Overcoming Key Technological Challenges in Using Mass Spectrometry for Mapping Cell Surfaces in Tissues
2010; Elsevier BV; Volume: 10; Issue: 2 Linguagem: Inglês
10.1074/mcp.r110.000935
ISSN1535-9484
AutoresNoelle M. Griffin, Jan E. Schnitzer,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoPlasma membranes form a critical biological interface between the inside of every cell and its external environment. Their roles in multiple key cellular functions make them important drug targets. However the protein composition of plasma membranes in general is poorly defined as the inherent properties of lipid embedded proteins, such as their hydrophobicity, low abundance, poor solubility and resistance to digestion and extraction makes them difficult to isolate, solubilize, and identify on a large scale by traditional mass spectrometry methods. Here we describe some of the significant advances that have occurred over the past ten years to address these challenges including: i) the development of new and improved membrane isolation techniques via either subfractionation or direct labeling and isolation of plasma membranes from cells and tissues; ii) modification of mass spectrometry methods to adapt to the hydrophobic nature of membrane proteins and peptides; iii) improvements to digestion protocols to compensate for the shortage of trypsin cleavage sites in lipid-embedded proteins, particularly multi-spanning proteins, and iv) the development of numerous bioinformatics tools which allow not only the identification and quantification of proteins, but also the prediction of membrane protein topology, membrane post-translational modifications and subcellular localization. This review emphasis the importance and difficulty of defining cells in proper patho- and physiological context to maintain the in vivo reality. We focus on how key technological challenges associated with the isolation and identification of cell surface proteins in tissues using mass spectrometry are being addressed in order to identify and quantify a comprehensive plasma membrane for drug and target discovery efforts. Plasma membranes form a critical biological interface between the inside of every cell and its external environment. Their roles in multiple key cellular functions make them important drug targets. However the protein composition of plasma membranes in general is poorly defined as the inherent properties of lipid embedded proteins, such as their hydrophobicity, low abundance, poor solubility and resistance to digestion and extraction makes them difficult to isolate, solubilize, and identify on a large scale by traditional mass spectrometry methods. Here we describe some of the significant advances that have occurred over the past ten years to address these challenges including: i) the development of new and improved membrane isolation techniques via either subfractionation or direct labeling and isolation of plasma membranes from cells and tissues; ii) modification of mass spectrometry methods to adapt to the hydrophobic nature of membrane proteins and peptides; iii) improvements to digestion protocols to compensate for the shortage of trypsin cleavage sites in lipid-embedded proteins, particularly multi-spanning proteins, and iv) the development of numerous bioinformatics tools which allow not only the identification and quantification of proteins, but also the prediction of membrane protein topology, membrane post-translational modifications and subcellular localization. This review emphasis the importance and difficulty of defining cells in proper patho- and physiological context to maintain the in vivo reality. We focus on how key technological challenges associated with the isolation and identification of cell surface proteins in tissues using mass spectrometry are being addressed in order to identify and quantify a comprehensive plasma membrane for drug and target discovery efforts. Plasma membranes (PM) 1The abbreviations used are:PMplasma membraneECendothelial cellERendoplasmic reticulumSILACstable isotope labeling by amino acids in cell cultureNHSN-hydroxysuccinimideIMPintegral membrane proteinGPIglycosylphosphatidylinositol2DEtwo-dimensional electrophoresisPTMpost-translational modificationIDAWGisotopic detection of amino sugars with glutamineSIspectral indexSINnormalized spectral indexAMExaccurate mass exclusion-based data-dependent acquisition. and their associated proteins are part of a key biological interface between the outside and the inside of the cell. They are implicated in important cellular functions, such as small molecule transport, cell communication, and signaling. Such proteins are critical in sensing changes in the external environment and in transmitting signals into and out of the cell. Impaired cellular signaling, often involving PM proteins, is apparent in many cancers (1.Dhillon A.S. Hagan S. Rath O. Kolch W. MAP kinase signalling pathways in cancer.Oncogene. 2007; 26: 3279-3290Crossref PubMed Scopus (1786) Google Scholar, 2.Lallet-Daher H. Roudbaraki M. Bavencoffe A. Mariot P. Gackière F. Bidaux G. Urbain R. Gosset P. Delcourt P. Fleurisse L. Slomianny C. Dewailly E. Mauroy B. Bonnal J.L. Skryma R. Prevarskaya N. Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry.Oncogene. 2009; 28: 1792-1806Crossref PubMed Scopus (102) Google Scholar). Membrane proteins represent one-third of the proteins encoded by the human genome (3.Wallin E. von Heijne G. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms.Protein Sci. 1998; 7: 1029-1038Crossref PubMed Google Scholar) but represent more than two-thirds of the known protein targets for existing drugs (4.Rabilloud T. Membrane proteins ride shotgun.Nat. Biotechnol. 2003; 21: 508-510Crossref PubMed Scopus (85) Google Scholar). Thus, defining the proteome of PMs is critical for understanding cellular functions and fundamental biological processes and for finding new targets for drug discovery efforts. plasma membrane endothelial cell endoplasmic reticulum stable isotope labeling by amino acids in cell culture N-hydroxysuccinimide integral membrane protein glycosylphosphatidylinositol two-dimensional electrophoresis post-translational modification isotopic detection of amino sugars with glutamine spectral index normalized spectral index accurate mass exclusion-based data-dependent acquisition. In an ideal world, the PM proteome of all cell types would be analyzed comprehensively to (i) better understand why and where different membrane proteins are expressed, (ii) reveal new functions for the PM in various cell types, (iii) identify cell-specific surface-accessible markers as targetable proteins for local drug delivery, and (iv) identify diagnostic or prognostic indicators in healthy and diseased cell or tissue states. This can be attempted relatively easily in cell culture using a homogenous cell population as compared with the heterogeneity of cells within any tissue or organ from which they are derived. Unfortunately, once cells are isolated from different organs and even more so when cultured and grown in vitro, the cells can change dramatically in appearance, structure, and responsiveness, and protein expression and distribution within the cell can change (5.Madri J.A. Williams S.K. Capillary endothelial cell cultures: phenotypic modulation by matrix components.J. Cell Biol. 1983; 97: 153-165Crossref PubMed Google Scholar, 6.Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R. Testa J.E. Oh P. Schnitzer J.E. Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture.Nat. Biotechnol. 2004; 22: 985-992Crossref PubMed Scopus (366) Google Scholar). Biomarkers readily disappear, and the cultured cells do not reflect the in vivo reality. Most importantly, they lose expression of tissue-specific proteins and dedifferentiate into a more common phenotype. Mass spectrometry (MS) analysis revealed that as much as 40% of the proteins expressed by endothelial cells in vivo are not found in vitro (6.Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R. Testa J.E. Oh P. Schnitzer J.E. Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture.Nat. Biotechnol. 2004; 22: 985-992Crossref PubMed Scopus (366) Google Scholar). Thus, several groups have tried to address this problem by capturing the PMs in as close to an in vivo situation as possible as discussed below. PM proteins in general may not serve as suitable targets for drug treatment regimens mainly because of their inaccessibility to intravenously injected agents. Although most small molecule drugs can readily penetrate and accumulate in almost any tissue, often a high dose must be administered for the therapeutic dosage to accumulate in the diseased tissue of interest. However, major problems can arise when such drugs are toxic to both normal and healthy tissue, e.g. chemotherapeutic agents, which can accumulate in healthy tissue, resulting in the unwanted and often severe side effects associated with these drugs. Thus, even if a protein expressed on the surface of a cell is indicative of a disease state, it would be relatively impossible to target this cell specifically while avoiding the accumulation in healthy tissue using current drug delivery approaches, especially if the cell of interest is embedded within a tissue or organ. This is simply because multiple barriers, e.g. endothelium, epithelium, etc., must be crossed to access the cell regardless of the route of administration. As a result, great effort is now being focused on developing more targeted approaches where the toxic agents are specifically delivered to the organ or tissue of interest (7.Kaspar M. Trachsel E. Neri D. The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis.Cancer Res. 2007; 67: 4940-4948Crossref PubMed Scopus (0) Google Scholar, 8.Oh P. Li Y. Yu J. Durr E. Krasinska K.M. Carver L.A. Testa J.E. Schnitzer J.E. Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy.Nature. 2004; 429: 629-635Crossref PubMed Scopus (427) Google Scholar, 9.Sugahara K.N. Teesalu T. Karmali P.P. Kotamraju V.R. Agemy L. Girard O.M. Hanahan D. Mattrey R.F. Ruoslahti E. Tissue-penetrating delivery of compounds and nanoparticles into tumors.Cancer Cell. 2009; 16: 510-520Abstract Full Text Full Text PDF PubMed Scopus (736) Google Scholar). With such targeting approaches, dosages can be significantly reduced, thus increasing the therapeutic efficacy while minimizing the side effects (10.Carver L.A. Schnitzer J.E. Caveolae: mining little caves for new cancer targets.Nat. Rev. Cancer. 2003; 3: 571-581Crossref PubMed Scopus (214) Google Scholar, 11.Neri D. Bicknell R. Tumour vascular targeting.Nat. Rev. Cancer. 2005; 5: 436-446Crossref PubMed Scopus (509) Google Scholar). Consequently, proteins that are expressed on the surface of endothelial cells (ECs) that line the luminal surface of all vasculature are attractive targets for drugs and imaging agents as these proteins are in direct contact with the circulating blood and are thus inherently more accessible to intravenously administered agents than PM proteins of cells residing deep inside tissues and organs. Proteins expressed on the outer luminal EC surface can readily bind antibodies and other agents that are circulating in the blood. Thus, identifying and characterizing the proteins that line the vasculature of each organ and tissue is highly desirable for drug delivery and diagnostic imaging. PM proteins, regardless of the cell or tissue of origin, have generally been under-represented in proteomics analysis mainly because of their low abundance. In addition, the inherent insolubility of membrane proteins due to their hydrophobic nature has rendered them difficult to isolate and identify compared with their counterparts in the soluble cytosol and nuclear fractions. In many high throughput protein identification approaches, soluble proteomes are readily characterized, and it is fairly common for thousands of proteins to be identified in such samples. However, when more challenging proteomes, such as those of the PM, are of interest, the numbers of proteins identified are significantly lower. There are many technical reasons for such a dramatic decrease in protein identification when membrane proteomes are of interest. This review focuses on how we and other laboratories are overcoming key technological challenges associated with using traditional MS-based approaches, which were initially developed for the identification of more soluble proteins, for the mapping of membrane proteomes and in particular the in vivo cell surface proteome of endothelial cells. With the advent of mass spectrometry for shotgun protein analysis, key challenges associated with the large scale analysis of PM proteins became readily apparent. These include 1) membrane isolation and enrichment methods; 2) the subsequent solubilization of the membrane proteins, which although possible with detergents may hinder downstream MS analysis; 3) identification of membrane proteins using traditional MS approaches; 4) determining the topology of membrane proteins, which is very important for the development of antibodies, drugs, or targeting reagents; and 5) achieving comprehensive coverage of the protein content of the membrane sample, which is critical for 6) quantification and relative enrichment but requires 7) proper normalization for meaningful comparisons to be made. The following sections discuss these challenges in more detail and outline how these challenges are being addressed. PM protein enrichment through subfractionation or isolation aims to not only reduce the complexity of the sample and improve the overall dynamic range of detectable proteins but also helps overcome the low abundance issue as PMs only represent a small fraction of the cell. In addition, many PM separation techniques based on subcellular fractionation principles (e.g. density gradient centrifugation and two-phase partitioning), which use the whole cultured cell as starting material, do not appear to have the resolution to provide highly purified PM fractions without substantial contaminating membranes from other subcellular organelles, such as nuclei, Golgi, ER, and mitochondria (12.Lawson E.L. Clifton J.G. Huang F. Li X. Hixson D.C. Josic D. Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes.Electrophoresis. 2006; 27: 2747-2758Crossref PubMed Scopus (73) Google Scholar, 13.Stasyk T. Huber L.A. Zooming in: fractionation strategies in proteomics.Proteomics. 2004; 4: 3704-3716Crossref PubMed Scopus (170) Google Scholar). Therefore, many newly published PM "capture" techniques center around a similar theme where the goal is to capture, specifically label, or alternatively "shave off" the proteins protruding from the PM into the extracellular space. Some groups have explored the use of proteases floating free in solution around intact cells in culture to cleave off surface-exposed peptides. The cleaved peptides are then concentrated in the cell solution and analyzed by mass spectrometry (14.Elortza F. Mohammed S. Bunkenborg J. Foster L.J. Nühse T.S. Brodbeck U. Peck S.C. Jensen O.N. Modification-specific proteomics of plasma membrane proteins: identification and characterization of glycosylphosphatidylinositol-anchored proteins released upon phospholipase D treatment.J. Proteome Res. 2006; 5: 935-943Crossref PubMed Scopus (97) Google Scholar, 15.Rodríguez-Ortega M.J. Norais N. Bensi G. Liberatori S. Capo S. Mora M. Scarselli M. Doro F. Ferrari G. Garaguso I. Maggi T. Neumann A. Covre A. Telford J.L. Grandi G. Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome.Nat. Biotechnol. 2006; 24: 191-197Crossref PubMed Scopus (337) Google Scholar, 16.Speers A.E. Blackler A.R. Wu C.C. Shotgun analysis of integral membrane proteins facilitated by elevated temperature.Anal. Chem. 2007; 79: 4613-4620Crossref PubMed Scopus (69) Google Scholar, 17.Tjalsma H. Lambooy L. Hermans P.W. Swinkels D.W. Shedding & shaving: disclosure of proteomic expressions on a bacterial face.Proteomics. 2008; 8: 1415-1428Crossref PubMed Scopus (93) Google Scholar). Although the method appears intuitive, there may be problems relating to the lysis of the cells by the proteases, thus contaminating the membrane "peptide" solution with intracellular proteins. An alternative approach is the cross-linking of PM protein complexes where specific reagents are used to maintain protein complexes in their close to native state (18.Freed J.K. Smith J.R. Li P. Greene A.S. Isolation of signal transduction complexes using biotin and crosslinking methodologies.Proteomics. 2007; 7: 2371-2374Crossref PubMed Scopus (11) Google Scholar, 19.Gubbens J. Ruijter E. de Fays L.E. Damen J.M. de Kruijff B. Slijper M. Rijkers D.T. Liskamp R.M. de Kroon A.I. Photocrosslinking and click chemistry enable the specific detection of proteins interacting with phospholipids at the membrane interface.Chem. Biol. 2009; 16: 3-14Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The cells are subsequently lysed, and the non-complexed proteins can be removed by various methods, including size exclusion chromatography. Over the past 30 years, numerous chemical techniques have been developed to label surface proteins of cells. The majority of these methods were developed in cell culture systems before the advent of mass spectrometry permitted large scale protein identification. In one such approach, lactoperoxidase-catalyzed iodination was carried out with epithelial cells to label cell sheets with 125I. The iodination was carried out under conditions that allowed little penetration of lactoperoxidase into the cells, which restricted iodination to the cell surface, and thus, membrane-labeled 125I could therefore provide an effective marker for following PM fragments through subcellular fractionation. This labeling strategy was used to differentiate between apical and basal-lateral PMs in epithelial cell polarization studies (20.Lewis B.A. Elkin A. Michell R.H. Coleman R. Basolateral plasma membranes of intestinal epithelial cells. Identification by lactoperoxidase-catalysed iodination and isolation after density perturbation with digitonin.Biochem. J. 1975; 152: 71-84Crossref PubMed Google Scholar, 21.Rodriguez H.J. Edelman I.S. Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium.J. Membr. Biol. 1979; 45: 215-232Crossref PubMed Scopus (0) Google Scholar). This approach was later applied to label surface proteins of EC PMs in vitro and was most often used in combination with lectin affinity chromatography to isolate and characterize glycoproteins (22.Schnitzer J.E. Carley W.W. Palade G.E. Albumin interacts specifically with a 60-kDa microvascular endothelial glycoprotein.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 6773-6777Crossref PubMed Google Scholar, 23.Schnitzer J.E. gp60 is an albumin-binding glycoprotein expressed by continuous endothelium involved in albumin transcytosis.Am. J. Physiol. Heart Circ. Physiol. 1992; 262: H246-H254Crossref PubMed Google Scholar, 24.Schnitzer J.E. Shen C.P. Palade G.E. Lectin analysis of common glycoproteins detected on the surface of continuous microvascular endothelium in situ and in culture: identification of sialoglycoproteins.Eur. J. Cell Biol. 1990; 52: 241-251PubMed Google Scholar). This approach was adapted for the first mapping of the EC surface in tissue (24.Schnitzer J.E. Shen C.P. Palade G.E. Lectin analysis of common glycoproteins detected on the surface of continuous microvascular endothelium in situ and in culture: identification of sialoglycoproteins.Eur. J. Cell Biol. 1990; 52: 241-251PubMed Google Scholar, 25.Merker M.P. Carley W.W. Gillis C.N. Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung.FASEB J. 1990; 4: 3040-3048Crossref PubMed Scopus (9) Google Scholar). In situ radioiodination of pulmonary endothelial plasma membranes was performed by perfusing intact lungs via the pulmonary artery with lactoperoxidase-conjugated beads. This method could be used to verify protein expression in vivo versus cell culture. Although this radioiodination approach helped identify a small number of PM proteins, its utility for large scale protein mapping is significantly limited as there is no simple way to separate and identify radiolabeled proteins. Nonetheless, lectin affinity principles were still used to pull out membrane glycoproteins (26.Ghosh D. Beavis R.C. Wilkins J.A. The identification and characterization of membranome components.J. Proteome Res. 2008; 7: 1572-1583Crossref PubMed Scopus (28) Google Scholar, 27.Kullolli M. Hancock W.S. Hincapie M. Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins.J. Sep. Sci. 2008; 31: 2733-2739Crossref PubMed Scopus (69) Google Scholar, 28.Vercoutter-Edouart A.S. Slomianny M.C. Dekeyzer-Beseme O. Haeuw J.F. Michalski J.C. Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells.Proteomics. 2008; 8: 3236-3256Crossref PubMed Scopus (62) Google Scholar) in the absence of radiolabeling as the vast majority of proteins expressed on the surface of the PM are post-translationally modified by the addition of glycans to asparagine residues. This fact has been exploited by many groups to isolate glycoproteins prior to mass spectrometry analysis. Other methods for the selective isolation, identification, and quantification of peptides that contain N-linked glycoproteins have emerged and are based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides, and the specific release of formerly N-linked glycosylated peptides via peptide-N-glycosidase F. The recovered peptides are then identified and quantified by MS/MS (29.Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1195) Google Scholar). Alternative glycolabeling enrichment chemistries include the oxidation of cell surface polysaccharides on living cells combined with subsequent biocytin hydrazide labeling (30.Wollscheid B. Bausch-Fluck D. Henderson C. O'Brien R. Bibel M. Schiess R. Aebersold R. Watts J.D. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.Nat. Biotechnol. 2009; 27: 378-386Crossref PubMed Scopus (374) Google Scholar), but such approaches have been developed for cell culture and have not yet been applied in vivo. Several approaches have been developed that introduce a stable isotope to a given glycan via chemical derivatization (31.Bowman M.J. Zaia J. Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry.Anal. Chem. 2007; 79: 5777-5784Crossref PubMed Scopus (89) Google Scholar, 32.Xie Y. Liu J. Zhang J. Hedrick J.L. Lebrilla C.B. Method for the comparative glycomic analyses of O-linked, mucin-type oligosaccharides.Anal. Chem. 2004; 76: 5186-5197Crossref PubMed Scopus (33) Google Scholar). Glycans are typically derivatized prior to analysis either by tagging the reducing terminus with a chromophore when subsequent analyses are chromatographic or by permethylation when the sample is to be analyzed by MS. A typical work flow for these in vitro labeling approaches involves the parallel release of glycans from the sample populations under investigation and derivatization with an isotopic label after which the samples are mixed and then analyzed by MS. In vivo labeling strategies have also been described. One such approach is called IDAWG, isotopic detection of amino sugars with glutamine (33.Orlando R. Lim J.M. Atwood 3rd, J.A. Angel P.M. Fang M. Aoki K. Alvarez-Manilla G. Moremen K.W. York W.S. Tiemeyer M. Pierce M. Dalton S. Wells L. IDAWG: metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells.J. Proteome Res. 2009; 8: 3816-3823Crossref PubMed Scopus (91) Google Scholar), which is similar in principle to SILAC (34.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4225) Google Scholar). The main source of nitrogen for amino sugars in the production of sugar nucleotides is the side chain of glutamine; hence, isotopically labeled glutamine can be added as the sole source of glutamine for the cells, meaning that it will be incorporated into all N- and O-linked glycans. However, as with SILAC approaches, the cost of the reagents as well as incomplete incorporation of the heavy labeled amino acid into all the glycans can limit its application. The next development evolved to allow both labeling and capturing of surface-expressed proteins by exploiting biotin-avidin chemistry to isolate cell surface membrane proteins. The membrane proteins are biotinylated on amino acid residues located in extracellular domains and subsequently enriched using magnetic streptavidin beads. There were numerous reports in the early 1980s describing biotinylation of cell surface proteins from plants and microorganisms (35.Simpson G.L. Born J.L. Cain G. Growth of human malaria parasites in biotinylated erythrocytes.Mol. Biochem. Parasitol. 1981; 4: 243-253Crossref PubMed Scopus (3) Google Scholar, 36.Grimes H.D. Slay R.M. Hodges T.K. Plant plasma membrane proteins: II. biotinylation of Daucus carota protoplasts and detection of plasma membrane polypeptides after SDS-PAGE.Plant Physiol. 1988; 88: 444-449Crossref PubMed Google Scholar). This approach was later applied to mammalian cells in culture. Initial reports described its application to epithelial cell polarization studies to differentiate between apical and basal-lateral membranes. The cells were grown on filters and then incubated with the biotin reagents to facilitate apical surface labeling, and therefore determine polarized expression of proteins (37.Sargiacomo M. Lisanti M. Graeve L. Le Bivic A. Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells.J. Membr. Biol. 1989; 107: 277-286Crossref PubMed Scopus (0) Google Scholar). Zhang et al. (38.Zhang W. Zhou G. Zhao Y. White M.A. Zhao Y. Affinity enrichment of plasma membrane for proteomics analysis.Electrophoresis. 2003; 24: 2855-2863Crossref PubMed Scopus (81) Google Scholar) and others (39.Zhao Y. Zhang W. Kho Y. Zhao Y. Proteomic analysis of integral plasma membrane proteins.Anal. Chem. 2004; 76: 1817-1823Crossref PubMed Scopus (219) Google Scholar) (for a review, see Ref. 40.Elia G. Biotinylation reagents for the study of cell surface proteins.Proteomics. 2008; 8: 4012-4024Crossref PubMed Scopus (130) Google Scholar) later adapted this affinity enrichment method for proteomics analysis. It combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads for the isolation of cell surface proteins from cultured cells. The authors showed enrichment of PMs relative to other cellular organelles, Zhao et al. (39.Zhao Y. Zhang W. Kho Y. Zhao Y. Proteomic analysis of integral plasma membrane proteins.Anal. Chem. 2004; 76: 1817-1823Crossref PubMed Scopus (219) Google Scholar) were able to detect 898 proteins, including a significant number of proteins identified with only 1 peptide using this method, with 781 of them being annotated as PM-localized. Although this approach gave a good first approximation of proteins at the cell surface, cytoskeletal and intracellular proteins were also observed in these preparations. This is not surprising as biotin reagents are fairly small and can cross lipid membranes to get into cells, and thus proteins within the cells can become labeled. However, the use of charged or polar biotinylation reduces the entry of the reagents into the cells, but cytoskeletal proteins are still often detected (40.Elia G. Biotinylation reagents for the study of cell surface proteins.Proteomics. 2008; 8: 4012-4024Crossref PubMed Scopus (130) Google Scholar). Modifications to the biotin approach in which the probe is membrane-impermeable have also been described (41.Zhang H. Brown R.N. Qian W.J. Monroe M.E. Purvine S.O. Moore R.J. Gritsenko M.A. Shi L. Romine M.F. Fredrickson J.K. Pasa-Toliæ L. Smith R.D. Lipton M.S. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with (18)O labeling.J. Proteome Res. 2010; 9: 2160-2169Crossref PubMed Scopus (0) Google Scholar). In this study, a commercially available sulfo-NHS-SS-biotin probe was used in combination with 18O labeling for quantitative analysis of cell surface membrane proteins. There are various sulfo-NHS-biotin ester reagents available with varying properties and spacer arm lengths. They are all polar and water-soluble, thus reducing their ability to penetrate cell membranes. Alternatively, membrane-impermeable thiol-reactive biotins (e.g. maleimido-biotin) are also available for the labeling of membrane proteins where the usual amine labeling by biotin is undesirable. However, whether all PM proteins are biotinylated similarly remains unclear as different biotinylation reagents preferentially target different reactive groups; for example, some reagents target primary amino groups, which are abundant in the form of lysine side chain ε-amines, and others target free sulfhydryl groups found in cysteine residues (40.Elia G. Biotinylation reagents for the study of cell surface prote
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