid chromatography. J Chromatogr A, 893, 293-305.

25. Walsh, B.J. and Herbert, B.R. (1999) Casting and running vertical slab-gel electrophoresis for 2D-PAGE. Methods Mol Biol, 112, 245-253.

26. Washburn, M.P., Wolters, D. and Yates, J.R., 3rd. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol, 19, 242-247.

27. Whitelegge, J.P. and le Coutre, J. (2001) Proteomics. Making sense of genomic information for drug discovery. Am J Pharmacogenomics, 1, 29-35.

28. Wu, CC and MacCoss, MJ. (2002) Shotgun proteomics: tools for the analysis of complex biological systems. Curr Opin Mol Ther, 4, 242-250.

29. Wu, CC. and Yates, J.R. (2003) The application of mass spectrometry to membrane proteomics. Nat Biotechnol, 21, 262-267.

Claims

WHAT IS CLAIMED IS:

1. A method of characterizing proteins present in a plasma membrane of a cell, comprising: (a) subjecting a cell having an intact plasma membrane to a protease treatment to thereby obtain peptide fragments derived from proteins present in the plasma membrane of the cell; (b) determining a composition or sequence of said peptide fragments thereby characterizing the plasma membrane proteins ofthe cell.

2. The method of claim 1, wherein said determining said composition of said peptide fragments is effected using mass spectrometry.

3. The method of claim 1 , wherein said determining said sequence of said peptide fragments is effected using protein sequencing.

4. The method of claim 1, further comprising a step of labeling said proteins present in said plasma membrane with a non-permeable in vivo label prior to step (a).

5. The method of claim 4, wherein said non-permeable in vivo label is selected from the group consisting of biotin and ICAT.

6. The method of claim 1, further comprising a step of subjecting said cell to a glycosidase treatment prior to and/or concurrently with said protease treatment.

7. The method of claim 6, wherein said glycosidase treatment is effected using a glycosidase selected from the group consisting of Ceramide Glycanase, Endo- b-galactosidase, Endo-a-N-acetylgalactosaminidase, b-Endo-chitinase, Endoglycoceramidase II ACT, Endoglycosidase D, Endoglycosidase FI, Endoglycosidase F2, Endoglycosidase F3, Endoglycosidase H, N-Glycosidase A, N- Glycosidase F, and N-Glycosidase F.

8. The method of claim 1, further comprising a step of denaturing and/or digesting said peptide fragments prior to step (b).

9. The method of claim 8, wherein said denaturing is effected using a urea-based denaturant and/or an ionic detergent.

10. The method of claim 9, wherein said urea-based denaturant is 8 M UREA.

11. The method of claim 9, wherein said ionic detergent is SDS.

12. The method of claim 11, wherein said SDS is provided at a concentration range of 0.01-0.5 %.

13. The method of claim 8, wherein said digesting is effected using trypsin, Lys-C, chemotrypsin, and/or Asp-N.

14. The method of claim 8, further comprising subjecting said peptide fragments to a glycosidase treatment prior to and/or concurrently with said digesting.

15. The method of claim 14, wherein said glycosidase treatment is effected using a glycosidase selected from the group consisting of Ceramide Glycanase, Endo- b-galactosidase, Endo-a-N-acetylgalactosaminidase, b-Endo-chitinase, Endoglycoceramidase II ACT, Endoglycosidase D, Endoglycosidase FI, 2005/052182

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Endoglycosidase F2, Endoglycosidase F3, Endoglycosidase H, N-Glycosidase A, N- Glycosidase F, and N-Glycosidase F.

16. The method of claim 1, further comprising a step of subjecting said peptides to a protease inhibitor following said protease treatment.

17. The method of claim 16, wherein said protease inhibitor is selected from the group consisting of AEBSF, Antithrombin III, Aprotinin, Benzamidine, Bestatin, Calpeptin, Chymostatin, E-64 Protease Inhibitor, EST, Leupeptin, a2- Macroglobulin, Pepstatin A, Na-Tosyl-Lys Chloromethyl Ketone, Na-Tosyl-Phe Chloromethyl Ketone, and Trypsin Inhibitor.

18. The method of claim 1, wherein the cell is selected from the group consisting of a mammalian cell, a plant cell, a cyanobacteria cell, a yeast cell and a gram negative bacterial cell.

19. The method of claim 1, wherein said protease treatment is effected using a protease selected from the group consisting of proline-endopeptidase, Staphylococcus aureus V8 protease, low specificity chymotrypsin, high specificity chymotrypsin, trypsin, Proteinase K (PK), and Asp-N.

20. The method of claim 2, wherein said mass spectrometry is LC-ESI- TOF-MS.

21. A peptide composition comprising a plurality of peptide fragments each derived from an extracellular portion of a cell membrane protein.

22. The peptide composition of claim 21, wherein said peptide is labeled using a non-permeable in vivo label.

23. . The peptide composition of claim 22, wherein said non-permeable in vivo label is selected from the group consisting of biotin and ICAT. O 2005/052182 44

24. The peptide composition of claim 21, wherein the cell is selected from the group consisting of mammalian cell, a plant cell, a cyanobacteria cell, a yeast cell and a gram negative bacterial cell.

25. The peptide composition of claim 21, wherein said cell is embryonic carcinoma P19 cells and whereas said peptide composition is set forth by SEQ ID NOs: 1-25 and 27-66.

26. The peptide composition of claim 21, wherein said cell is a yeast cell and whereas said peptide composition is set forth by SEQ ID NOs:67-84.