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Analysis of the Arabidopsis Cytosolic Ribosome Proteome Provides Detailed Insights into Its Components and Their Post-translational Modification

2007; Elsevier BV; Volume: 7; Issue: 2 Linguagem: Inglês

10.1074/mcp.m700052-mcp200

1535-9484

Adam J. Carroll, Joshua L. Heazlewood, Jun Ito, A. Harvey Millar,

Peptidase Inhibition and Analysis

Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical gene-specific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene- or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co- and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These site-specific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis. Finding gene-specific peptides by mass spectrometry analysis to pinpoint gene loci responsible for particular protein products is a major challenge in proteomics especially in highly conserved gene families in higher eukaryotes. We used a combination of in silico approaches coupled to mass spectrometry analysis to advance the proteomics insight into Arabidopsis cytosolic ribosomal composition and its post-translational modifications. In silico digestion of all 409 ribosomal protein sequences in Arabidopsis defined the proportion of theoretical gene-specific peptides for each gene family and highlighted the need for low m/z cutoffs of MS ion selection for MS/MS to characterize low molecular weight, highly basic ribosomal proteins. We undertook an extensive MS/MS survey of the cytosolic ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene- or gene family-specific peptides. This provided an in-depth analysis of the protein composition based on 1446 high quality MS/MS spectra matching to 795 peptide sequences from ribosomal proteins. These identified peptides from five gene families of ribosomal proteins not identified previously, providing experimental data on 79 of the 80 different types of ribosomal subunits. We provide strong evidence for gene-specific identification of 87 different ribosomal proteins from these 79 families. We also provide new information on 30 specific sites of co- and post-translational modification of ribosomal proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. These site-specific modification data provide a wealth of resources for further assessment of the role of ribosome modification in influencing translation in Arabidopsis. Ribosomes are large ribonucleoprotein complexes that catalyze the peptidyltransferase reaction in polypeptide synthesis and are thus responsible for the translation of transcripts encoded in cellular genomes. These complexes play the most fundamental role of any protein complex in the generation of the cellular proteome as a whole. Ribosomes consist of two subunits, large and small, but the internal composition of these subunits and their macromolecular size varies between bacteria, animals, fungi, and plants. Both these subunits are composed on rRNA and protein (r-protein) 1The abbreviations used are: r-protein, ribosomal protein; 2D, two-dimensional; 1D, one-dimensional; AGI, Arabidopsis Gene Index; EST, expressed sequence tag; RP, ribosomal protein; TAIR, The Arabidopsis Information Resource; ddH2O, double deionized water (MilliQ-purified); MGF, Mascot Generic Format; PHP, hypertext preprocessor. 1The abbreviations used are: r-protein, ribosomal protein; 2D, two-dimensional; 1D, one-dimensional; AGI, Arabidopsis Gene Index; EST, expressed sequence tag; RP, ribosomal protein; TAIR, The Arabidopsis Information Resource; ddH2O, double deionized water (MilliQ-purified); MGF, Mascot Generic Format; PHP, hypertext preprocessor. components. Among eukaryotes the 80 S cytosolic ribosomes of the yeast (Saccharomyces cerevisiae), rat (Rattus norvegicus), and human (Homo sapiens) have been the most extensively investigated. These studies have revealed four distinct rRNAs, the 18 S rRNA of the 40 S subunit and 5, 5.8, and 23 S rRNAs of the 60 S subunit. Up to 79 distinct proteins are part of these two subunits in eukaryotes, 32 small subunit and 47 large subunit proteins, compared with only 54 proteins in the bacterial ribosome subunits (1Doudna J.A. Rath V.L. Structure and function of the eukaryotic ribosome: the next frontier.Cell. 2002; 109: 153-156Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In eukaryotes a separate bacterial-like ribosome is present in the mitochondria and is referred to as a 70 S ribosome containing a large 50 S and small 30 S subunit; its smaller overall size is reflected in altered rRNA sizes and different protein subunits (2O'Brien T.W. Properties of human mitochondrial ribosomes.IUBMB Life. 2003; 55: 505-513Crossref PubMed Scopus (109) Google Scholar). Detailed analyses of yeast, rat, and human cytosolic ribosomes by peptide mass spectrometry have provided insights into the composition and post-translational modification of ribosomal proteins (3Arnold R.J. Polevoda B. Reilly J.P. Sherman F. The action of N-terminal acetyltransferases on yeast ribosomal proteins.J. Biol. Chem. 1999; 274: 37035-37040Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Lee S.W. Berger S.J. Martinovic S. Pasa-Tolic L. Anderson G.A. Shen Y. Zhao R. Smith R.D. Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5942-5947Crossref PubMed Scopus (165) Google Scholar, 5Louie D.F. Resing K.A. Lewis T.S. Ahn N.G. Mass spectrometric analysis of 40 S ribosomal proteins from Rat-1 fibroblasts.J. Biol. Chem. 1996; 271: 28189-28198Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 6Odintsova T.I. Muller E.C. Ivanov A.V. Egorov T.A. Bienert R. Vladimirov S.N. Kostka S. Otto A. Wittmann-Liebold B. Karpova G.G. Characterization and analysis of posttranslational modifications of the human large cytoplasmic ribosomal subunit proteins by mass spectrometry and Edman sequencing.J. Protein Chem. 2003; 22: 249-258Crossref PubMed Scopus (61) Google Scholar, 7Yu Y. Ji H. Doudna J.A. Leary J.A. Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes.Protein Sci. 2005; 14: 1438-1446Crossref PubMed Scopus (81) Google Scholar). Methionine removal, acetylation, methylation, and phosphorylation are known ribosomal protein modifications, and both phosphorylation and methylation have been linked to changes in ribosomal function or biosynthesis (8Bachand F. Silver P.A. PRMT3 is a ribosomal protein methyltransferase that affects the cellular levels of ribosomal subunits.EMBO J. 2004; 23: 2641-2650Crossref PubMed Scopus (131) Google Scholar, 9Mazumder B. Sampath P. Seshadri V. Maitra R.K. DiCorleto P.E. Fox P.L. Regulated release of L13a from the 60S ribosomal subunit as a mechanism of transcript-specific translational control.Cell. 2003; 115: 187-198Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Early studies of the cytosolic ribosome in plants revealed it was slightly smaller than that found in mammals, notably due to lower apparent mass of the 60 S subunit (10Cammarano P. Pons S. Romeo A. Galdieri M. Gualerzi C. Characterization of unfolded and compact ribosomal subunits from plants and their relationship to those of lower and higher animals: evidence for physicochemical heterogeneity among eucaryotic ribosomes.Biochim. Biophys. Acta. 1972; 281: 571-596Crossref PubMed Scopus (19) Google Scholar). Early biochemical analyses of the protein components of ribosomes have been undertaken using 2D gels in the monocotyledonous plants wheat, barley, and maize and the dicotyledonous plants soybean, tomato, and tobacco (11Bailey-Serres J. Freeling M. Hypoxic stress-induced changes in ribosomes of maize seedling roots.Plant Physiol. 1990; 94: 1237-1243Crossref PubMed Scopus (102) Google Scholar, 12Capel M.S. Bourque D.P. Characterization of Nicotiana tabacum chloroplast and cytoplasmic ribosomal proteins.J. Biol. Chem. 1982; 257: 7746-7755Abstract Full Text PDF PubMed Google Scholar, 13Gannt J. Key J. Auxin-induced changes in the level of translatable ribosomal protein messenger ribonucleic acids in soybean hypocotyl.Biochemistry. 1983; 22: 4131-4139Crossref Scopus (27) Google Scholar, 14Koyama K. Wada A. Maki Y. Tanaka A. Changes in the protein composition of cytoplasmic ribosomes during the greening of etiolated barley leaves.Physiol. Plant. 1996; 96: 85-90Crossref Google Scholar, 15Sikorski M.M. Przybyl D. Legocki A.B. Nierhaus K.H. Group fractionation of wheat germ ribosomal proteins.Plant Sci. Lett. 1983; 30: 303-320Crossref Scopus (5) Google Scholar, 16Scharf K.-D. Nover L. Control of ribosome biosynthesis in plant cell cultures under heat shock conditions. II Ribosomal Proteins.Biochim. Biophys. Acta. 1987; 909: 44-57Crossref Scopus (44) Google Scholar). Counting of distinct protein spots on these gels suggested that the 40 S subunit contained up to 40 proteins, whereas the 60 S subunit contained up to 59 proteins; however, without genomic sequences it was not possible to systematically assign these proteins to genes and gene families that would be required to resolve the composition of the complex in plants in a gene-specific manner. The sequencing of the Arabidopsis genome provided the first opportunity in plants to consider the number and arrangement of ribosomal protein-coding genes in plants. Using the strong sequence conservation of the eukaryotic r-proteins, Barakat et al. (17Barakat A. Szick-Miranda K. Chang I.F. Guyot R. Blanc G. Cooke R. Delseny M. Bailey-Serres J. The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome.Plant Physiol. 2001; 127: 398-415Crossref PubMed Scopus (252) Google Scholar) undertook an analysis of expressed sequence tags, and the early annotation of the complete genomic sequence identified 249 genes including 22 apparent pseudogenes that encoded 80 different types of r-proteins (32 small subunit and 48 large subunit proteins). The extra family of proteins not conserved in mammals was the plant-specific P3 component known to be in the 60 S subunit. Analysis of the r-protein gene families reveals that each family consists, on average, of three members. The sequences within these families can have very high conservation, leading to many paralogs with 97–100% sequence identity at the amino acid level, whereas other r-protein families contain significant sequence divergence. Based on public EST data and hybridization data on microarrays, most gene family members are expressed and could be present in the ribosome structure at different points of development, in different cell types, and under different conditions (18Schmid M. Davison T.S. Henz S.R. Pape U.J. Demar M. Vingron M. Scholkopf B. Weigel D. Lohmann J.U. A gene expression map of Arabidopsis thaliana development.Nat. Genet. 2005; 37: 501-506Crossref PubMed Scopus (1941) Google Scholar). Variation in ribosome composition by incorporation of different paralogs could be an important component in the regulation of transcript translation. This underpins the importance of a thorough understanding of the actual composition of ribosome protein complexes themselves and not just the set of genes that could encode ribosomes. Several studies have sought to investigate the ribosomal protein complement of experimental samples from Arabidopsis to answer these questions. Chang et al. (19Chang I.F. Szick-Miranda K. Pan S. Bailey-Serres J. Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes.Plant Physiol. 2005; 137: 848-862Crossref PubMed Scopus (126) Google Scholar) performed a study using ribosomes purified from Arabidopsis cell culture. They undertook a MALDI-TOF analysis of spots from 2D gel-separated protein samples and also a limited tandem MS analysis of 1D SDS-PAGE-separated protein bands. Giavalisco et al. (20Giavalisco P. Wilson D. Kreitler T. Lehrach H. Klose J. Gobom J. Fucini P. High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome.Plant Mol. Biol. 2005; 57: 577-591Crossref PubMed Scopus (99) Google Scholar) undertook a similar analysis of ribosome samples extracted from whole Arabidopsis leaves using 2D gel-separated samples and MALDI-TOF MS analysis. Chang et al. (19Chang I.F. Szick-Miranda K. Pan S. Bailey-Serres J. Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes.Plant Physiol. 2005; 137: 848-862Crossref PubMed Scopus (126) Google Scholar) identified proteins from 70 of the 80 gene families by their MALDI-TOF analysis and identified members from a further four gene families through their MS/MS analysis of 1D gel bands. In contrast Giavalisco et al. (20Giavalisco P. Wilson D. Kreitler T. Lehrach H. Klose J. Gobom J. Fucini P. High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome.Plant Mol. Biol. 2005; 57: 577-591Crossref PubMed Scopus (99) Google Scholar) could only identify 63 of the 80 gene families by their MALDI-TOF analysis. Both these studies made claims of products from particular genes within a large number of the gene families. This was often based on the presence of single MS ion masses, but often not even these could be found, and the proteins could only redundantly be linked back to gene families of two to seven members. We used a combination of approaches to advance the MS/MS-based insight into Arabidopsis ribosomal composition and its post-translational modifications. First, we used an in silico digestion of all ribosomal proteins to define targets for data acquisition and to drive a strategy of data collection to maximize recognition of ribosomal protein-derived peptides. Second, we undertook an extensive MS/MS survey of the ribosome using trypsin and, when required, chymotrypsin and pepsin. We then used custom software to extract and filter peptide match information from Mascot result files and implement high confidence criteria for calling gene-specific identifications based on the highest quality unambiguous spectra matching exclusively to certain in silico predicted gene-specific peptides. This provided a much richer and more detailed analysis of the protein composition and also identified peptides from five gene families of r-proteins not identified in previous studies. Further we acquired strong MS/MS data to support gene-specific protein identification in 32 cases not revealed in the previous study by Chang et al. (19Chang I.F. Szick-Miranda K. Pan S. Bailey-Serres J. Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes.Plant Physiol. 2005; 137: 848-862Crossref PubMed Scopus (126) Google Scholar). In addition, we provide a wealth of information on co- and post-translational modification of r-proteins in Arabidopsis by initiator methionine removal, N-terminal acetylation, N-terminal methylation, lysine N-methylation, and phosphorylation. Predicted sequences for Arabidopsis ribosomal proteins were obtained from the file "ATH1_pep_20051108," which was downloaded from The Arabidopsis Information Resource (TAIR) website (www.Arabidopsis.org) as part of the TAIR6 Arabidopsis Genome Release. A modified version of the published Perl script, Proteogest (21Cagney G. Amiri S. Premawaradena T. Lindo M. Emili A. In silico proteome analysis to facilitate proteomics experiments using mass spectrometry.Proteome Sci. 2003; 1: 5Crossref PubMed Scopus (81) Google Scholar), and custom hypertext preprocessor (PHP) scripts were used to populate peptide database tables containing rows for every tryptic, chymotryptic, or peptic peptide sequence predicted from the set of 409 protein sequences annotated as ribosomal proteins or ribosomal protein-like proteins (allowing one missed cleavage) whereby each row represented an individual peptide-to-gene association. In addition to columns for peptide sequence and Arabidopsis Gene Index (AGI), the tables also included a column containing the name of the r-protein family to which each gene belonged according to the nomenclature of Barakat et al. (17Barakat A. Szick-Miranda K. Chang I.F. Guyot R. Blanc G. Cooke R. Delseny M. Bailey-Serres J. The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome.Plant Physiol. 2001; 127: 398-415Crossref PubMed Scopus (252) Google Scholar). These tables were used to map Mascot-reported peptide match information onto r-proteins and r-protein families and were processed by additional PHP scripts to generate lists of peptide sequences that were only derived from single genes (gene-specific) and calculate the predicted m/z ratios of [M + 2H+]2+ ions of total and gene-specific peptides for r-protein peptide ion m/z distribution analysis. All steps were carried out on ice or at 4 °C. Approximately 100 g fresh weight of Arabidopsis cell culture was homogenized in 300 ml of extraction buffer (0.45 m mannitol, 30 mm Tris, 0.5% (w/v) polyvinylpyrrolidone 40, 100 mm KCl, 20 mm MgCl2, 0.5% (w/v) bovine serum albumin, 20 mm cysteine) in a Waring blender for 2 min, and the homogenate was filtered through four layers of muslin. The filtrate was centrifuged for 5 min at 1500 × g, and the supernatant was again centrifuged at 16,000 × g for 15 min. The supernatant of this 16,000 × g sample was then centrifuged at 30,000 × g for 30 min. In 70-ml polycarbonate bottle assemblies (Beckman part number 355655), 50-ml portions of the 30,000 × g supernatant were then layered over 20-ml cushions of 1.5 m sucrose dissolved in Ribosome Buffer (30 mm Tris, 100 mm KCl, 20 mm MgCl2, 5 mm β-mercaptoethanol) and centrifuged at 180,000 × g for 14.5 h to form a crude ribosomal pellet. Each crude ribosomal pellet was resuspended in 1 ml of Ribosome Buffer and centrifuged at 20,800 × g for 30 min to pellet insoluble material. The supernatants were combined, brought to a volume of 50 ml with Ribosome Buffer, and ultracentrifuged through 1.5 m sucrose as above. The pure ribosomal pellet was resuspended to a protein concentration of 6.5 mg/ml in Ribosome Buffer, snap frozen in liquid nitrogen, and stored at −80 °C until use. This protocol yielded 2–3 mg of ribosomal protein from 100 g fresh weight of cells. For the isolation of ribosomes from mitochondria, mitochondria were first purified from Arabidopsis by differential centrifugation and density gradient centrifugation essentially as described by Millar et al. (22Millar A.H. Liddell A. Leaver C.J. Isolation and subfractionation of mitochondria from plants.Methods Cell Biol. 2001; 65: 53-74Crossref PubMed Google Scholar). A concentrated suspension of mitochondria containing ∼40 mg of mitochondrial protein was resuspended to a total volume of 50 ml in Ribosome Buffer containing 2% (w/v) Triton X-100. The suspension was incubated on ice for 30 min with occasional gentle mixing. The suspension was then clarified by centrifugation at 30,000 × g for 30 min, and the supernatant was layered over a 20-ml cushion of 1.5 m sucrose in Ribosome Buffer and centrifuged at 180,000 × g for 20 h. After removal of the supernatant, the ribosomal pellet was resuspended in a minimal volume of Ribosome Buffer, snap frozen in liquid nitrogen, and stored at −80 °C until use. Ribosome dissociation was essentially carried out according to Lin and Key (23Lin C. Key J. Dissociation of N2 gas-induced monomeric ribosomes and functioning of the derived subunits in protein synthesis in pea.Plant Physiol. 1971; 48: 547-552Crossref PubMed Google Scholar). Briefly 100 μg of purified cytosolic ribosomes were resuspended in 50 μl of either modified ribosome buffer (30 mm Tris, 100 mm KCl, 5 mm MgCl2, 1 mm DTT, pH 7.5) or modified ribosome buffer containing 0.5 m KCl. Resuspended samples were loaded onto a 10-ml linear sucrose gradient (15–30%) in either modified ribosome buffer or modified ribosome buffer containing 0.5 m KCl and subjected to ultracentrifugation at 260,800 × g at 4 °C for 4 h using a Beckman SW41 Ti rotor. Fractions of ∼200 μl were collected directly into 96-well plates using a peristaltic pump. Absorbance readings (A260 and A280) were conducted on a POLARstar OPTIMA microplate reader (BMG Labtech) using 200 flashes/well. SDS-PAGE gels used were 4% acrylamide stacking gels above 12% (w/v) acrylamide, 0.1% (w/v) SDS or 5–16% (w/v) acrylamide, 0.1% (w/v) SDS in a large gel format (0.1 × 16 × 16 cm) and were run with a Tris-glycine buffering system. Gel electrophoresis was performed at 25 mA/gel and completed in 3 h. Approximately 25 μg of ribosomal proteins were solubilized in SDS-PAGE sample buffer and loaded onto a 12% polyacrylamide gel (14 cm × 16 cm × 0.75 mm) overlaid with a 4% stacking gel. The sample underwent electrophoresis for 4 h at 30 mA and upon completion was fixed in a solution consisting of 10% acetic acid and 50% methanol overnight. Following three 10-min washes in ddH2O each, gels were stained with 100 ml of Pro-Q Diamond (Invitrogen) for 90 min and destained using three successive 30-min washes with 100 ml of destain solution containing 50 mm sodium acetate (pH 4.0) and 20% acetonitrile. After two washes in ∼100 ml of ddH2O for 5 min at a time, fluorescent images of in-gel phosphorylated proteins were acquired using a Typhoon fluorescence scanner (GE Healthcare) with 532 nm excitation, a 580-nm band pass emission filter, and the photo multiplier tube set at 500 for optimal Pro-Q dye detection. ImageQuant™ software was used to view Pro-Q staining of the 1D PAGE gel. The gel was then stained with colloidal Coomassie overnight with gentle rocking. Following staining, the solution was discarded, and the gel was destained in 0.5% phosphoric acid for 4 h. TiO2 tips (NuTip) were supplied by Glygen Inc., and phosphopeptide enrichment procedures were essentially those outlined by Larsen et al. (24Larsen M.R. Thingholm T.E. Jensen O.N. Roepstorff P. Jorgensen T.J.D. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.Mol. Cell. Proteomics. 2005; 4: 873-886Abstract Full Text Full Text PDF PubMed Scopus (1328) Google Scholar) with some modifications. Briefly TiO2 tips were conditioned prior to binding of phosphopeptides by aspirating/expelling 10 μl of 0.1% TFA solution in ddH2O (pH ∼1.9) five times using a pipette and expelling all of the TFA solution from the tip. Before binding, the pH of a 5-μl sample containing 10 μg of trypsin-digested ribosomal peptide lysate mixture was adjusted to pH ∼1.9 by adding one part 1% TFA solution in ddH2O to four parts peptide mixture. The peptide mixture was aspirated/expelled 50 times using a pipette to ensure maximal binding of phosphorylated peptides to TiO2. The binding solution was then completely expelled from the tip. Bound samples were washed with 10 μl of 50% acetonitrile, 0.1% TFA aspirating/expelling the wash solution 10 times through the tip. This step was repeated two more times for a total of three wash steps. At the end of each wash step, all of the wash solution was expelled from the tip. Finally the bound phosphopeptides were eluted from the TiO2 tip by aspirating/expelling 10 μl of 0.3 m NH4OH solution in ddH2O (pH 10.5) 10 times through the tip. Portions of this eluate were used for nano-ESI-MS/MS analysis. For gel-arrayed proteins, samples to be analyzed were cut from the gels and destained twice for 45 min in 10 mm NH4CO3 and 50% (v/v) acetonitrile. Samples were dehydrated at 50 °C in a dry block heater for 30 min and rehydrated with 15 μl of digestion solution, which for trypsin and chymotrypsin consisted of 25 mm NH4CO3 and 25 μg/ml protease in 0.01% (v/v) trifluoroacetic acid and for pepsin consisted of 25 μg/ml pepsin in 7% (v/v) formic acid, and incubated overnight at 37 °C. Peptides were extracted by adding 15 μl of acetonitrile and vigorous shaking for 15 min, removing liquid, and adding 15 μl of 50% (v/v) acetonitrile and 5% (v/v) formic acid to the gel plugs followed by another 15 min of shaking (this step was repeated); washes were pooled after each extraction step. Samples were loaded onto self-packed Microsorb (Varian Inc.) C18 (5-μm, 100-Å) reverse phase columns (0.5 × 50 mm) using an Agilent Technologies 1100 series capillary liquid chromatography system and eluted into a QStar Pulsar i MS/MS system fitted with an IonSpray source (Applied Biosystems). Peptides were eluted from the C18 reverse phase column at 8 μl/min using a 9-min acetonitrile gradient (5–60%) in 0.1% (v/v) formic acid. Ions were selected automatically for the N2 collision cell utilizing the information-dependent acquisition capabilities of Analyst QS version 1.1 (Applied Biosystems) and the rolling collision energy feature for automated collision energy determination based on the m/z of the ions. The method used a 1-s TOF MS scan that automatically switched (using information-dependent acquisition) to a 2-s product ion scan (MS/MS) when a target ion reached an intensity of greater that 30 counts and its charge state was identified as 2+, 3+, or 4+. TOF MS scanning was undertaken on an m/z range of 200–900 m/z using a Q1 transmission window of 180 amu (100%). Product ion scans were undertaken at m/z ranges of 70–2000 m/z at low resolution utilizing Q2 transmission windows of 50 (33%), 190 (33%), and 650 amu (34%). For ribosomal protein lysates, samples were digested with trypsin (1:10, w/w) in 10 mm NH4CO3 overnight at 37 °C. For initial analyses digested samples of 1–10 μg were analyzed directly or after TiO2 selection as above except bound peptides were eluted over a 30-min period (5–60% acetonitrile in 0.1% (v/v) formic acid) at 10 μl/min. The analysis method used was similar to that described above except a TOF MS scan range of 400–1200 m/z was used with a Q1 transmission window of 380 amu (100%). For more detailed analyses samples of ∼0.2–1 μg were analyzed on a QStar Pulsar i MS/MS system (Applied Biosystems) fitted with a NanoES source (Protana Inc.) outfitted with a New Objective adapter (ADPT-PRO) to allow direct liquid chromatographic coupling to the source. Samples were loaded using an 1100 series capillary LC system (Agilent Technologies) onto a capillary sample trap column containing a 100-μm × 2.5-cm C18 insert (Upchurch Scientific) at 1 μl/min in 5% (v/v) acetonitrile and 0.1% (v/v) formic acid. Bound peptides were eluted at 500 nl/min into the mass spectrometer after automatically switching a retrofitted 10-port nanobore valve (Valco Inc.) on the QStar Pulsar i by an 1100 series Nano Pump (Agilent Technologies). Peptides were eluted over a 30-min period and analyzed using parameters similar to those outlined above for LC-MS/MS analysis of lysates. Resulting MS/MS-derived spectra were analyzed against an in-house Arabidopsis database comprising ATH1.pep (release 6) from TAIR and the mitochondrial and plastid protein sets (TAIR). This sequence database contained a total of 30,700 protein sequences (12,656,682 residues). Mascot Generic Format (.MGF) MS/MS peak lists were generated from raw Sciex Analyst format (.WIFF) data files with Mascot Daemon using the "mascot.dll 1.6b21 for Analyst QS 1.1" data import filter available from Matrix Science. The settings used for MS/MS peak list generation were: centroid survey scan ions (TOF MS) at a height percentage of 50% and a merge distance of 0.1 amu (for charge state determination); centroid MS/MS data at a height percentage of 50% and a merge distance of 2 amu; reject a CID if less than 10 peaks or if precursor mass less than 50 Da or greater than 10,000 Da; precursor mass tolerance for grouping, 1 Da; maximum number of cycles between groups, 10; minimum number of cycles per group, 1; default precursor charge states, 2+ and 3+. Searches were conducted using Mascot Search Engine version 2.1.04 (Matrix Science) with the following parameters: mass error tolerances of ±75 ppm for MS and ±0.6 Da for MS/MS; "Max missed cleavages" set to 2; variable modification, oxidation (Met); instrument set to ESI-Q-TOF. Results were filtered using "Standard scoring," "Maximum number of hits" was set to 200, "Significance threshold" was set at p < 0.05. For initial sea