The Chloroplast Grana Proteome Defined by Intact Mass Measurements from Liquid Chromatography Mass Spectrometry
2002; Elsevier BV; Volume: 1; Issue: 1 Linguagem: Inglês
10.1074/mcp.m100007-mcp200
ISSN1535-9484
AutoresStephen M. Gómez, John N. Nishio, Kym F. Faull, Julian P. Whitelegge,
Tópico(s)Algal biology and biofuel production
ResumoProteomics seeks to address the entire complement of protein gene products of an organism, but experimental analysis of such complex mixtures is biased against low abundance and membrane proteins. Electrospray-ionization mass spectrometry coupled with reverse-phase chromatography was used to separate and catalogue all detectable proteins in samples of photosystem II-enriched thylakoid membrane subdomains (grana) from pea and spinach. Around 90 intact mass tags were detected corresponding to approximately 40 gene products with variable post-translational covalent modifications. Provisional identity of 30 of these gene products was proposed based upon coincidence of measured mass with that calculated from genomic sequence. Analysis of isolated photosystem II complexes allowed detection and resolution of a minor population of D1 (PsbA) that was apparently palmitoylated and not detected in less purified preparations. Based upon observed +80-Da adducts, D1, D2 (PsbD), CP43 (PsbC), two Lhcbs, and PsbH were confirmed to be phosphorylated, and a new phosphoprotein was proposed to be the product of psbT. The appearance of a second +80-Da adduct on PsbH provides direct evidence for a second phosphorylation site on PsbH, complicating interpretation of its role in regulation of thylakoid membrane organization and function, including light-state transitions. Adducts of +32 Da, presumably arising from oxidative modification during illumination, were associated with more highly phosphorylated forms of PsbH implying a relationship between the two phenomena. Intact mass proteomics of organellar subfractions and more highly purified protein complexes provides increasingly detailed insights into functional genomics of photosynthetic membranes. Proteomics seeks to address the entire complement of protein gene products of an organism, but experimental analysis of such complex mixtures is biased against low abundance and membrane proteins. Electrospray-ionization mass spectrometry coupled with reverse-phase chromatography was used to separate and catalogue all detectable proteins in samples of photosystem II-enriched thylakoid membrane subdomains (grana) from pea and spinach. Around 90 intact mass tags were detected corresponding to approximately 40 gene products with variable post-translational covalent modifications. Provisional identity of 30 of these gene products was proposed based upon coincidence of measured mass with that calculated from genomic sequence. Analysis of isolated photosystem II complexes allowed detection and resolution of a minor population of D1 (PsbA) that was apparently palmitoylated and not detected in less purified preparations. Based upon observed +80-Da adducts, D1, D2 (PsbD), CP43 (PsbC), two Lhcbs, and PsbH were confirmed to be phosphorylated, and a new phosphoprotein was proposed to be the product of psbT. The appearance of a second +80-Da adduct on PsbH provides direct evidence for a second phosphorylation site on PsbH, complicating interpretation of its role in regulation of thylakoid membrane organization and function, including light-state transitions. Adducts of +32 Da, presumably arising from oxidative modification during illumination, were associated with more highly phosphorylated forms of PsbH implying a relationship between the two phenomena. Intact mass proteomics of organellar subfractions and more highly purified protein complexes provides increasingly detailed insights into functional genomics of photosynthetic membranes. With many genomes completed and many more in the pipeline it is clear that the post-genomic era has arrived. Considerable attention is now being directed toward defining the function of individual gene products and the inter-relationships between them (functional genomics). Proteomics seeks to catalogue the full complement of the gene products of a cell and the effect of development, environment, and disease upon their expression. Mass spectrometry is driving proteomics, most commonly as a tool to identify proteins separated and visualized on two-dimensional gels. However, such strategies are insensitive to low abundance proteins 1Aebersold R. Rist B. Gygi S.P. Quantitative proteome analysis: methods and applications.Ann. N. Y. Acad. Sci. 2000; 919: 33-47Google Scholar, 2Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9390-9395Google Scholar, proteins that are not fully represented on two-dimensional gels (for example, some classes of intrinsic membrane proteins) and to subtle changes in covalent modifications that do not appreciably alter isoelectric point or electrophoretic mobility. To address some of these shortcomings, intact mass proteomics has been proposed 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar, 4Whitelegge J.P. Faull K.F. Gundersen C. Gómez S.M. Garab G. Photosynthesis: Mechanisms and Effects. Vol. V. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 4381-4384Google Scholar, 5Whitelegge J.P. le Coutre J. Lee J.C. Engel C.K. Privé G.G. Faull K.F. Kaback H.R. Toward the bilayer proteome, electrospray-ionization mass spectrometry of large intact transmembrane proteins.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10695-10698Google Scholar, 6Whitelegge J.P. Penn B. To T. Waring A. Sherman M. Stevens R.L. Fluharty C.B. Faull K.F. Fluharty A.L. The effect of methionine oxidation upon the structure and function of the cerebroside-sulfate activator protein.Protein Sci. 2000; 9: 1618-1630Google Scholar, 7le Coutre J. Whitelegge J.P. Gross A. Turk E. Wright E.M. Kaback H.R. Faull K.F. Proteomics on full-length membrane proteins using mass spectrometry.Biochemistry. 2000; 39: 4237-4242Google Scholar, 8Whitelegge J.P. le Coutre J. Proteomics: making sense of genomic information for drug discovery.Am. J. Pharmacogenomics. 2001; 1: 29-35Google Scholar.The ideal analysis of any protein includes a mass spectrum of the intact molecule to define the native covalent state and its heterogeneity 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar, 4Whitelegge J.P. Faull K.F. Gundersen C. Gómez S.M. Garab G. Photosynthesis: Mechanisms and Effects. Vol. V. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 4381-4384Google Scholar. A versatile procedure has been developed for effective electrospray-ionization mass spectrometry (MS) 1The abbreviations used are: MS, mass spectrometry; MS-MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; PS, photosystem; MSH, membrane-spanning α-helices; LC-MS, liquid chromatography-mass spectrometry; IMT, intact mass tag. 1The abbreviations used are: MS, mass spectrometry; MS-MS, tandem mass spectrometry; HPLC, high-performance liquid chromatography; PS, photosystem; MSH, membrane-spanning α-helices; LC-MS, liquid chromatography-mass spectrometry; IMT, intact mass tag. of intact intrinsic membrane proteins purified by reverse-phase chromatography in aqueous formic acid/isopropanol 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar. An advantage of this technique is the ability to accurately measure proteins greater than 30 kDa, thereby allowing analysis of the majority of the gene products from any genome. The mass measurements of spinach PS II D1 (38,022 Da; 5 membrane-spanning α-helices (MSH)), D2 (39,419 Da; 5 MSH), and Halobacterium halobium bacteriorhodopsin holoprotein (27,052 Da; 7 MSH) were within 0.01% of calculated theoretical values, setting a benchmark standard for analysis of intrinsic membrane proteins 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar. The high mass accuracy of this technique for larger hydrophobic proteins has been demonstrated by analysis of a His6-tagged Escherichia coli lactose permease (lacY; 47,357 Da; 12 MSH) 5Whitelegge J.P. le Coutre J. Lee J.C. Engel C.K. Privé G.G. Faull K.F. Kaback H.R. Toward the bilayer proteome, electrospray-ionization mass spectrometry of large intact transmembrane proteins.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10695-10698Google Scholar, 7le Coutre J. Whitelegge J.P. Gross A. Turk E. Wright E.M. Kaback H.R. Faull K.F. Proteomics on full-length membrane proteins using mass spectrometry.Biochemistry. 2000; 39: 4237-4242Google Scholar, His6-tagged Vibrio parahaemolyticus Na+/galactose cotransporters (sglT; 60,676 and 90,544 Da; 14/15 MSH) 9Turk E. Kim O. le Coutre J. Whitelegge J.P. Eskandari S. Lam J.T. Kreman M. Zampighi G. Faull K.F. Wright E.M. Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.J. Biol. Chem. 2000; 275: 25711-25716Google Scholar, and the α-subunit of the rat Na+/K+-ATPase (atn2; 112,344 Da; 10 MSH). 2J. P. Whitelegge and S. J. Karlish, unpublished data. 2J. P. Whitelegge and S. J. Karlish, unpublished data.Strict translation of a published gene sequence is usually not sufficient to match a mass to a gene, and genome sequence manipulation is required before agreement with measured masses is achieved. Varietal differences, DNA sequencing errors, post-transcriptional and post-translational modifications, as well as protein damage, must all be considered. Spectra frequently reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of the dominant proteins 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar, 4Whitelegge J.P. Faull K.F. Gundersen C. Gómez S.M. Garab G. Photosynthesis: Mechanisms and Effects. Vol. V. Kluwer Academic Publishers, Dordrecht, The Netherlands1999: 4381-4384Google Scholar.Toward a complete description of the thylakoid proteome, the closely appressed membranes of the granal stacks were prepared using their known resistance to solubilization by Triton X-100, resulting in a subfraction highly enriched in the polypeptides of PS II. Using this simplified starting material it was possible, using LC-MS, to record the masses of all detectable polypeptides in PS II-enriched membrane preparations from spinach and pea. Proteins were identified provisionally based upon coincidence of measured mass with that calculated from sequences in the data base and their predicted elution from the HPLC column based upon calculated hydrophobicity. The heterogeneity of the larger PS II subunits was revealed illustrating the accuracy and resolution afforded by electrospray-ionization-MS. The observation of light-de-pendent double phosphorylation of PsbH demonstrates potential advantages and pitfalls of the approach for examining relative expression and steady-state native modifications. Analysis of subfractions of the proteome using this technique decreased the genomic coverage but allowed resolution of several, otherwise unrecognized, native modifications that may be functionally significant.EXPERIMENTAL PROCEDURESLeaves from 3-week-old greenhouse-grown pea (Pisum sativum cv. Alaska) plants, 6-month-old tobacco (Nicotiana tabacum cv. Samsung) plants, and spinach obtained from local market sources were used for preparation of PS II-enriched membranes 10Peter G.F. Thornber J.P. Biochemical composition and organization of higher plant photosystem II light-harvesting pigment proteins.J. Biol. Chem. 1991; 266: 16745-16785Google Scholar. Samples (80 μg of chlorophyll or 250 μg of protein; pea, n = 3; spinach, n = 7; tobacco n = 2) were prepared by acetone precipitation prior to dissolution in 60% HCOOH (diluted from 90% ACS grade; Fisher). Primary reverse-phase chromatography was performed as described previously 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar. The poly(styrene-divinylbenzene) coploymer (Polymer Labs PLRP/S; 5 μm × 300 Å; 2.1 × 150 mm) stationary-phase column was eluted at a flow rate of 100 μl/min at 40 °C. The primary gradient (Buffer A, 0.1% trifluoroacetic acid/water; Buffer B, 0.1% trifluoroacetic acid/acetonitrile) eluted extrinsic polypeptides and predominantly small to moderately sized intrinsic proteins. The column was equilibrated in 5% Buffer B followed by a stepped linear gradient from 5 to 25% Buffer B between 5 and 10 min after injection, 25 to 75% Buffer B between 10 and 130 min, and 75 to 100% Buffer B between 130 and 150 min.The spinach D1 spectra (see Fig. 2A) was obtained by acetone precipitation of PS II reaction centers (100 μg of protein, prepared according to Ref. 11Whitelegge J.P. Jewess P. Pickering M.G. Gerrish C. Camilleri P. Bowyer J.R. Sequence analysis of photoaffinity-labeled peptides derived by proteolysis of photosystem 2 reaction centers from thylakoid membranes treated with [14C]azidoatrazine.Eur. J. Biochem. 1992; 207: 1077-1084Google Scholar that were dissolved in 60% formic acid and subjected to reverse-phase chromatography with the gradient described above, except using Buffer A and Buffer C (0.05% trifluoroacetic acid in 1:1 acetonitrile/2-propanol, v/v). The addition of 2-propanol improves the elution efficiency for larger intrinsic membrane proteins.A secondary gradient (Buffer D, 60% HCOOH; Buffer E, 2-propanol) was used to elute the very hydrophobic PS II polypeptides that remained bound to the column after the primary chromatographic elution in the AB buffer system described above 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar, 11Whitelegge J.P. Jewess P. Pickering M.G. Gerrish C. Camilleri P. Bowyer J.R. Sequence analysis of photoaffinity-labeled peptides derived by proteolysis of photosystem 2 reaction centers from thylakoid membranes treated with [14C]azidoatrazine.Eur. J. Biochem. 1992; 207: 1077-1084Google Scholar. The column was equilibrated in 95% Buffer D, 5% Buffer E, prior to linear gradient elution to 100% Buffer E over 55 min. Six primary runs were used to accumulate material for each secondary elution.Mass spectra were recorded on a PerkinElmer Life Sciences Sciex API III+ triple-quadrupole mass spectrometer with an Ionspray™ source (Applied Biosystems, Foster City, CA) as described 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar. The instrument was scanned from 600–2300 m/z with a step size of 0.3 m/z and a dwell of 1 ms giving a total scan time of 6 s. An orifice potential of 65 V was used in all experiments. The computations of measured protein molecular mass were made using MacSpec 3.3 and zero-charge molecular mass reconstructions using BioMultiView 1.3.1 software (Applied Biosystems, Foster City, CA). Calculated average molecular masses were generated from translated published gene sequences (GenBank™) or published protein sequences (PIR or Swiss-Prot) using PeptideMass (expasy.cbr.nrc.ca/tools/peptide-mass.html). Post-translational modifications of thylakoid proteins were predicted by comparison of published modifications of orthologs.Predictions of the HPLC retention time of proteins under 40 kDa were made by searching the ARATH (Arabidopsis thaliana) subset of the Swiss-Prot and TrEMBL data bases with TagIdent (ca.expasy.org/tools/tagident.html). The ProtParam tool at ExPASy was used to calculate a predicted hydrophobicity (GRAVY; grand average of hydropathicity) of each of the proteins found in the TagIdent search. The GRAVY results from Arabidopsis thaliana were plotted against the predicted masses and compared with the observed pea HPLC retention times. The results of the TagIdent search (performed in July 2001) represent a crude data set because of the lack of, or incorrect, annotation of the A. thaliana genomes, especially of nuclear-encoded chloroplast proteins that are proteolytically trimmed of a substantial N-terminal leader sequence prior to thylakoid insertion in vivo.RESULTSThylakoid membrane proteins were stripped of lipids, chlorophyll, and other pigments by precipitation with acetone and dissolved in 60% formic acid for the primary reverse-phase HPLC analysis coupled with electrospray-ionization mass spectrometry (LC-MS; see Ref. 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar. Many polypeptides elute efficiently under standard conditions of 0.1% trifluoroacetic acid with increasing acetonitrile concentration. Averaging the elution profiles of several experiments allows a generalized elution map to be generated (Fig. 1). The trace shown follows total ion production across the scanned mass range such that peaks appear as molecules of different masses elute, rather like a protein UV elution profile except that relative abundance can be biased by the potential for different proteins to have different ionization efficiencies. Generally, the abundance of smaller proteins is exaggerated, because they ionize more efficiently than larger ones.The very hydrophobic proteins elute from the column with lowered efficiency such that a substantial proportion remain bound to the column. Thus more hydrophobic PS II core polypeptides, including larger ones such as D2, CP43, and CP47, and small ones such as PsbM, are variably detected in the primary elution profile for appressed membrane fractions. The addition of isopropanol to acetonitrile (Buffer C) improved elution efficiency, though optimal spectra of D1 and D2 were obtained only by loading larger quantities of highly purified PS II reaction-center preparations (a subfraction of the appressed thylakoids used for the primary elution described above) to reduce the total number of proteins in the sample (Fig. 2A). Efficient elution of the more hydrophobic PS II polypeptides was achieved in 60% formic acid with elution by isopropanol (Buffers D and E), but chromatographic resolution is impaired, and less complex mixtures are preferable 3Whitelegge J.P. Gundersen C. Faull K.F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.Protein Sci. 1998; 7: 1423-1430Google Scholar. The generalized primary elution profile for appressed membranes (Fig. 1) was annotated with peak identities assigned by comparison of measured intact masses to values calculated based upon gene sequences and available information on post-transcriptional and post-translational modifications. There are few proteins for which all this information is available. However, specific genes in related species (orthologs) are generally highly conserved in sequence, and known modifications in one ortholog were useful for predicting the same modification in another.Fig. 2Electrospray-ionization mass spectrometry of minor thylakoid membrane protein isoforms.A, mass spectrum of spinach D1 protein. A segment of the mass spectrum showing four multiply charged ions derived from D1 is shown to illustrate signal to noise and the detection of a minor palmitoylated species. Each ion is labeled with its charge state and the measured m/z of the minor species. The molecular weight of the uncharged molecule is calculated by multiplying m/z by the charge state and subtracting the mass of the charges (protons). Measured molecular weight is averaged from all detectable signals across the complete mass spectrum (Hypermass, PE Sciex; Applied Biosystems, Foster City, CA). B, zero-charge molecular weight spectrum of spinach D1 was reconstructed from corresponding mass spectrum by computation (BioMultiView, PE Sciex; Applied Biosystems, Foster City, CA).View Large Image Figure ViewerDownload (PPT)Extrinsic polypeptides, including those of the oxygen-evolving complex, elute early whereas more hydrophobic intrinsic polypeptides elute later in the primary elution profile. The masses of detected proteins and their proposed identities are presented in Table I in the order of their elution. Each significant mass spectrometric signal yielding a molecular weight is considered an intact mass tag (IMT) and compared with the data base. Some IMTs are easily assigned, because they correspond closely to well studied proteins whose mass can be calculated with great confidence based upon previous characterization. The native masses of some of the proteins that have been previously identified match very well with the IMT and are so indicated in Table I. Others are assigned based upon the pure coincidence of measured and calculated masses, with confidence being modulated by the number of other proteins of similar mass and similar hydrophobicity within the sample. In special cases it was necessary to compare all known orthologs of a particular protein and use phylogenetic conservation of sequence to identify probable sequence errors. 3J. P. Whitelegge, S. M. Gómez, and K. F. Faull, manuscript in preparation. The calculated masses presented here are based upon sequences from data base entries without such sequence correction. Of the 90 IMTs recognized in the primary/secondary elution profiles (see Tables I and II), 40 genes were assigned, leaving 30 IMTs unassigned. Some of these likely correspond to the products of genes whose function remains as yet unidentified or proteins for which there is no gene sequence data in pea or spinach.TABLE IGeneralized primary reverse-phase elution map of intact mass tags from appressed thylakoid membrane subfractionsIMTaIMTs assembled in order of elution during the primary reverse-phase chromatographic elution. The mean ± S.D. of n experiments is presented. pea (± S.D.) (n = 4 experiments)Calculated massesbCalculated average mass of the uncharged assigned gene product shown in ID column. (Da)ΔcΔ, % difference between expected and observed masses; X, Δ < 0.01%; Y, Δ = 0.01–0.02%; Z, Δ = 0.02–0.07%. 1, IMT 26532.1 is in the mass range and retention time for PsbO, except the mass does not match the published sequence. The mass difference of 129 Da could reasonably be expected to be because of one or more amino acid changes. Removal of a single Q from the C terminus (26533.7 calculated) results in excellent agreement, for example. 2, the pea PsbH sequence has at least two amino acid changes from the published sequence. We have identified one (A18V) by MS/MS (not shown). 3, the pea petD sequence does not include the first exon and erroneously starts with an AUG codon in the second exon. A chimeric protein translated from the first exon of the spinach sequence and the second exon from pea gives a calculated mass of 17369.7 Da. The mass difference is consistent with changing either Pro8 or Pro12 encoded in exon 1 of spinach petD to Ser. 4, the published pea psbT gene sequence appears to have an in-frame deletion that is currently being confirmed.IMTaIMTs assembled in order of elution during the primary reverse-phase chromatographic elution. The mean ± S.D. of n experiments is presented. spinach (± S.D.) (n = 7 experiments)Calculated massesbCalculated average mass of the uncharged assigned gene product shown in ID column. (Da)ΔcΔ, % difference between expected and observed masses; X, Δ < 0.01%; Y, Δ = 0.01–0.02%; Z, Δ = 0.02–0.07%. 1, IMT 26532.1 is in the mass range and retention time for PsbO, except the mass does not match the published sequence. The mass difference of 129 Da could reasonably be expected to be because of one or more amino acid changes. Removal of a single Q from the C terminus (26533.7 calculated) results in excellent agreement, for example. 2, the pea PsbH sequence has at least two amino acid changes from the published sequence. We have identified one (A18V) by MS/MS (not shown). 3, the pea petD sequence does not include the first exon and erroneously starts with an AUG codon in the second exon. A chimeric protein translated from the first exon of the spinach sequence and the second exon from pea gives a calculated mass of 17369.7 Da. The mass difference is consistent with changing either Pro8 or Pro12 encoded in exon 1 of spinach petD to Ser. 4, the published pea psbT gene sequence appears to have an in-frame deletion that is currently being confirmed.IDdProtein IDs in quotes indicate assignments made by retention times consistent with membrane localization and similar masses from published sequences from other plants. IDs in italic type indicate that a tobacco IMT (not shown) was correlated with a published tobacco gene sequence and that the protein eluted at a similar time to the pea and spinach orthologs. O, oxygen; P, phosphate; Ac, acetyl; F, formyl; fM, N-formylmethionine. Five unassigned post-translationally modified masses are labeled in lower case letters (a–e) in order of elution. All accession numbers are from GenBank™, unless otherwise noted. Pea: psaC, X13157; psbP, X15552; psbO, X15350; petC, X63065; psbF, psbJ, psbE, psbL, X15767; Ihcb1*4, X56338; Ihcb1*2, K02067; Ihcb2*1, X57082; Ihcb3*1, X69215; psbD, M27309; psbB, psbH, psbT, AF153442; petD, X00535; psbA, M11005; psbC, M27309; psbM, D12535; PsbI (see Ref. 39). Spinach: PsbX, pir# SO3277 (unidentified X changed to Cys); psaE, X14018; psaD, X14617; psbP, X05511; psbQ, X05512; psbO, X05548; psaH, X16858; petC, X06244; psbF, psbE, psbL, M35673; psbR, J03887; psaF, X13133; Ihcb1*1, X14341; Ihcb6*1, Z25886; psbS, X68552; psbY2, AF060198; psbB, NP054960; psbH, NP054963 amino acids 8–79 (earlier entries initiate from the second methionine 7, but internal sequence errors are corrected in this sequence); psbT, P37259; psbM, NP054926; psbI, NP054916; psbW, X85038; petD, X07106.3296.5 ± 0.23297.9ZPsbX + O3187.3 ± 0.23279.7 ± 0.43281.9ZPsbX3485.2 ± 0.43163.2 ± 0.33000.0 ± 0.72088.7 ± 0.29894.3 ± 1.29728.9 ± 0.99729.0XPsaE9824.4 ± 1.19657.7 ± 2.09657.9XPsaEΔAla4028.8 ± 0.59721.3 ± 0.9"PsaN"9150.5 ± 1.38641.0 ± 1.68848.4 ± 0.98849.3XPsaC17936.3 ± 2.317871.8 ± 1.417872.6XPsaD + O17921.3 ± 1.417856.7 ± 2.117856.6XPsaD20225.4 ± 1.520225.6XPsbP + O20265.0 ± 1.620264.6X20209.6 ± 2.020209.6XPsbP16375.2 ± 2.016522.7 ± 2.916521.8XPsbQ17829.4 ± 2.1a + 2F17801.7 ± 2.7a + F17774.6 ± 2.3a16143.4 ± 2.6b + Ac16102.0 ± 1.0b12361.4 ± 1.326525.0 ± 1.826525.7X26532.1 ± 2.926661.81PsbO10276.3 ± 0.910381.6 ± 1.210381.8XPsaH15564.7 ± 2.012941.4 ± 2.513815.4 ± 1.822037.2 ± 2.018956.5 ± 3.218954.6XPetC + O19074.7 ± 2.419074.8X18940.4 ± 2.318938.6XPetC11320.3 ± 1.310764.8 ± 0.529474.1 ± 2.68596.3 ± 0.86927.9 ± 1.36924.0 ± 1.1c + Ac + O6911.6 ± 0.76907.3 ± 0.9c + Ac6869.2 ± 0.26869.0 ± 1.5c4411.1 ± 1.24411.2X4424.7 ± 0.54425.2XPsbF + O4394.9 ± 0.34395.2X4409.2 ± 0.64409.2XPsbF15178.6 ± 1.015171.8 ± 2.422230.6 ± 1.821781.0 ± 1.8d + Ac21677.3 ± 1.6d + O21739.0 ± 1.321661.2 ± 3.6d21441.8 ± 2.84854.8 ± 0.725285.3 ± 3.325298.6 ± 2.910205.7 ± 1.410234.9 ± 1.410235.7XPsbR8389.0 ± 0.49685.3 ± 0.921782.7 ± 2.822345.6 ± 2.517255.3 ± 1.217272.3 ± 3.017275.1YPsaF3983.3 ± 0.34585.6 ± 0.624574.8 ± 1.324839.7 ± 2.824972.0 ± 2.224972.3X24760.2 ± 2.3Ac-Lhcb1*Ps424981.1 ± 1.925019.1 ± 3.425020.4XAc-Lhcb1*So125036.4 ± 2.225036.3XAc-Lhcb1*Ps2 + P24959.1 ± 1.524956.3Y24946.3 ± 2.9Ac-Lhcb1*Ps224836.4 ± 2.124837.1XAc-Lhcb2*Ps124750.7 ± 1.924936.2 ± 2.624839.1 ± 1.425083.7 ± 3.024774.2 ± 1.925005.2 ± 3.222913.4 ± 1.622856.1 ± 3.422842.5 ± 1.622814.0 ± 1.922813.0XLhcb6*So122302.9 ± 1.822233.8 ± 1.624332.7 ± 2.324330.8X24323.3 ± 2.3Lhcb3*Ps122952.9 ± 3.124345.9 ± 2.028690.8 ± 2.328071.8 ± 3.9"Lhcb4"26262.5 ± 2.426562.8 ± 1.927067.4 ± 2.7"Lhcb5"22169.8 ± 1.822568.6 ± 2.122568.3XAc-PsbS4540.3 ± 0.44960.4 ± 0.54960.8XPsbY24482.0 ± 0.0"PsbK"23345.8 ± 2.023324.0 ± 2.927379.2 ± 2.623279.4 ± 2.99283.5 ± 0.99283.4X9255.4 ± 0.99255.4XPsbE17934.6 ± 2.218056.5 ± 1.8e + O17919.6 ± 1.118040.2 ± 1.6e4409.3 ± 0.53972.1 ± 0.44272.7 ± 0.63866.0 ± 0.24367.1 ± 0.14366.0Y4365.9 ± 0.84366.0XPsbL4759.7 ± 0.239481.7 ± 2.839475.9YAc-PsbD39439.6 ± 2.539437.5XPsbD56032.2 ± 6.756064.0Ac-PsbB10101.1 ± 1.67774.0 ± 0.67774.9YPsbH + 2P + O7792.4 ± 0.57823.027694.3 ± 0.67694.9XPsbH + P + O7713.9 ± 0.87743.027613.9 ± 1.17614.9YPsbH + O7857.4 ± 1.17887.027758.8 ± 0.67758.9XPsbH + 2P7777.3 ± 0.97807.027679.3 ± 0.97678.9XPsbH + P7697.3 ± 1.07727.027599.5 ± 1.27598.9XPsbH5913.2 ± 0.45927.7 ± 0.45927.7XPsbW17329.3 ± 1.617329.6XPetD + O17359.1 ± 1.215136.0317313.7 ± 2.017313.6XPetD4140.6 ± 0.64112.54fM-PsbT + P4060.8 ± 0.74032.543849.0 ± 0.43849.7YfM-PsbT4284.9 ± 0.350205.9 ± 8.350204.9XAc-PsbC3809.8 ± 0.63810.6YfM-PsbM4212.0 ± 0.64211.9XfM-PsbI + O4210.3 ± 0.64209.8Y4195.8 ± 0.54195.9XfM-PsbIa IMTs assembled in order of elution during the primary reverse-phase chromatographic elution. The mean ± S.D. of n experiments is presented.b Calculated average mass of the uncharged assigned gene product shown in ID column.c Δ, % difference between expected and observed masses; X, Δ < 0.01%; Y, Δ = 0.01–0.02%; Z, Δ = 0.02–0.07%. 1, IMT 26532.1 is in the mass range and retention time for PsbO, except the mass does not match the published sequence. The mass difference of 129 Da could reasonably be expected to be because of one or more amino acid changes. Removal of a single Q from the C terminus (26533.7 calculated) results in excellent agreement, for example. 2, the pea PsbH sequence has at least two amino acid changes from the published sequence. We have identified one (A18V) by MS/MS (not shown). 3, the pea petD sequence does not include the first exon and erro
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