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Proteomic Analysis of Surface and Endosomal Membrane Proteins from the Avian LMH Epithelial Cell Line Lei Zhang,†,^ George S. Katselis,‡,^,§ Roger E. Moore,‡ Kossi Lekpor,‡ Ronald M. Goto,† Terry D. Lee,‡ and Marcia M. Miller*,† †
Department of Molecular and Cellular Biology and ‡Department of Immunology, Beckman Research Institute, City of Hope, 1500 E. Duarte Road, Duarte, California 91010-3000, United States
bS Supporting Information ABSTRACT: Proteins at the cell surface and within the endocytic pathway are increasingly being recognized for their roles in a wide variety of intercellular interactions. Here we used the inherent hydrophobicity and N-glycosylation of membrane proteins to enrich these proteins from the surface and endosome of avian LMH epithelial cells for mass spectrometric analysis. The cycling of many different types of proteins from the cell surface into the endosome and sometimes back to the surface again makes it appropriate to analyze these two membranous cellular components together. Stringent searches of the International Protein Index (IPI) entries for Gallus gallus identified 318 unique integral membrane proteins (IMPs) (201 bearing N-glycosylation sites), 265 unique membrane-associated proteins (MAPs), and an additional group of 784 non-membrane proteins (NMPs) among TX-114 detergent and aqueous phase-enriched proteins. Capture of N-glycosylated tryptic peptides revealed 36 additional glycoproteins most of which were CD antigens, receptors, and molecules for cell adhesion and immune response. IMPs and MAPs present at the surface and within the endosome included proteins involved in transport (255), metabolism (285), communication (108), adhesion (47), and immune responses (42). Among these were 355 putative uncharacterized and hypothetical IMPs, MAPs, and NMPs for which highly similar annotated sequences were found in standard protein protein BLAST searches. KEYWORDS: Gallus gallus, membrane proteins, cell surface, endosome, TX-114 extraction, tryptic glycopeptide capture
’ INTRODUCTION Proteins located at the surface and within the tubulo-vesicular network of the cellular endosome (endoplasmic reticulum, early endosomes, late endosomes, multivesicular bodies, recycling endosomes, autophagic vesicles, lysosomes, and Golgi apparatus) are vital to cellular functions. Glycosylated integral membrane proteins (IMPs) and membrane-associated proteins (MAPs) are common at these locations, serving as transporters, receptors, and ligands sustaining cellular processes and tissue functions, including immune responses. Invading pathogens often use membrane proteins as tethers and portals to gain access to the endosome and the cytoplasm. Detailed knowledge of the proteins displayed at the cell surface and within the endosome of various cell types is of importance in understanding the molecular basis of cell interactions under normal and disease challenge conditions.1 Knowledge of which proteins are present on the surface and in the endosome of cell lines used in laboratory investigations can be valuable in experimental design and interpretation. IMPs and MAPs vary in abundance between tissues and will likely be found to vary between individuals as the result of genetic and epigenetic modifications. Because of the low relative abundance of IMPs and MAPs compared to the quantity r 2011 American Chemical Society
of cytoplasmic and nuclear proteins in many cell types, it is challenging to experimentally define membrane protein proteomes for any given cell type or tissue. A variety of enrichment methods are applied to the study of membrane proteins originating from the cell surface and intracellular organelles. Top-down proteomics approaches for separating and analyzing individual spots from two-dimensional gels have proven not particularly well-suited for defining arrays of membrane proteins.2 Shot-gun approaches are often more inclusive in enriching membrane proteins from whole cells or subcellular fractions.3 6 Some shot-gun methods focus only on cell surface IMPs with multiple steps taken to remove or shave away nuclear and other internal IMPs (from endosome and mitochondria) and membrane-associated proteins prior to mass determinations.7 9 Others enrich cell surface IMPs and associated proteins by biotinylation and capture on streptavidincoated resins.10 Lectins are often used in combination with additional fractionation methods for enrichment of N-glycosylated proteins.11 16 Filter-aided sample preparation allows solubilization Received: February 27, 2011 Published: July 21, 2011 3973
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IPI as either hypothetical or putative uncharacterized to identify the most similar NCBI protein reference sequence.
’ EXPERIMENTAL PROCEDURES Cells Culture and Disruption
One million chicken LMH cells37 were seeded in complete medium (30 mL; DMEM containing 10% FBS, 1% Pen/Strep, 1% glutamine) in each of ten 150 cm2 Corning T flasks (Costar Corning, New York, NY). After incubation (72 h, 39 C) in a humidified 5% CO2 environment, the ten flasks yielded 4 108 cells as the starting material in each preparation (Figure 1). Cells were gently loosened from flask surfaces using a Cell Lifter (Costar Corning, NY) and were collected, washed with PBS, and pelleted (500 g for 10 min, 4 C). The cell pellet was resuspended in 4 mL homogenization buffer (10 mM acetic acid, 1 mM EDTA, 190 mM sucrose, 10 mM triethanolamine, pH 7.4, containing 1 mM PMSF, and protease inhibitors [Complete Protease Inhibitor Cocktail Tablets, Roche Applied Science, Mannheim, Germany, prepared as directed]). Cells were sheared using a 10 mL syringe fitted with a 25-gauge needle by repeatedly drawing up and expelling the cell suspension. Aliquots were mixed with equal volumes of trypan blue and examined by light microscopy to monitor cell breakage. Disruption was deemed complete when few intact cells remained (typically after 30 passes). Any remaining intact cells, nuclei, and large cell fragments were removed by centrifugation (3000 g for 10 min, 4 C) and postnuclear supernatant (PNS) collected. Enrichment by TX-114 Detergent Phase Separation Figure 1. Work flow for the proteomic analysis of LMH cell membrane proteins acquired through the inherent properties of relative hydrophobicity (TX-114 extraction and fractionation) and N-glycosylation (resin capture of tryptic glycopeptides).
of entire cells in SDS, providing the potential for comprehensive analysis of the cellular membrane protein proteome.17,18 Surface membrane protein-specific methods, such as using hydrazide chemistry and bifunctional linkers, allow capture of intact glycoproteins and tryptic glycopeptides.19 23 The inherent hydrophobicity of amphipathic membrane proteins is another means for partitioning membrane proteins that is particularly well suited for the study of membrane proteins originating from the cell surface and intracellular organelles, including the endosome.24 32 LMH cells are often used in studies of avian and human viral and bacterial pathogens.33 36 With the goal of identifying cell surface and endosomal membrane proteins of the LMH cell line, we used TX-114 phase partitioning24 to enrich inherently hydrophobic transmembrane domains. In parallel preparations we captured N-glycosylated tryptic peptides through hydrazidebased chemistry (proteins were proteolyzed prior to periodate oxidation and resin coupling)23 to identify N-glycosylated proteins destined for the endosome and cell surface. Tandem mass spectra data for the peptides isolated by these two methods were generated and matched with proteins using the X! Tandem modeler and a custom database generated from the International Protein Index (IPI) entries for Gallus gallus. IMPs and MAPs were classified structurally with regards to N-glycosylation sites, transmembrane helices, and gene ontology. Attributes of the individual proteins provided insights into the subcellular origin of each. BLAST searches were performed for all proteins listed in
The PNS was extracted by adding precondensed TX-114 (poly(ethylene glycol) tert-octylphenyl ether, Sigma-Aldrich, St. Louis, MO; stock no. X114) containing EDTA-free protease inhibitor cocktail at 0 C, as previously described.24,25 The volume was adjusted to bring the final concentration of TX-114 to 2%. The mixture was stirred gently (30 min, on ice), then centrifuged (10,000 g for 10 min, 4 C) to remove insoluble components, and the PNS-TX-114 supernatant was removed to a new tube. Postextraction buffer (PEB) (0.2 mM EDTA, 5 mM MgCl2, 200 mM NaCl, and 40 mM Tris-HCl, pH 7.5) was mixed at a 0.45:1 ratio with the PNS-TX-114 supernatant. To induce phase separation, the mixture was incubated (5 min, 37 C) and then centrifuged (4,500 g, 3 min, 25 C). The upper, aqueous phase was removed to another tube without disturbing the interface. The lower, detergent phase was re-extracted five times using the same (0.45:1) ratio of PEB to detergent phase for each re-extraction. After the fifth re-extraction, proteins in the detergent phase were precipitated by adding trichloroacetic acid (TCA) in acetone to a final concentration of 20% (w/v) and storing the mixture overnight at 20 C. The aqueous phases for each re-extraction were pooled, and the proteins were precipitated by adding TCA to bring the final TCA concentration to 20%. After centrifugation (13,000 g, 20 min, 4 C) the TCA-precipitated proteins formed a visible pellet in both the detergent and aqueous phase samples. The pellets were washed twice with 90% v/v acetone, spun in a Savant SpeedVac concentrator until nearly dry, and then dissolved in NuPAGE LDS sample buffer containing dithiothreitol (Invitrogen, Carlsbad, CA). Protein concentrations were determined using the BCA (bicinchoninic acid) Protein Assay (Thermo Fisher Scientific, Rockford, IL). 3974
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Proteins were fractionated on one-dimensional gels (NuPAGE 4 12% Bis-Tris precast gels [1.5 mm thick]) with a maximum of 100 μg protein loaded per lane in an XCell Surelock Mini-Cell (Invitrogen). Gels were run (100 V, 90 min) in NuPAGE MES buffer (Invitrogen). Proteins were visualized by staining gels overnight in SimplyBlue SafeStain (Invitrogen) followed by destaining in water. Individual lanes were cut into strips (∼1.5 mm 10 mm), cut again crosswise, and placed in separate wells in a Montage Zip Kit-96 Well Plate (Millipore, Billerica, MA). The strips were further destained (room temperature, 30 min) first in 25 mM ammonium bicarbonate containing 5% acetonitrile and then in 25 mM ammonium bicarbonate containing 50% acetonitrile (150 μL/well). The destained gel fragments were incubated (10 min) in 100% acetonitrile (200 μL/well). The immobilized proteins were reduced with 10 mM dithiothreitol (50 μL/well) at 56 C for 60 min and alkylated with 100 mM iodoacetamide (50 μL/well) in the dark for 60 min at room temperature. The gel fragments were then incubated at room temperature with 100 mM ammonium bicarbonate (100 μL/well) followed by incubation with two changes of acetonitrile (100 μL/well, 5 min each time). A gentle vacuum (∼10 in. Hg) was applied to completely remove excess solution at each of these steps. Then sequencing grade modified trypsin (15 μL, 11 ng/μL trypsin in 100 mM ammonium bicarbonate; Promega, Madison, WI) was added to each well, and the proteins were digested (overnight, 30 C). Following this step, ∼10 μL/well acetonitrile was added to wet the bottom of each well. The resulting peptides were then extracted from the gel fragments by addition of 1% formic acid (130 μL/well) and incubation (at room temperature, 30 min). The peptides were captured on C18 media at the bottom of the wells by applying vacuum (∼8 in. Hg) for 10 min. Captured peptides were washed twice with 1% formic acid (100 μL/well) and then eluted under low vacuum into a receiver plate using 1% formic acid/50% methanol solution (25 μL/well). Methanol was completely removed from the peptide solution by evaporation (3 min) in a SpeedVac concentrator. Enough 1% formic acid was then added to bring the final volume to ∼20 μL. The peptides were then stored for not more than 3 days at 20 C before LC MS/MS analysis.
reaction, and the mixture was diluted 10-fold with 40 mM Tris buffer, pH 8.3. Sequencing grade trypsin was added at a 1:25 ratio (trypsin to protein), and the solution was incubated (37 C, overnight). The PPS surfactant was then hydrolyzed by adding HCl to a final concentration of 250 mM and incubating at room temperature for 1 h. Insoluble components were pelleted by centrifugation (6000 g, 20 min), and the supernatant was removed. The supernatant was then cleared of urea, DTT, and Tris buffer by passage through a Sep-Pak C18 column (Waters, Milford, MA). Tryptic peptides were eluted from the column with 80% acetonitrile and 0.1% trifluoroacetic acid (TFA) in water and dried in a SpeedVac concentrator. The dried tryptic peptides were dissolved at an estimated final concentration of 1 mg/50 μL in coupling buffer (100 mM sodium acetate, 150 mM NaCl, pH 5.5), and any insoluble matter was removed by centrifugation. The supernatant was incubated in the dark with sodium periodate at a final concentration of 10 mM to oxidize the cis-diol groups of carbohydrates to aldehydes. After the peptide solution was mixed (end-over-end rotation, 30 min, room temperature), sodium sulfite was added to a final concentration of 20 mM, and the incubation was continued for 10 min to remove excess sodium periodate. The coupling reaction was started by adding hydrazide resin (Bio-Rad, Hercules, CA) into the quenched peptide solution at ratio of 1 mL resin to 20 mg peptide. Additional coupling buffer was then added to bring the final ratio of resin to liquid to 1:5. The coupling reaction was allowed to continue at 37 C overnight with end-over-end rotation. The resin was washed first twice with deionized water (600 μL), twice with 1.5 N NaCl (600 μL), twice with methanol (600 μL), and finally twice with acetonitrile (600 μL). The resin was then resuspended in 100 mM ammonium bicarbonate, pH ∼8.0 (600 μL), and the peptides released from the resin by digestion (overnight, 37 C) with PNGase F (peptide: N-glycosidase F [EC 3.5.15.2, N-linked-glycopeptide-(N-acetylβ-D-glucosaminyl)-L-asparagine amidohydrolase], Sigma-Aldrich, St. Louis, MO; 1 μL [500 units] PNGase F solution per 2 mg of peptide). The resin was pelleted by centrifugation and rinsed twice with 100 mM ammonium bicarbonate (100 μL). The supernatant and the two washes containing the released peptides were combined and then concentrated to near dryness in a SpeedVac concentrator and stored for not more than 3 days at 20 C until LC MS/MS analysis.
Tryptic Glycopeptide Capture
Liquid Chromatography and Mass Spectrometry Analysis
Digestion and Fractionation of the TX-114 Detergent and Aqueous Phase Proteins
PNS were prepared as described above from 4 108 cells. The PNS was centrifuged (140,000 g, 2.5 h) (Beckman Ti 50 rotor) to pellet the small membranous components (the microsomal fraction) produced during cell disruption and to separate these from soluble proteins. The yield was ∼1.0 mg of crude microsomal membrane proteins, as determined by BCA protein assay. Using previously described methods23 with a few modifications, the pelleted sample was resuspended in denaturing buffer (200 μL 5 mM EDTA, 10 mM TCEP, 0.2% PPS Silent Surfactant [sodium 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate; Protein Discovery, Inc., Knoxville, TN] in 40 mM Tris buffer, pH 8.3). The sample was heated (100 C, 10 min) and allowed to cool to room temperature. Dry urea was added to a final concentration of 8 M, and the sample was incubated (37 C, 30 min). Iodoacetamide was added to a final concentration of 20 mM, and the sample was incubated in the dark (room temperature, 30 min). Dithiothreitol (DTT) was then added to a final concentration of 10 mM to quench the alkylation
Peptides generated by both preparative methods were analyzed in single trials using an Eksigent NanoLC-2D HPLC (Eksigent Technologies, Dublin, CA) coupled to a LTQ-FT hybrid ion-trap/ICR mass spectrometer equipped with a 7 T magnet (Thermo Electron, San Jose, CA). Aliquots (10 μL) of each sample were loaded onto a Zorbax 300SB-C18, 5 μm, 5 mm 0.3 mm enrichment column (Agilent Technologies, Palo Alto, CA), and peptides were washed for 5 min with 99% Solvent A (water with 0.1% formic acid) and 1% Solvent B (90% acetonitrile with 0.1% formic acid) at 10 μL/min. Peptide separation was performed in a fused silica capillary tubing (11.5 cm, 75 μm i.d. 363 μm o.d.; Polymicro Technologies, Phoenix, AZ) with an integrated electrospray emitter and packed with Pursuit C18 reverse phase packing material (3 μm, Varian, Inc., Palo Alto, CA). Peptides were eluted, at a flow rate of 200 nL/min, using a linear ramp from 1% solvent B to 10% Solvent B over 3 min, followed by ramps to 40% Solvent B over 42 min, 55% solvent B over 4 min, and 98% Solvent B over 2 min, followed by 3 min at 3975
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Journal of Proteome Research 98% Solvent B, a rapid ramp back to 1% Solvent B and a reequilibration time of 5 min. A 1.5 kV electrospray voltage was applied upstream of the column, and the eluate was sprayed directly from the end of the column into the mass spectrometer. Survey spectra were collected in profile mode over 400 2000 m/z. Automated peak recognition, dynamic exclusion, and selection of the remaining top five most intense precursor ions for tandem MS in the ion trap from each high-resolution ICR spectrum at 35% normalization collision energy were performed using Xcalibur software (v 2.0.7, Thermo Fisher Scientific Inc., San Jose, CA). Mass Spectrometry Database Searches
The spectra raw files were converted to mzXML format using ReAdW (http://sashimi.sourceforge.net) and processed sequentially through X! Tandem Tornado to assign peptide sequences using the Global Proteome Machine (GPM; v 2009.04.01.1, ftp://ftp.thegpm.org). Peptide identification was determined using a 0.3 Da fragment mass error and a parent mass error of (10 ppm assuming full tryptic specificity with up to one missed cleavage. Carbamidomethylation of cysteine was considered as a complete modification. Methionine oxidation, pyroglutamic acid formation at the peptide amino terminus, and formylation of the protein amino terminus were also allowed as potential modifications. When searching for glycopeptides, deamidation of asparagine was also allowed as a potential modification. Spectra were searched against a custom database that included Gallus gallus proteins in the IPI chicken v3.66 database38 (22,194 entries; April 26, 2010) and a database of 112 common contaminant proteins. The reverse sequences of the custom database (called the decoy database) were appended for calculation of the false discovery rate (FDR). Spectra from each gel band were merged into a bigger set, and the output data set was searched with the X! Tandem search engine. The FDRs within the detergent-rich and aqueous data sets were estimated using the formula, FDR = (2 decoy matches/total matches) 100. Protein matches are reported at an estimated FDR of 5%, except for the glycopeptide searches. Glycopeptide identifications were validated if the N-glycosylation motif (Asn-Xaa-Ser/Thr, where Xaa is not proline) was present within a tryptic peptide of suitable m/z for MS analysis. All peptides obtained by glycopeptide capture and used in protein identifications are provided in Supplementary Table 3. GPM search results were analyzed using Scaffold (Scaffold_2_05_02, Proteome Software Inc., Portland, OR) to organize the results and validate the MS/MS-based peptide and protein identifications.
Prediction of N-Glycosylation Sites in Identified Proteins
The NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/ NetNGlyc/) was used to predict potential N-glycosylation sites in all predicted proteins by searching for the Asn-Xaa-Ser/Thr triplet with no proline at the Xaa position.39 Those defined as IMPs on the basis of transmembrane helix (TMH) domains were assumed to have signal peptide sequences and were scored as glycosylated if their Jury scores were 9/9 and ++ or +++ without considering the simultaneously provided SignalP prediction. When IMPs received scores less than 9/9 and ++ for predicted glycosylation, the SignalP prediction was considered and annotations were consulted. Those with post-transcriptional modification annotations listing “glycoprotein” were scored as glycosylated. For the proteins classified as MAPs, proteins lacking evidence of TMH domains, were scored positive for N-glycosylation only if a signal peptide was predicted and if the Jury score
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was (9/9) and ++ or +++. In a few instances, MAPs with lower NetNglyc 1.0 scores for glycosylation were scored as N-glycosylated when annotation indicated these were glycoproteins. Prediction of Transmembrane Domains
Four topology prediction algorithms were used to predict the presence of transmembrane R-helices in the identified proteins: HMMTOP2.1 (http://www.enzim.hu/hmmtop/),40 TMHMM2 (http://www.cbs.dtu.dk/services/TMHMM/),41 PHOBIUS (http://servers.binf.ku.dk/phobius/),42 and SOSUI v1.10 (http://bp.nuap.nagoya-u.ac.jp/sosui/sosuiG/sosuigsubmit. html).43 Data Interpretation and Classification of Identified Proteins
To define the functions of the proteins gathered, we used protein and gene descriptions and functional classifications from six sources: Gene Ontology terms (GO, http://amigo. geneontology.org), UniProt Knowledgebase (http://www.uniprot.org) on the ExPASy proteomics server, the description line of proteins in FASTA format, GeneCards annotations (http:// www.genecards.org), ProteinCenter (Proxeon, Thermo Scientific), and published literature. Initially we used an in-house automatic fetching program to identify membrane proteins and collected the subcellular location/cellular component annotations for each of these so that this information could be used with data for predicted transmembrane domains (based on four prediction algorithms) to sort IMPs, MAPs, and non-membrane proteins (NMP) into separate files. To further verify assignments, annotations for subcellular location/subcellular component were obtained for all proteins and each protein was assigned to the appropriate data set. Proteins were described by categories within the GO terms: Biological Process (GO: 0008150), Molecular Function (GO: 0003674), and Cellular Component (GO: 0005575). Three additional terms (cell adhesion, immune response and GTPase activity) were added along with standard GO terms describing membrane proteins. Cluster of differentiation (CD) annotations were included.
’ RESULTS AND DISCUSSION Avian LMH cells were fractionated as shown in Figure 1. Peptides in the TX-114 detergent-rich and detergent-poor (aqueous) phases following 1D-SDS-PAGE (Supplementary Figure S1) and tryptic glycopeptides captured by hydrazide chemistry were identified by LC MS/MS (Figure 2). The MS data were analyzed under stringent conditions to identify 1399 nonredundant LMH cell proteins. This included 1061 proteins identified from the TX-114 detergent-rich phase, 604 from the TX-114 aqueous phase, and 55 from capture of tryptic glycopeptides along with 851, 465, and 34 additional indistinguishable homologues, respectively. The data for these proteins were compiled into three nonredundant data sets describing IMPs, MAPs and NMPs (Supplementary Tables S1a, S1b, and S1c). Nearly all proteins isolated by TX-114 extraction were identified via multiple peptides and/or through more than two spectra. Data and mass spectra for the proteins identified by one unique peptide or two or fewer spectra are provided in Supplementary Tables S2a (peptide data) and S2b (spectra). A summary of the tryptic glycopeptides captured and the proteins they defined is provided in Supplementary Table S3. In the glycopeptide capture method identifications of most proteins were made on the basis of single N-glycosylated peptides. 3976
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Figure 2. Overview of the yield of LMH cell integral membrane proteins (IMPs), membrane-associated proteins (MAPs), and nonmembrane proteins (NMPs) identified through TX-114 extraction and capture of tryptic glycopeptide capture. Shown are TX-114 detergent-rich (D), TX-114 detergent-poor, aqueous (A), and captured tryptic glycopeptide (G) fractions. Bars show the yields of IMPs, MAPs, and NMPs in each.
We used membrane protein topology prediction algorithms and annotations to identify membrane proteins and to assign them to the IMPs and MAPs categories (Supplementary Tables S1a and S1b). Four TMH prediction algorithms were used to gain confidence in the predictions since there are some differences among the algorithms in how soluble and integral membrane proteins are scored and in how the TMH retained in mature IMPs are distinguished.3 In addition, the classification of each protein was checked manually through annotations in IPI, UniProt, GeneCards, and ProteinCenter. Confirmation of Gallus Putative Uncharacterized and Hypothetical Proteins
It is desirable in proteomic studies of genomically wellcharacterized species to avoid inclusion of putative uncharacterized and hypothetical proteins (PUHP). Here inclusion of PUHP was unavoidable because of a lag in annotation of the Gallus gallus genome. Many chicken proteins in the IPI and NCBI databases are presently described as PUHP.44,45 A substantial portion of the matches in our X! Tandem analyses were with these. Overall 355 putative uncharacterized and hypothetical IMPs, MAPs, and NMPs were tallied with GPM scores ranging from 539 to 1.9 (Supplementary Table S4). All were above the 5% FDR cutoff. Through further analysis with BLASTp we found highly significant matches with well-annotated proteins and thereby established probably identities for all 355 unidentified proteins (Supplementary Table S4). Among these are 88 IMPs and 49 MAPs including surface-associated CD antigens and members of the Ras superfamily of monomeric G proteins that regulate trafficking of proteins from the Golgi through the endosome to the cell surface membrane and further recycling. TX-114 Detergent-Rich Phase Enriched IMPs and MAPs
As expected, membrane proteins were concentrated in the TX-114 detergent-rich fraction. Of the 1061 proteins in the TX114 detergent-rich phase, nearly half were membrane proteins (49%, 519/1061) (Figure 3 and Supplementary Tables S1a and S1b).
Figure 3. Analysis of the LMH cell IMPs and MAPs identified through TX-114 detergent-rich (D), TX-114 aqueous (A), and tryptic glycopeptide capture (G) fractions. (A) Venn diagrams illustrate the yields of all IMPs (left) and MAPs (right) identified in the D, A, and G data sets. The numbers of proteins identified in common between the fractions are illustrated by circle overlap. (B) Venn diagrams illustrate the yields of glycosylated IMPs (left) and MAPs (right) predicted from NetNGlyc 1.0 for the D and A data sets and by direct occupancy for the G data set peptides. Overlaps illustrate the numbers of proteins identified in common among the fractions. (C) Yield of glycosylated IMPs with one, two and three or more transmembrane helices (TMH) identified in the D, A, and G data sets. Percent TMH data were obtained from averaging the numbers of TMH predicted for each glycoprotein by the TMHMM2, HMMTOP2, PHOBIUS, and SOSUI algorithms.
The membrane proteins were, in turn, nearly equally split between IMPs (58%, 299/519) and MAPs (42%, 220/519). The IMPs included many types of transporters (including transporters for ions, amino acids, cholesterol, glucose, channel-like proteins, VAMP, and many more), enzymes (e.g., cytochromes, cathespin D, peptidases, ATPases, etc.), and class I antigen presenting molecules (BF1 and BF2). Also present were receptors (e.g., EGF receptor, transferrin receptor, gicerin, LAMP1, LAMP2, integrin, a collectin scavenger receptor, and others) variously originating from the cell surface, endocytic pathway ,and cellular organelles. Other proteins known to be expressed by LMH, such as the chicken MHC BG1 proteins,46 were missing, perhaps because of low abundance or poor solubility in TX-114. A number of LMH proteins partitioned to the TX-114 detergent-rich phase despite having no transmembrane helices. Although partitioning to the TX-114 detergent phase and initially scored as having one or two TMHs (usually by THMMTOP2), we found through examining annotations that these were not truly integral membrane proteins. As noted in similar studies25,47,48 some proteins are tethered to membranes by post-translational lipid-anchor modifications or combinations of other noncovalent interactions, such as hydrophobic and electrostatic interactions, 3977
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Journal of Proteome Research and partition in the TX-114 detergent-rich phase in the absence of transmembrane helices. All such proteins were reassigned to the MAPs data set (Supplementary Table S1b). Overall, approximately one-third of the LMH cell MAPs were anchored by posttranslational lipid modification. Annotation revealed that some of these proteins were GPI-anchored to the extracellular plasma membrane surface (including alkaline phosphatase, melanotransferrin/eos47, a 71 kDa protein, a protein similar to rat bone marrow stromal cell protein, complement regulatory GPI-anchor protein, and the major prion protein homologue), while others, such as the RAS-related proteins RAB-14 and RAB-43, were lipid-anchored at the cytoplasmic surface. Still others were lipidanchored to endosomes (e.g., RAS-related proteins RAB-11A, RAB26, and Rab27A) or other organelles (e.g., VDAC2 protein and choline dehydrogenase from mitochondria). Overall 91 proteins were identified as modified at the N-terminus by myristoylation, prenylation, and other forms of post-translational lipidation (Supplementary Table S1b). In addition to the IMPs and MAPs in the TX-114 detergent-rich fraction, there were 542 proteins with no apparent membrane association. These were categorized as NMPs. The TX-114 aqueous phase contained primarily NMPs (79%, 477/604); however, 33 IMPs and 94 MAPs partitioned into this detergent-poor phase (Figure 3 and Supplementary Tables S1a and S1b). Nearly half of the IMPs (14/33) identified in the aqueous phase were also identified in the TX-114 detergent-rich phase, as were more than half the MAPs (49/94) (Figure 3a and Supplementary Table S1a). By including the aqueous phase in the analysis we identified 64 membrane proteins (19 IMPs and 45 MAPs) that would have otherwise been excluded from the LMH membrane proteome. Partitioning of amphipathic IMPs and MAPs proteins into the aqueous phase occurs perhaps through the residual detergent that remains in the postextraction buffer after phase separation.24 Alternatively, heavy glycosylation or an unusually high content of hydrophilic amino acids might result in partitioning to the detergent-poor aqueous phase.49 Compared to earlier studies using TX-114 phase separation, we obtained more proteins overall and a higher yield of membrane proteins. For example, in a fractionation similar to the one described here, TX-114 extraction of powdered brain tissue (100 mg), followed by 1-D gel fractionation and trypsin-digestion of gel bands, provided 331 unique proteins, of which 89 (27%) were membrane proteins.50 In another study, in which TX-114extracted heart muscle membrane proteins were precipitated and then digested in solution with trypsin, the yield was 413 proteins overall.51 Of these, 88 (21%) were membrane proteins. In a study focusing on a subcellular component, natural killer cell secretory lysosomes, the yield of membrane proteins IMPs and MAPs made up 135 (61% of total) of the 221 proteins identified.25 In the present study of the LMH cell microsomal fraction, 1399 unique proteins were identified, of which 610 (41%) were IMPs and MAPs. Of these, 216 proteins predicted to be N-glycosylated were identified without further selective purification. Each of these studies, including this one, differed somewhat in experimental detail and aims, but some factors that likely contributed to the differences in yields can be deduced. Higher yields of membrane proteins in our study and that of natural killer cell secretory lysosomes25 may result from cell disruption and fractionation of subcellular components prior to dissolving in TX-114. Removal of any remaining whole cells, nuclei and cellular debris (this likely includes cytoskeletal elements) would increase the frequency of ionization of peptides
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originating from membrane proteins simply by reducing the concentration of competing proteins. The greater sensitivity of the LTQ-FT hybrid ion-trap/ICR mass spectrometer in our study also likely contributed to the greater yield by identifying more peptides present in low abundance. Further optimization of the isolation steps prior to and/or following TX-114 fractionation might provide even better yields. For example, depleting the preparation of mitochondria by differential centrifugation and pelleting the membranes from the postnuclear supernatant by high speed centrifugation might be effective in reducing sample complexity and further enriching for membrane proteins prior to TX-114 fractionation. Increased yield might be also achieved if TX-114 fractionation were combined with other methods for handling the TX-114 soluble proteins prior to trypsin digestion. For example, TX-114 fractionation followed with glycoprotein capture by lectin affinity and hydrazine chemistry is one approach that clearly enriches for membrane proteins.49 Substituting a means other than PAGE gels for protein immobilization prior to trypsin digestion might be another way to increase the yield of membrane proteins identified through TX-114 fractionation. Only a small fraction of peptides obtained from digestion of gel-trapped proteins may be available for mass spectrometry since adsorptive losses in subsequent steps can be considerable.52 Such losses might be avoided or minimized by retaining the TX-114 extracted proteins instead in size-exclusion filtration devices, as has been recently described in filter-assisted sample preparation methods.18 We would expect higher yields of peptides from TX-114 extracted single- and multipass transmembrane proteins and that MAPs would be obtained were the TX-114 extracted proteins deposited on a filtration membrane rather than in a polyacrylamide gel matrix for reduction, alkylation and enzymatic digestion. Lectinaffinity capture53 might then be used to further enrich for N-glycosylated proteins extracted by TX-114 and to confirm occupancy of predicted N-glycosylation sites. Glycopeptide Capture Is Highly Specific in Identifying N-Glycosylated Proteins
Capture of glycopeptides from the LMH cell microsomal fraction and release by PNGase F deglycosylation provided 86 unique N-glycosylated peptides (Supplementary Table S3), which allowed identification of 55 distinct proteins (Figure 3a). Although it would be possible to recover O-glycosylated peptides through an independent or secondary digestion, we did not attempt this purification since IMPs and MAPs are rarely O-glycosylated. Most of the proteins identified through glycopeptide capture were also predicted to be IMPs (75%, 41/55) (Supplementary Table S1a). Of the remaining 14 proteins, five were glycosylated MAPs (melanotransferrin, endoplasmin, ER oxidoreductin-like protein, the major prion protein homologue, and a putative uncharacterized protein, likely β-glucuronidase), and nine were glycosylated NMPs that are present within lysosomes or secreted proteins (e.g., cathespin H, lysosomal beta-galactosidase, osteonidogen and GGH protein) (Figure 3a and Supplementary Tables S1b and S1c). Notably, the 75% yield of membrane glycoproteins in our study is significantly higher than the membrane glycoprotein yield in the initial study where tryptic glycopeptide capture was first used to describe the glycoproteins of an ovarian carcinoma cell line (IGROV-1/CP).23 Although the total yield of glycoproteins from IGROV-1/CP cell line was greater overall, only 52% of these glycoproteins were membrane glycoproteins. This difference 3978
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Figure 4. Annotations for each protein identified in the D, A, and G data sets were examined for Gene Ontology terms describing biological processes and molecular functions. Terms included were all Gene Ontology terms except for the additional terms “cell adhesion”, “immune response,” and “GTPase activity” obtained from UniProt and GeneCards. Results are displayed in percent within each data set.
emerged when we analyzed the ovarian cell glycoproteins (listed in the Supplementary Tables23) to define the number of transmembrane glycoproteins using TMAP (integrated into ProteinCenter) following assignment of IPI accession numbers using the Gene ID Conversion Tool (DAVID 6.7). The reason for this difference in yield of glycosylated membrane proteins is not clear, but it might be related to differences in the types of proteins expressed by the two cells lines (ovarian carcinoma versus liver carcinoma differences). Ovarian carcinomas are often highly secretory so cell lines derived from them, such as IGROV-1/ CP, might retain this capacity. The reduced yield of membrane proteins from IGROV-1/CP cells may reflect capture of a relatively larger number of secreted N-glycoproteins in the IGROV-1/CP data set as compared to that of LMH cells. Further analysis of the data from Sun and colleagues23 showed that in their study, as in ours, the peptides captured originate predominantly from proteins with only a single TMH. Overlap Is Low between the Proteins Identified by TX-114 and Those Identified by Tryptic Glycopeptide Capture
Considering the larger number of glycosylated IMPs identified through TX-114 detergent extraction, the overlap with the proteins identified by glycopeptide capture is modest. Only 19 of the 55 proteins identified by glycopeptide capture were also identified through TX-114 extraction (Figure 3a) even though most of the IMPs identified in the TX-114 extraction contained canonical N-glycosylation motifs (63%, 201/318) (Figure 3b).
This indicates that neither preparation captured all the membrane proteins and suggests that mostly different protein populations were sampled by TX-114 fractionation and tryptic glycopeptide capture even though nearly all cell surface IMPs by nature contain both hydrophobic regions and bear N-linked glycans. To further examine the difference between the protein revealed by glycopeptide capture and TX-114 extraction, we first analyzed all IMPs bearing glycosylation motifs (predicted or occupied) to determine whether proteins in the two populations had similar numbers of predicted TMHs. The relative distributions of glycoproteins with one, two, and three or more TMHs were strikingly different. IMPs with three or more TMHs were predominant in the TX-114 detergent-rich fraction (47% (89/ 189) if the proteins had g3 TMHs compared to about one-third bearing a single TMH) (Figure 3c). In contrast, the proteins identified by tryptic glycopeptide capture were mostly (54%) single TMH proteins. Thus it appears that glycoproteins containing g3 TMHs were especially enriched in the TX-114 detergentrich fraction and available for subsequent LC MS/MS analysis. Why a lower percentage of proteins with g3 TMHs were found via tryptic glycopeptide capture is not apparent but could depend upon the solubility of multi-TMH proteins in cleavable surfactants, efficiency of trypsin digestion in these preparations, or other factors. Nearly all MAPs lack N-glycosylation, so only five MAPs were represented in the tryptic glycopeptide capture data set. Three of 3979
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Journal of Proteome Research these were found only by glycopeptide capture (major prion protein homologue, a protein similar to endoplasmic reticulum oxidoreductin 1-Lbeta and a putative uncharacterized protein, likely β-glucuronidase). The other two (melanotransferrineOS47 and endoplasmin) were also identified through TX-114 extraction. Of the tryptic glycopeptide captured MAPs only two were GPIanchored proteins (melanotransferrineOS47 and major prion protein homologue) and both of these were also found in the TX-114 detergent phase. Since both of these and four additional GPIanchored proteins were also identified by TX-114 there is little evidence in this study that glypiated membrane-associated proteins were selected in tryptic glycopeptide capture. The other MAPs captured only in the TX-114 detergent-rich phase included many members of the Ras superfamily of small monomeric G signaling proteins. Among these were 25 Rab GTPases that control steps in membrane vesicle formation, trafficking and vesicle transport. Examples include Arf GTPases (ARF1, ARF5 and ARF6) involved in regulating vesicular transport, Rho GTPases that regulate many aspects of cytoskeletal dynamics, Ras GTPases (HRAS, KRAS and DIRAS2) that control cell growth/differentiation/survival, Rap proteins (RAP2a and RAP1B) involved in controlling cell adhesion, RANP1 that controls nuclear transport, and Rheb, a recently identified protein within the mTOR pathway. Subunits of the large heterotrimeric G proteins, including GNAS, GNAQ, GNAI1, GNAI3, and GNAO1, were also found in the TX-114 detergent-rich phase, as were 18 kinases. We examined the functional compartments from which the isolated proteins originated to obtain an overview of the proteins identified and to look for additional differences that might exist between proteins captured by TX-114 partitioning and tryptic glycopeptide capture. The proteins were compared using subsets of GO terms generally understood to describe processes and functions involving proteins at the cell surface or localized within the endocytic pathway. We included three additional keywords found in UniProt and GeneCards annotation used to describe features ascribed to cell surface proteins. These were “cell adhesion,” “immune response” and “GTPase activity” (Supplementary Tables S5a and S5b). Proportionally more proteins described by the terms “cell adhesion,” “immune response,” “protein binding,” and “receptor activity,” were identified through glycopeptide capture (Figure 4). TX-114 enriched IMPs and MAPs associated especially with the “cellular organization and biogenesis,” “metabolic process,” “transport,” “catalytic activity,” “nucleotide binding,” and “transporter activity”. Not unexpectedly some of these proteins are associated with hepatocellular function (e.g., aminopeptidase EY, cathespin D, cytochrome B5, NADPH-cytochrome P450 oxidoreductase, and canalicular multispecific organic ion transporter) and demonstrate that the LMH cells retain some features of normal hepatocytes. The proteins defined by using TX-114 extraction and tryptic glycopeptide capture also differed in the proportion of cluster of differentiation (CD) antigens present. The greatest number of matches corresponding to CD antigens was found in the TX-114 IMPs and MAPs data sets (derived from both detergent and aqueous phases). A total of 31 were identified. Overall, this was, however, only 5% of the IMPs and MAPs (31/583) in the TX114 extract (Supplementary Table S6). In contrast, the proportion of CD antigens among the proteins identified by tryptic glycopeptide capture was 6.6 times greater (33%, 18/55). Cell adhesion proteins were similarly more common among the proteins isolated by tryptic glycopeptide capture. Cell adhesion proteins (some of which were also CD antigens) comprised only
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2% of the proteins identified through using TX-114 extraction, but 24% of proteins defined by glycopeptide capture. Notably, one ubiquitous glycosylated protein with a single TMH was missing among the proteins found by glycopeptide capture. While several classical (BF2) and nonclassical (BF1) MHC antigen-presenting molecules were identified in the TX-114 preparation, none were identified through tryptic glycopeptide capture.
’ CONCLUSION This study provides an overview of integral and membraneassociated proteins located at the cell surface and within the endocytic pathway of the avian LMH hepatocellular carcinoma cell line. The proteins include transporters, enzymes, receptors, membrane trafficking molecules, cell adhesion molecules, CD antigens, and molecules involved in immune response. Data are provided for the expression and probable identities of 355 avian proteins listed as hypothetical or putative uncharacterized in the present annotation of the Gallus gallus genome (v2.1). The study further demonstrates isolation of membrane proteins by their inherent properties of hydrophobicity and N-glycosylation, through TX-114 extraction and tryptic glycopeptide capture, respectively, provided access to mostly different populations of proteins located at the cell surface and within the endosome. ’ ASSOCIATED CONTENT
bS
Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Ph: (626) 301-8264. Fax: (626) 301-8280. E-mail: mamiller@ coh.org. Present Addresses §
Present address: Mass Spectrometry Facility, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5 Canada Author Contributions ^
These authors contributed equally to this work.
’ ACKNOWLEDGMENT The authors thank Christian Ravnsborg and Katianna Pihakari for access to ProteinCenter, Keely Walker for helpful comments on the manuscript, and Kathy Reisinger for help with the figures. We also thank Yujun Wang and John Hardy for the LMH cell scanning electron micrograph provided for the synopsis artwork. We are grateful for support provided by National Science Foundation grant MCB-05-24167. ’ ABBREVIATIONS USED BCA, bicinchoninic acid; Bis-Tris, 2,2-bis(hydroxymethyl)2,2A,2B nitrilotriethanol; CD, cluster of differentiation; DMEM, Dulbecco’s minimum essential medium; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; FDR, false discovery rate; GO, Gene Ontology; GPM, Global Proteome Machine; GPI, glycosylphosphatidylinositol; 3980
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Journal of Proteome Research IMPs, integral membrane proteins; IPI, International Protein Index; LDS, lithium dodecyl sulfate; LMH, LM strain hepatoma; LTQ-FT/ICR, linear trap quadrupole-Fourier transform/ion cyclotron resonance; MAPs, membrane-associated proteins; MES, 2-(N-morpholino) ethanesulfonic acid; NMPs, nonmembrane proteins; PBS, phosphate buffered saline; PEB, postextraction buffer; PMSF, phenylmethylsulfonyl fluoride; PNGase F, peptide: N-glycosidase F [EC 3.5.15.2, N-linkedglycopeptide-(N-acetyl-β-D-glucosaminyl)-L-asparagine amidohydrolase]; PNS, postnuclear supernatant; PPS, 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate; TCA, trichloroacetic acid TCEP, tris(2-carboxyethyl)phosphine hydrochloride; TFA, trifluoroacetic acid; TMH, transmembrane helices; PUHP, putative uncharacterized and hypothetical proteins
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