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Enhancing Identifications of Lipid-embedded Proteins by Mass Spectrometry for Improved Mapping of Endothelial Plasma Membranes in Vivo

2009; Elsevier BV; Volume: 8; Issue: 6 Linguagem: Inglês

10.1074/mcp.m800215-mcp200

1535-9484

Yan Li, Jingyi Yu, Yipeng Wang, Noelle M. Griffin, Fred Long, Sabrina Shore, Phil Oh, Jan E. Schnitzer,

Metabolomics and Mass Spectrometry Studies

Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits. Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits. The plasma membrane provides a fundamental physical interface between the inside and outside of any cell. Beyond creating a protected compartment with a segregated, distinct, and well controlled internal milieu for the cell, it also mediates a wide variety of basic biological functions including signal transduction, molecular transport, membrane trafficking, cell migration, cell-cell interactions, intercellular communication, and even drug resistance. Plasma membrane-associated proteins, especially integral membrane proteins (IMPs) 1The abbreviations used are:IMPintegral membrane proteinLCQLCQ DecaXP mass spectrometerLTQLTQ mass spectrometer2Dtwo-dimensional1Done-dimensional3Dthree-dimensional2DCgel-free 2D LC/MS/MS on line with the LCQ DecaXP mass spectrometerG2DCgel-based 2D LC/MS/MS SDS-PAGE on line with the LCQ DecaXP mass spectrometerGRPCgel-based 1D (reversed-phase) LC/MS/MS on line with the LCQ DecaXP mass spectrometerGRPTgel-based 1D (reversed-phase) LC/MS/MS on line with the LTQ mass spectrometerECendothelial cellTMHtransmembrane helicesSTMsingle transmembrane helixMTMmultiple transmembrane helicesCLBcell lysis bufferSCXstrong cation exchangeSIspectral indexRPQrelative protein quantityRPreversed-phaseCVcoefficient of variation.1The abbreviations used are:IMPintegral membrane proteinLCQLCQ DecaXP mass spectrometerLTQLTQ mass spectrometer2Dtwo-dimensional1Done-dimensional3Dthree-dimensional2DCgel-free 2D LC/MS/MS on line with the LCQ DecaXP mass spectrometerG2DCgel-based 2D LC/MS/MS SDS-PAGE on line with the LCQ DecaXP mass spectrometerGRPCgel-based 1D (reversed-phase) LC/MS/MS on line with the LCQ DecaXP mass spectrometerGRPTgel-based 1D (reversed-phase) LC/MS/MS on line with the LTQ mass spectrometerECendothelial cellTMHtransmembrane helicesSTMsingle transmembrane helixMTMmultiple transmembrane helicesCLBcell lysis bufferSCXstrong cation exchangeSIspectral indexRPQrelative protein quantityRPreversed-phaseCVcoefficient of variation. that traverse the lipid bilayer, are key elements mediating these vital biological processes. Consistent with its fundamental importance in both normal cellular functions and pathophysiology, the plasma membrane has also been targeted extensively for biomarker discovery and drug development. In fact, more than two-thirds of known targets for existing drugs are plasma membrane proteins (1Josic D. Clifton J.G. Mammalian plasma membrane proteomics.Proteomics. 2007; 7: 3010-3029Crossref PubMed Scopus (111) Google Scholar).Despite the potential benefits, profiling the proteome of plasma membranes comprehensively using standard large scale methods including MS-based strategies has been limited and technically quite challenging. Intrinsic hydrophobicity, a wide concentration range of proteins, and other factors have hampered IMP resolution and identification using conventional two-dimensional gel electrophoresis. Gel and gel-free protein separations, including combinations of both, have been reported as an alternative to two-dimensional gel electrophoresis (2Chen E.I. Hewel J. 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Even when organellar membranes are enriched through isolation by subcellular fractionation, the yield of proteins identified has been below expectation, especially for multipass transmembrane proteins such as G-protein-coupled receptors.Here we systematically characterize four analytical approaches to enhance the identification of proteins, specifically those embedded in plasma membranes isolated directly from vascular endothelium in rat lung. Endothelial cells (ECs) constitute the tissue-blood interface that controls many important physiological functions, including tissue homeostasis, nutrition, vasomotion, and even drug delivery. In vivo mapping of the EC plasma membrane proteome provides unique opportunities for extending basic understanding in vascular biology and for directing the delivery of therapeutic and imaging agents in vivo (23Schnitzer J.E. Vascular targeting as a strategy for cancer therapy.N. Engl. J. Med. 1998; 339: 472-474Crossref PubMed Scopus (85) Google Scholar, 24Oh 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 (439) Google Scholar, 25Oh P. Borgstrom P. Witkiewicz H. Li Y. Borgstrom B.J. Chrastina A. Iwata K. Zinn K.R. Baldwin R. Testa J.E. Schnitzer J.E. Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.Nat. Biotechnol. 2007; 25: 327-337Crossref PubMed Scopus (256) Google Scholar). But it also presents distinct challenges beyond those generally associated with extraction, solubilization, and identification of IMPs in cells and tissues. ECs form a thin monolayer lining each blood vessel. They constitute a very small fraction of all the cells existing in tissue, thereby making it difficult to isolate sufficiently pure EC plasma membrane fractions for proteomics analysis using conventional subcellular fractionation techniques. Although relatively simple to isolate from tissue and grow in culture, ECs require cues from the tissue microenvironment to maintain their tissue-specific qualities and thus undergo rapid and considerable phenotypic drift after isolation (26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar).We have developed a specialized coating procedure using colloidal silica nanoparticles perfused through the blood vessels of the tissue to isolate luminal plasma membranes of the vascular endothelium as they exist natively in tissue (26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar, 27Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins.Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 28Oh P. Caver L. Schnitzer J.E. Isolation and subfractionation of plasma membranes to purify caveolae separately from lipid rafts.in: Celis J.E. Cell Biology: a Laboratory Handbook. 3rd. Elsevier, Amsterdam2006: 11-26Crossref Scopus (3) Google Scholar). Our initial survey of these plasma membranes isolated directly from rat lungs used primarily three standard analytical techniques of the time: two-dimensional electrophoresis, Western analysis, and the shotgun method of two-dimensional liquid chromatography-tandem mass spectrometry (24Oh 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 (439) Google Scholar, 26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar). We identified 450 proteins of which only ∼15% were IMPs. Although at the time this was a notable total number of proteins, more IMPs are expected. In fact, this large scale 2DC study did not identify several well known EC surface marker proteins, including specific enzymes, adhesion molecules, and growth factor receptors.Here we comparatively analyze four different MS-based strategies involving two- and three-dimensional separation by combining protein prefractionation via SDS-PAGE with in-gel digestion to produce peptides separated by one- and two-dimensional nano-HPLC before seamless and continuous MS analysis. Each method used multiple replicate measurements to comprehensively identify proteins, especially IMPs, and in doing so achieved a clear statistical definition of completeness that permits meaningful comparisons. Ultimately this analysis greatly expanded the EC plasma membranome to 1,833 proteins of which nearly 30% are membrane-embedded.EXPERIMENTAL PROCEDURESChemicals and ReagentsSequencing grade modified trypsin was purchased from Promega (Madison, WI), dithiothreitol and iodoacetamide were obtained from Pierce, ammonium bicarbonate was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ), HPLC grade Burdick & Jackson acetonitrile was purchased from VWR (Westchester, PA), and formic acid was from EMD Chemicals Inc. (Gibbstown, NJ). All other chemical reagents were obtained from Sigma-Aldrich.Isolation of Luminal Plasma Membranes from Vascular Endothelium in Rat LungSprague-Dawley female rats (150–250 g; Charles River Laboratories) were used for all experiments. Animal procedures were carried out in accordance with the Sidney Kimmel Cancer Institutional Animal Care and Usage Committee standards. Luminal vascular EC plasma membranes were isolated directly from rat lung tissues using a nanoparticle coating procedure as described previously (26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar, 27Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins.Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 28Oh P. Caver L. Schnitzer J.E. Isolation and subfractionation of plasma membranes to purify caveolae separately from lipid rafts.in: Celis J.E. Cell Biology: a Laboratory Handbook. 3rd. Elsevier, Amsterdam2006: 11-26Crossref Scopus (3) Google Scholar). Quality controls using Western analysis showed >20-fold enrichment for several known EC and plasma membrane markers as well as >20-fold depletion of markers of other cell types and subcellular organelles (26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar, 27Schnitzer J.E. McIntosh D.P. Dvorak A.M. Liu J. Oh P. Separation of caveolae from associated microdomains of GPI-anchored proteins.Science. 1995; 269: 1435-1439Crossref PubMed Scopus (454) Google Scholar, 28Oh P. Caver L. Schnitzer J.E. Isolation and subfractionation of plasma membranes to purify caveolae separately from lipid rafts.in: Celis J.E. Cell Biology: a Laboratory Handbook. 3rd. Elsevier, Amsterdam2006: 11-26Crossref Scopus (3) Google Scholar, 29Oh P. Schnitzer J.E. Segregation of heterotrimeric G proteins in cell surface microdomains. Gq binds caveolin to concentrate in caveolae, whereas Gi and Gs target lipid rafts by default.Mol. Biol. Cell. 2001; 12: 685-698Crossref PubMed Scopus (349) Google Scholar).Mass Spectrometric Analysis of Membrane ProteinsSample PreparationProteins (40 µg unless otherwise noted) in lung plasma membranes and tissue homogenates were solubilized in cell lysis buffer (CLB; 2 m urea, 0.17 m Tris base, 3 mm EDTA, 1.2% β-mercaptoethanol, and 3% SDS). The solubilized samples were boiled for 5 min and were resolved by SDS-PAGE (PAGEr gel, 8–16% T, 10 × 10 cm; Cambrex Bio Science, Inc., Rockland, ME) and visualized by colloidal Coomassie Blue staining (Invitrogen).In the early sample preparations, the gel lanes were cut into ∼50 slices for manual digestion performed in a 0.5-ml microcentrifuge tube as described in Wilm et al. (30Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.Nature. 1996; 379: 466-469Crossref PubMed Scopus (1503) Google Scholar). After purchasing a robot system (MassPrep Station II from Waters Corp., Milford, MA), ∼70 slices were cut to facilitate the in-gel digestion performed in 96-well plates according to the manufacturer's instructions. All of the gel slices were cut into ∼1-mm2 pieces for the digestions. Digested peptides were extracted from the gel slices three times with 20% acetonitrile and 10% formic acid solution. The extracted peptide fractions were transferred into 0.5-ml microcentrifuge tubes individually or combined into seven fractions before lyophilizing (Heto Vacuum Centrifuge, Appropriate Technical Resources, Inc., Laurel, MD).Strategies of MS AnalysisGRPCFor gel plus reversed-phase LC/MS/MS analysis using LCQ (Thermo Fisher Scientific, Inc., Waltham, MA) (see Fig. 1), the lyophilized peptides from each gel slice were resuspended in 10 µl of buffer A (0.1% formic acid and 5% acetonitrile) and loaded onto a self-packed C18 microcapillary column (see supplemental information) manually under a helium pressure cell with approximately 600 p.s.i. The bound peptides were eluted with 5–80% acetonitrile gradients containing 0.1% formic acid over a 60-min period as controlled by Agilent 1100 HPLC quaternary pumps (Agilent, Palo Alto, CA) directly coupled to the LCQ equipped with an ESI nanospray ion source (Micro Sprayer, Mass Evolution, TX). The flow rate was maintained at 200–250 nl/min by a precolumn flow splitter, a 50-µm fused silica capillary tube.GRPTFor gel plus reversed-phase LC/MS/MS analysis using the LTQ (Thermo Fisher Scientific, Inc.) (see Fig. 1), the lyophilized peptides from each gel slice were resuspended in 5 µl of buffer A and injected into a 5-mm trap cartridge (Dionex Corp., Sunnyvale, CA) for desalting using a FAMOS autosampler and a Switchos II system (Dionex Corp.). The desalted peptides were then back-eluted onto the analytical column (PepMap 100, C18) for the separation steps. The bound peptides were separated by a 110-min acetonitrile gradient (5–80% containing 0.1% formic acid) controlled by an UltiMate HPLC system (Dionex Corp.) directly coupled to an LTQ equipped with a Nanospray I ion source (Thermo Fisher Scientific, Inc.). The flow rate was maintained at 200–250 nl/min by an internal precolumn flow splitter.G2DCFor gel plus 2D LC/MS/MS using LCQ, the peptides extracted from each gel slice were first pooled into seven groups before lyophilization. The dried digests were resuspended with 30 µl of buffer A and then loaded manually into a two-dimensional (strong cation exchange (SCX) and reversed-phase; see supplemental information) self-packed microcapillary column under a helium pressure cell with approximately 600 p.s.i. The loaded samples were directly introduced into an LCQ equipped with ESI nanospray ion source by eluting the bound peptides with a 2D LC/MS/MS scheme (31Washburn M.P. Wolters D. Yates III, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4051) Google Scholar) controlled by Agilent 1100 HPLC quaternary pumps. Briefly 17 salt steps (ammonium acetate) were applied stepwise as 0, 10, 17.5, 25, 37.5, 50, 62.5, 75, 100, 125, 150, 175, 200, 225, 250, 375, and 500 mm over 2 min to elute the bound peptides from the SCX column. The initial step was a 100-min run with the gradient of 80 min to 60% buffer B (0.1% formic acid and 80% acetonitrile), 10 min to 100% buffer B, and 10 min to 100% buffer A. The gradients from steps 2 to 16 were 3 min at 100% buffer A, 2 min at 2 to 75% buffer C, 10 min to 15% buffer B, and a 97-min gradient to 45% buffer B. The gradient for step 17 was 2 min at 100% buffer A, 20 min at 100% buffer C, 8 min to 15% buffer B, and 110 min to 45% buffer B. The flow rate was maintained at 200–250 nl/min by a precolumn flow splitter of a 50-µm fused silica capillary.2DCFor gel-free 2D LC/MS/MS analysis using an LCQ, plasma membrane and lung tissue homogenate proteins (150 µg) were analyzed as described previously (26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar). Briefly the SDS-solubilized samples were precipitated by methanol/chloroform to remove the detergent and then resolubilized in 8 m urea buffer. In-solution enzymatic digestions were performed by both Lys-C and trypsin. The resultant peptides were desalted before lyophilization. The dried peptide mixture was resuspended in ∼30 µl of buffer A and then loaded manually into a two-dimensional (SCX and reversed-phase; see supplemental information) self-packed microcapillary column under a helium pressure cell with approximately 600 p.s.i. The loaded samples were directly introduced into an LCQ equipped with an ESI nanospray ion source by eluting the bound peptides for 2D LC/MS/MS analysis (26Durr E. Yu J. Krasinska K.M. Carver L.A. Yates J.R.I. 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 (376) Google Scholar) similar to G2DC described above.Data AcquisitionFor both the LCQ and the LTQ, MS analysis was carried out in data-dependent mode. A full MS scan of data acquisition was performed at the range of 400–1400 m/z, and one MS scan was followed by three MS/MS scans on the most abundant ions. The temperature of the ion transfer tube of both mass spectrometers was set at 180 °C, and the spray voltage was 2.0 kV. The normalized collision energy was set at 35% for both the LCQ and the LTQ. A dynamic exclusion window was applied for a duration of 3 min for reversed-phase LC/MS/MS and 10 min for 2D LC/MS/MS.BioinformaticsDatabase SearchMS/MS spectra were converted into peak lists using the default settings of Extract_msn in Xcalibur 2.0 (Thermo Fisher Scientific, Inc.), and the protein database search was performed with Sequest algorithms in Bioworks 3.1 (Thermo Fisher Scientific, Inc.) using Linux cluster. The forward and reversed databases used in the searches contained human, rat, and mouse sequences downloaded from UniProt and NCBI RefSeq (non-redundant) protein databases April 2006 (see Table I for entry details). The accession numbers for all proteins identified in the final data set were updated based on the January 2009 databases of UniProt, UniParc, and RefSeq. The false positive rate for protein identifications was determined by the ratio of the number of peptides found only in the reversed database searches to the total number of peptides in both forward and reversed database searches. Searches were performed as tryptic peptides only with two missed cleavages and precursor mass tolerance of 1.5 Da for LTQ data, 2.0 Da for LCQ data, and 0.0 Da for fragment ions in both LCQ and LTQ. No modifications were applied to our database searching. Accepted peptide identifications were based on a minimum ΔCn score of 0.1 and minimum cross-correlation scores of 1.8 (z = 1), 2.5 (z = 2), and 3.5 (z = 3). The peptides identified using these criteria were shown to have much lower mass errors compared with other Sequest scores tested (see supplemental information and supplemental Fig. 3). Protein identification results were extracted from Sequest .out files, filtered, and grouped with DTASelect software (32Tabb D.L. McDonald W.H. Yates III, J.R. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics.J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1126) Google Scholar) using the above criteria and a minimum of two unique peptides from the same measurement.Table IProteins identified using different databasesTotal entries in database usedTotal number of proteins identifiedNumber of groups of sorted proteinsNumber of unique proteins in rat databaseNumber of unique proteins in mouse databaseNumber of unique proteins in human databaseFinal identified prote