Analysis of the Oryza sativa Plasma Membrane Proteome Using Combined Protein and Peptide Fractionation Approaches in Conjunction with Mass Spectrometry Siria H. A. Natera,*,†,‡ Kristina L. Ford,‡ Andrew M. Cassin,‡ John H. Patterson,‡ Edward J. Newbigin,† and Antony Bacic†,‡ Plant Cell Biology Research Centre and Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, VIC 3010, Australia Received May 02, 2007
To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LCMS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membranespanning β-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally unclassified despite being predicted to be membrane proteins. Keywords: Oryza sativa (rice) • integral membrane proteins • plasma membrane proteins • mass spectrometry
Introduction The plasma membrane (PM) is a highly differentiated structure surrounding all living cells and acting as the primary interface between the cellular contents (cytoplasm) and the extracellular environment. Aside from its role as a physical barrier, the PM is a scaffold for numerous proteinaceous channelsstransporters, pores, and receptorssthat mediate chemical, nutrient, and signal exchanges between the cell and the surrounding environment. In plants, the PM acts as a major site for cell wall polysaccharide biosynthesis.1 Despite the obviously significant roles that glycoproteins residing in the PM play in cellular biology, PM proteins only represent a small fraction of the total protein within a cell. * Corresponding author. E-mail:
[email protected]. Fax number: +61 3 9347 1071. † Plant Cell Biology Research Centre. ‡ Australian Centre for Plant Functional Genomics. 10.1021/pr070255c CCC: $40.75
2008 American Chemical Society
Between 20 and 30% of the genomes of sequenced organisms have been predicted to encode membrane proteins.2 Although predicting the subcellular location of a membrane protein can help with identifying its possible function, the absence of sequence motifs shared by proteins targeted to the PM makes it difficult to estimate the number of PM proteins encoded by a genome. Nevertheless, the PM subproteome for Arabidopsis was recently predicted to contain 750 proteins, based on an estimate of about 3% of predicted proteins being PM-located.3 Recent proteomic studies of the Arabidopsis PM using a combination of SDSPAGE, RP-HPLC separation, and MS/MS analysis identified between 100 and 238 PM proteins in suspension cultured (SC) cells,3 leaves, and petioles.4 If the estimate of 3% of genes coding for PM proteins is generally true for all species, this would suggest that in rice >1000 genes encode PM proteins compared to >2500 for humans. Journal of Proteome Research 2008, 7, 1159–1187 1159 Published on Web 02/09/2008
research articles The inherent properties of this subset of proteins (low abundance, hydrophobicity, basic isoelectric points, high molecular weight) render them relatively intractable to traditional separation technologies and proteomic analyses such as twodimensional gel electrophoresis (2-DE).5,6 Proteomic approaches that do not incorporate 2-DE have been used to identify membrane proteins from the Arabidopsis chloroplast envelope7 and PM.3,4 Two alternative approaches to 2-DE that have been used for their ability to separate membrane proteins are 1D-SDS-PAGE of intact proteins and multi-dimensional LC of sample-derived tryptic peptides either in-line (MudPIT8) or off-line.7,9 In this study, we describe the preparation and assessment of highly enriched PM fractions from rice suspension cultured cells and analysis of the proteins in these fractions using 1DSDS-PAGE (Gel-LC-MS/MS) or strong cation exchange chromatography in combination with LC-MS/MS (2D-LC-MS/MS). While previous attempts to analyze rice PMs have been made using 2-DE,10 to our knowledge this study represents the first non-2-DE analysis of PM proteins from monocots. This has allowed us to identify numerous proteins not identified using the 2-DE methodology that greatly expand the previously described set of rice PM proteins from 464 resolved proteins (74 identified)11 to 805 identified proteins.
Experimental Procedures Plant Material. Oryza sativa (L. C5924) Oc suspension cultured (SC) cells12 (kindly provided by Prof. H. Kende, Plant Research Laboratory, Michigan State University, U.S.A.) were grown with shaking (120 rpm) in the dark at 23 °C in MS medium13 and supplemented with 1.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6% (w/v) sucrose. Fresh cultures were established on a weekly basis by adding 50 mL of cultured cells to 50 mL of fresh medium. Plasma Membrane (PM) Isolation. Rice SC cells were collected after 7 days of growth for PM isolation. Plant material (100 g) was homogenized in 200 mL of 500 mM sucrose, 50 mM KPO4 buffer (pH 7.5), and 20 mM KCl, containing 1 mL of protease inhibitor cocktail for plant cell extracts (P9599; Sigma, St. Louis, MO, U.S.A.), using a hand-held glass homogenizer. DTT was added to a final concentration of 10 mM before the homogenate was filtered though 50 µm nylon mesh (Australian Filter Specialists, Maddington, WA, Australia). The homogenate was then centrifuged at 6000g for 10 min, and the supernatant was recentrifuged at 50 000g for 35 min. The resulting microsomal pellet (MM) was resuspended in 5 mM KPO4 buffer. The resuspended membranes were added (10 g) to a 30 g phase system to produce a 40 g aqueous two-phase system with a final composition of 5.95% (w/w) dextran T500 (Pharmacosmos A/S, Holbaek, Denmark), 5.95% (w/w) polyethylene glycol (PEG) 3350 (Sigma, St. Louis, MO, U.S.A.), 5 mM KPO4 buffer, and 3 mM KCl. The PM was then enriched by aqueous twophase partitioning as previously described.14 The PM was collected following a greater than 2-fold dilution of the final upper phase using 10 mM Tris-MES, pH 7.5, and ultracentrifugation at 100 000g. All steps were done at 4 °C. The PM pellet was resuspended in 250 mM sucrose in 10 mM Tris-MES, pH 7.5, and either carbonate-washed immediately or snap-frozen in liquid N2 and stored at -80 °C. Protein concentrations were estimated using the Bradford assay (Bio-Rad, Hercules, CA, U.S.A.) with BSA as a standard. High pH, carbonate (100 mM sodium carbonate, pH 11) washing was used to open membrane vesicles and remove 1160
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soluble proteins. The carbonate-washed PMs (WPM) were collected as described above and either retained as a pellet or resuspended in 200 µL of 250 mM sucrose in 10 mM Tris-MES and stored as described above. Enzyme Marker Assays. The sensitivity of Mg2+-dependent ATPase activity to vanadate was used as a marker for PM activity.16,17 Cytochrome-c oxidase activity was used as a marker of mitochondria; antimycin A-insensitive NADH cytochrome-c reductase activity was used as a marker of endoplasmic reticulum; and latent inosine diphosphatase (IDPase) activity was used as a marker of the Golgi apparatus. Assays were done as previously described.18 SDS-PAGE and Western Blot Aanalysis. Protein samples were separated on 10% SDS-PAGE gels essentially according to Laemmli (1970)19 with the exception that samples were solubilized by heating at 60 °C. Proteins for MS analyses were visualized with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO, U.S.A.). Western blot analyses were performed after SDSPAGE as previously described20 and developed with antibodies directed towards markers of different subcellular compartments. An anti-H+-ATPase (P-type) antibody raised against the PM H+-ATPase of Nicotiana plumbaginifolia21 was kindly provided by Dr. M. Boutry (Universite Catholique de Louvain, Belgium); an antibody raised against the Arabidopsis endoplasmic reticulum (ER) protein SEC1222 was purchased from Rose Biotechnology (Palo Alto, CA, U.S.A.); an anti-R-TIP23 (tonoplast integral protein) antibody and an anti-γ-TIP23 antibody were from Dr. John Rogers (Washington State University, WA, U.S.A.); an antibody to yeast HSP60 was kindly provided by Dr. T. Lithgow (University of Melbourne, Australia); an anti-RGP1 antibody24 was kindly provided by Dr. K. Dhugga (Pioneer Hi-Bred International, U.S.A.); and anti-CESA antibodies25 were kindly provided by Dr. C. Somerville (Stanford University, U.S.A.). Antibodies were detected using goat antirabbit antibodies, horseradish peroxidase staining, and chemiluminescent enhancement (Pierce, Rockford, IL, U.S.A.). In-Gel and In-Solution Digestion. In-gel digestion was done essentially as described26 with some minor modifications. Segments (2 mm wide) from a large format (20 cm × 20 cm) 1-D SDS-PAGE gel were manually excised, and the proteins in the 60 resultant bands were digested with sequencing grade modified trypsin (Promega, Madison, WI, U.S.A.) in the following manner. The gel segments were cut into roughly 1 mm2 pieces and washed with 100 mM NH4HCO3. The gel pieces were destained using several washes of 50% (v/v) acetonitrile in 50 mM NH4HCO3 and rinsed twice with 100 mM NH4HCO3. Following dehydration in a Speed-Vac centrifuge, the proteins were reduced by addition of 10 mM DTT (1 h, 56 °C) and alkylated with 55 mM iodoacetamide (45 min, rt (22 °C)). After dehydration using acetonitrile and a Speed-Vac, the gel pieces were rehydrated on ice for 45 min in a buffered trypsin solution (12.5 ng trypsin per 1 µL 100 mM NH4HCO3). The proteins were digested overnight at rt. Peptides were extracted by incubating the gel pieces at rt with shaking, twice with 50% acetonitrile in 5% formic acid (v/v) and twice with 25% acetonitrile in 5% formic acid. Supernatants were pooled, and the final volume was reduced to 10–20 µL under a vacuum. The protein digests were then reconstituted in 0.1% (v/v) formic acid and filtered through a 0.20 µm Minisart RC4 single use syringe filter (Sartorius, Göttingen, Germany) prior to LC-MS/MS analysis. Henceforth, this approach will be referred to as Gel-LC-MS/ MS.
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Oryza sativa Plasma Membrane Proteomics For in-solution digests, WPM pellets were resuspended in 8 M urea before being reduced and alkylated as described for the in-gel digest. Sufficient 100 mM ammonium bicarbonate was then added to reduce the urea concentration to less than 1 M before overnight trypsin digestion at 37 °C. Neat formic acid was added to give a final pH of 3 before the tryptic peptides were passed over a C18 SepPak column (Waters, Milford, MA, U.S.A.) and concentrated under vacuum to a final volume of 100 µL. The concentrated tryptic peptides were diluted with 25 mM ammonium formate in 5% (v/v) acetonitrile and then separated on a PolySULFOETHYL Aspartamide SCX column (4.6 mm × 200 mm, 5 µm, 300 Å-1, PolyLC Inc., Columbia, U.S.A.) attached to an Agilent 1100 series HPLC system (Agilent Technologies, Palo Alto, CA, U.S.A.) with the following separation gradient: buffer A (25 mM ammonium formate in 5% (v/v) acetonitrile) for 10 min, then up to 100% buffer B (500 mM ammonium formate in 25% (v/v) acetonitrile) over 30 min, 100% buffer B for another 30 min, then down to 100% buffer A over 10 min at a flow rate of 0.7 mL/min with 0.5 min fractions being collected in deep (3.1 cm) 96-well plates. The fractions were reduced to 10–20 µL under a vacuum. Each fraction was then resuspended in 60 µL of 0.1% formic acid and analyzed by LC-MS/MS. Henceforth, this approach will be referred to as 2D-LC-MS/MS. Reversed-Phase Liquid Chromatography Electrospray Tandem Mass Spectrometry (LC-ESI-MS/MS). For LC-MS/MS, the sample-derived tryptic peptides were injected into an LCPackings (Amsterdam, The Netherlands) UltiMate capillary LC system and first preconcentrated on a 300 µm × 5 mm C18 precolumn (GraceVydac, Hesperia, California, U.S.A.). Following desalting by washing with 0.1% formic acid, the peptides were eluted onto a Vydac C18 column (75 µm i.d. × 15 cm, 3 µm 100 Å-1), eluting directly into the MS. The peptides were then separated using a series of gradients consisting of 100% solution A (0.1% formic acid) to 50% solution B (0.1% formic acid in 70% acetonitrile) over 60 min; 50% B to 100% B over 10 min; and 100% B for 10 min at a flow rate of 0.25 µL/min. MS and MS/MS data were acquired using a nanospray source on a QStar XL hybrid quadrupole-TOF LC-MS/MS (Applied Biosystems/MDS Sciex, Foster City, CA, U.S.A.) MS using the AnalystQS software (Applied Biosystems/MDS Sciex) operating in a data-dependent acquisition mode. This software was also used to process the spectra prior to database searching. Data Analysis and Informatics. Protein identification was done using MS/MS spectra to search against the O. sativasubset of the National Center for Biotechnology Information nonredundant protein database (NCBInr) and consensus sequences from the Institute for Genome Research (TIGR) rice gene indices (http://www.tigr.org/tigrscripts/tgi/ T_index.cgi?species)rice, downloaded June 2005). Database searching was done using Mascot software27 (Matrix Science, London, U.K.) run on an in-house server. The parameters used for initial Mascot searches were as follows: database, NCBInr protein; taxonomy, Oryza sativa rice or Mammalia (to check for trypsin and keratin contamination); enzyme, trypsin; fixed modifications, carbamidomethyl; variable modifications, oxidized methionine; peptide tolerance, 0.25 Da; peptide charge, 2+, 3+; number of missed cleavage sites, up to 1 missed cleavage site; MS/MS tolerance, 0.15 Da. If identification against the NCBInr database was unsuccessful, the MS/MS spectra were researched against the TIGR rice gene indices database.28 To reduce the subjectivity of data interpretation, the MS/ MS spectra were also searched against the same databases
Table 1. Enzymatic Characterization of the PM-Enriched Fraction enzyme
vanadate-sensitive ATPase cyt.-c oxidase IDPase NADH antimycin A-insensitive cyt.-c reductase % vanadate-sensitive ATPase/ total ATPase activity
subcellular location
PM/MM activity ratio
PM MT GA ER
9.09 ( 1.04a (n ) 6) 0.53 ( 0.3a (n ) 4) 0.08 ( 0.06a (n ) 6) 0.03 ( 0.04a (n ) 5) 92.2% (n ) 3)
a Ratio between the activity in the plasma membrane (PM) fraction and the activity in the microsomal (MM) fraction. MT ) mitochondria, GA ) Golgi apparatus, ER ) endoplasmic reticulum, cyt.-c ) cytochrome-c, n ) number of independent experiments.
using a second algorithm, X!Tandem (www.thegpm.org/ TANDEM/),29 which was downloaded and run on an in-house server. The parameters for the X!Tandem searches were the same as for MASCOT except for the following: fragment mass error, 0.2 Da; and parent mass error, (100 ppm. Peptides listed in this study are those from the filtered Mascot search output that were also reported by X!Tandem. An in-house filtering program (Cassin et al., in preparation) was used to report all significant peptides (as determined by Mascot, p < 0.05) that were also identified by X!Tandem (log(e) < -1). The peptide list was then compiled into a set consisting of the minimum number of proteins that accounted for all the identified peptides. For example, if a group of peptides could be assigned to two accession numbers, as it was not possible to distinguish which of these two proteins (or if both) was present, this was counted as a single protein in the final compilation. BLAST (www.ncbi.nlm.nih.gov/BLAST/) searches were used to determine the similarity of unknown proteins to known proteins or proteins of predicted function. For proteins of unknown function, putative function was assigned based on a sequence match to known proteins and on the presence of functional domains (as per ref 3). Transmembrane domain (TMD) predictions were made using the TMD prediction program, TMHMM Server v.2.0 (http://www.cbs.dtu.dk/services/ TMHMM-2.0/).30 PSORT (http://psort.nibb.ac.jp/form.html) and TargetP (http://www.cbs.dtu.dk/services/TargetP/), along with available literature, were used to predict subcellular locations for the identified proteins. The presence of GPIanchoring, prenylation or farnesylation motifs was confirmed using prediction programs available at the IMP Bioinformatics Group website (http://mendel.imp.ac.at/mendeljsp/index.jsp).
Results Preparation and Characterization of Highly Enriched Rice PM. Aqueous two-phase partitioning was used to prepare PM fractions from rice SC cells as previously described.14 After initial work to establish suitable polymer concentrations, SC cells were used as the PM source as they provide a large amount of relatively homogenous plant material for extraction and method optimization. Initial estimates of the purity of the membrane fractions were made using enzyme marker assays. These indicated that there was a 9-fold enrichment of the PM after two-phase partitioning relative to the microsomal fraction (MM, see Table 1). The majority of the total ATPase activity (92%) in the PM fractions was vanadate-sensitive, indicating low levels of contamination by tonoplast and other membrane types with different ATPases (endoplasmic reticulum, mitoJournal of Proteome Research • Vol. 7, No. 3, 2008 1161
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Natera et al. Using the two-phase system and carbonate washing step, yields of 150–200 µg of WPM were routinely obtained from 100 g (wet weight) of rice SC cells (0.15% yield).
Figure 1. (A) Immunological characterization of the enriched PM fraction. Membrane fractions isolated from rice suspension culture cells, mixed membrane (MM), plasma membrane (PM), and carbonate-washed plasma membrane (WPM), were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to specific membrane antigens. The anti-H+ATPase antibody was raised against the tobacco H+-ATPase of the PM (P-type) (2 µg, protein loaded in each lane). Anti-CESA antibodies were raised against CESA proteins from Arabidopsis (20 µg). Anti-TIP antibodies were raised against the tobacco γ-TIP and R-TIP located in the tonoplast (20 µg). Antibodies to yeast HSP60 were raised against a mitochondrial protein (5 µg). The anti-SEC12 antibodies were raised against the Arabidopsis SEC12 protein, an integral protein of the endoplasmic reticulum (10 µg). Anti-RGP1 antibodies were raised against the RGP1 protein from Pisum sativum (10 µg) (WPM not tested with this antibody). (B) SDS-PAGE separation of WPM proteins from rice suspension cultured cells. Approximately 150 µg of WPM proteins was separated on a large format 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue. Molecular weight markers (kDa) are shown on the left.
chondria, chloroplasts), a figure that is the same as that obtained in Arabidopsis PM fractions prepared by two-phase partitioning.3 The PM-enriched fraction also showed some contamination by cytochrome-c oxidase (mitochondria, MT) and limited contamination by other cellular membranes, as seen by the depletion of latent inosine diphosphatase (IDPase) (Golgi apparatus, GA) and antimycin-insensitive NADH cytochrome-c reductase (ER) activities compared to the MM fraction (Table 1). To reduce contamination further, the PM fraction was washed with a high pH carbonate buffer,15 a treatment that will remove soluble proteins from within the vesicles but will also inactivate marker enzymes. To follow enrichment after carbonate washing, specific marker proteins were detected by Western blotting. Figure 1A shows that proteins from the tonoplast (R-TIP and γ-TIP), ER membrane (SEC12), GA (RGP1), and mitochondrion (HSP60) that were initially present in MM fractions were either not detectable or present at greatly reduced levels in the PM and WPM. In contrast, antibodies to a PM H+-ATPase recognized a band of the expected size that was significantly enriched in the PM and WPM, further demonstrating the high level of enrichment and the low levels of contamination of these fractions by other endomembranes. As expected, antibodies to CESAs,25 proteins predicted to be in the PM,1,31,32 detected bands of the expected sizes that were enriched in the WPM fraction. 1162
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Extraction and Identification of Proteins from Rice WPM by Gel-LC-MS/MS and 2D-LC-MS/MS. Equivalent carbonate-washed PM (WPM) fractions (150 µg each) were prepared and analyzed using Gel-LC-MS/MS and 2D-LC-MS/MS. Figure 1B shows a protein profile from a 1D-SDS-PAGE separation of 150 µg of WPM proteins, after Coomassie staining, covering ∼10–240 kDa range. The entire lane was excised and was divided into 60 equal portions that were subjected to trypsin digestion and RP-LC-MS/MS analysis (Gel-LC-MS/MS). For off-line multidimensional liquid chromatography, the proteins in a WPM fraction were digested in solution after solubilization in urea. The resultant peptides were separated by SCX HPLC, and the peptide-containing fractions were analyzed by RP-LC-MS/MS (2D-LC-MS/MS). For both approaches, the criterion used for a protein identification was at least one peptide match with a Mascot significance level of p < 0.05 that was also found using the X!Tandem search program with a log(e) value of
