A New Approach for Plant Proteomics

A recently developed methodology for the characterization of complex proteomes, top-down Fourier transform mass spectrometry (FTMS), is applied for the first time to a plant proteome, that of the model plant Arabidopsis thaliana. Of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate “bottom-up” mass spectrometry studies in which the proteins were purified and digested and in which the mass spectrometry-measured mass values of the resulting peptides matched against those expected from the DNA-predicted proteins. In the top-down approach applied here, molecular ions from a protein mixture are purified, weighed exactly (±1 Da), and fragmented in the FTMS. Of the 22 molecular weight values found in three isolated mixtures, 7 were chosen, and their primary structures were fully characterized; in only one case was the bottom-up structure in full agreement. The top-down technique is not only efficient for identification of the DNA-predicted precursors, such as that of a protein present as a 5% mixture component, but also for characterization of the primary structure of the final protein. For two proteins the previously predicted cleavage site for loss of the signal peptide was found to be incorrect. Two 27-kDa proteins are fully characterized, although they are found to differ by only 12 residues and 6 Da in mass in a 3:1 ratio; the bottom-up studies did not distinguish these proteins. Direct tandem mass spectrometry dissociation of two 15-kDa molecular ions showed >90% sequence similarity, whereas three-stage mass spectrometry traced their +14-Da molecular mass discrepancies to an unusual N-methylation on the N-terminal amino group; the bottom-up approach identified only one precursor protein. The high potential of the top-down FTMS approach for characterization as well as identification of complex plant proteomes should provide a real incentive for its further automation. A recently developed methodology for the characterization of complex proteomes, top-down Fourier transform mass spectrometry (FTMS), is applied for the first time to a plant proteome, that of the model plant Arabidopsis thaliana. Of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate “bottom-up” mass spectrometry studies in which the proteins were purified and digested and in which the mass spectrometry-measured mass values of the resulting peptides matched against those expected from the DNA-predicted proteins. In the top-down approach applied here, molecular ions from a protein mixture are purified, weighed exactly (±1 Da), and fragmented in the FTMS. Of the 22 molecular weight values found in three isolated mixtures, 7 were chosen, and their primary structures were fully characterized; in only one case was the bottom-up structure in full agreement. The top-down technique is not only efficient for identification of the DNA-predicted precursors, such as that of a protein present as a 5% mixture component, but also for characterization of the primary structure of the final protein. For two proteins the previously predicted cleavage site for loss of the signal peptide was found to be incorrect. Two 27-kDa proteins are fully characterized, although they are found to differ by only 12 residues and 6 Da in mass in a 3:1 ratio; the bottom-up studies did not distinguish these proteins. Direct tandem mass spectrometry dissociation of two 15-kDa molecular ions showed >90% sequence similarity, whereas three-stage mass spectrometry traced their +14-Da molecular mass discrepancies to an unusual N-methylation on the N-terminal amino group; the bottom-up approach identified only one precursor protein. The high potential of the top-down FTMS approach for characterization as well as identification of complex plant proteomes should provide a real incentive for its further automation. Elucidation of the DNA sequences for bacterial, animal, and human genomes has revolutionized characterization of their expressed proteins (1Williams K.L. Hochstrasser D.F. Proteome Research: New Frontiers in Functional Genomics. 1997; : 1-12, Springer-Verlag, BerlinGoogle Scholar, 2Abbott A. A post-genomic challenge: learning to read patterns of protein synthesis.Nature. 1999; 402: 715-720Google Scholar). In a similar fashion, the recent sequencing of the genome for Arabidopsis thaliana (3Salanoubat M. Lemcke K. Rieger M. Ansorge W. Unseld M. Fartmann B. Valle G. Blocker H. Perez-Alonso M. Obermaier B. Delseny M. Boutry M. Grivell L.A. Mache R. Puigdomenech P. et al.Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.Nature. 2000; 408: 820-822Google Scholar, 4Tabata S. Kaneko T. Nakamura Y. Kotani H. Kato T. Asamizu E. Miyajima N. Sasamoto S. Kimura T. Hosouchi T. Kawashima K. Kohara M. Matsumoto M. Matsuno A. Muraki A. et al.Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.Nature. 2000; 408: 823-826Google Scholar) has made plant proteomics an exciting research field (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar, 6Schubert M. Petersson U.A. Haas B.J. Funk C. Schroder W.P. Kieselbach T. Proteome map of the chloroplast lumen of Arabidopsis thaliana.J. Biol. Chem. 2002; 277: 8354-8365Google Scholar). The plant cell has unique organelles such as the chloroplast, which is essential not only in photosynthesis but also in biosynthesis, for example, of lipids and amino acids (7Leister D. Chloroplast research in the genomic age.Trends in Genetics. 2003; 19: 47-56Google Scholar). Of the ∼3000 proteins predicted for the chloroplast of Arabidopsis, identification of 97 as the precursors of isolated proteins has been achieved with two studies (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar, 6Schubert M. Petersson U.A. Haas B.J. Funk C. Schroder W.P. Kieselbach T. Proteome map of the chloroplast lumen of Arabidopsis thaliana.J. Biol. Chem. 2002; 277: 8354-8365Google Scholar) utilizing the bottom-up mass spectrometry (MS) 1The abbreviations used are: MS, mass spectrometry; MS/MS, tandem mass spectrometry; MS2, tandem mass spectrometry; MS3, three-stage mass spectrometry; MSn, multistage mass spectrometry; ESI, electrospray ionization; FTMS, Fourier transform mass spectrometry; SWIFT, stored waveform inverse Fourier transform; CAD, collisionally activated dissociation; IRMPD, infrared multiphoton dissociation; ECD, electron capture dissociation; SEC, size exclusion chromatography; rubisco, ribulose-bisphosphate carboxylase/oxygenase. technique (8Andersen J.S. Svensson B. Roepstorff P. Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: Powerful analytical tools in recombinant protein chemistry.Nat. Biotechnol. 1996; 14: 449-457Google Scholar, 9Qin J. Chait B.T. Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry.Anal. Chem. 1997; 69: 4002-4009Google Scholar, 10Pandey A. Mann M. Proteomics to study genes and genomes.Nature. 2000; 405: 837-846Google Scholar); the study in the laboratory of one of the present authors (K. J. vW.) identified 81 proteins (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar). However, this is far less effective for characterization of the primary structure of an isolated protein, such as determining the cleavage site for loss of the signal peptide and locating posttranslational modifications, as well as distinguishing highly similar proteins. For this, the recently developed technique of “top-down” MS has uniquely valuable attributes (11Kelleher N.L. Taylor S.V. Grannis D. Kinsland C. Chiu H.J. Begley T.P. McLafferty F.W. Efficient sequence analysis of the six gene products (7–74 kDa) from the by tandem mass Scholar, N.L. McLafferty F.W. protein characterization by tandem mass Chem. 1999; Scholar, F.W. mass 1999; Scholar, N.L. primary structure to from mass Biol. 2000; Scholar, B.J. N.L. and of protein identification in and the Biotechnol. 19: Scholar, B.J. N.L. complex of proteins for analysis by mass spectrometry.Anal. Chem. 2002; Scholar, Y. M. E. J. Begley T.P. McLafferty F.W. characterization of proteins kDa) by electron capture dissociation mass Chem. 2002; Scholar, Y. M. Begley T.P. McLafferty F.W. H. characterization of proteins from by electron capture dissociation mass 2003; 14: Scholar). the two MS for the identification and characterization of chloroplast proteins. In the bottom-up approach the protein is first purified and peptides with molecular weight values are by MS (8Andersen J.S. Svensson B. Roepstorff P. Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: Powerful analytical tools in recombinant protein chemistry.Nat. Biotechnol. 1996; 14: 449-457Google Scholar, 9Qin J. Chait B.T. Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry.Anal. Chem. 1997; 69: 4002-4009Google Scholar, 10Pandey A. Mann M. Proteomics to study genes and genomes.Nature. 2000; 405: 837-846Google Scholar). The resulting mass is matched against those expected from the DNA-predicted proteins to the of peptides provide for of valuable for proteins of J. E. Direct analysis of protein mass Biotechnol. 1999; Scholar). However, to the structure of the isolated protein, to the predicted and protein sequence, from peptides of the protein, which is made by the sequence of (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar, J.S. Svensson B. Roepstorff P. Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: Powerful analytical tools in recombinant protein chemistry.Nat. Biotechnol. 1996; 14: 449-457Google Scholar, 9Qin J. Chait B.T. Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry.Anal. Chem. 1997; 69: 4002-4009Google Scholar, J.B. Friso G. Kalume D.E. Roepstorff P. van Wijk K.J. Proteomics of the identification and analysis of lumenal and peripheral thylakoid Cell. 2000; Scholar). peptide mass to be to signal peptide and posttranslational modifications also be to (1Williams K.L. Hochstrasser D.F. Proteome Research: New Frontiers in Functional Genomics. 1997; : 1-12, Springer-Verlag, BerlinGoogle Scholar, 2Abbott A. A post-genomic challenge: learning to read patterns of protein synthesis.Nature. 1999; 402: 715-720Google Scholar, J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar). The signal peptide cleavage site be determined by an of (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar) predicted O. H. Heijne G. a for chloroplast peptides and their cleavage 1999; Scholar). However, the high identification of the well bottom-up has to its for In the top-down approach (11Kelleher N.L. Taylor S.V. Grannis D. Kinsland C. Chiu H.J. Begley T.P. McLafferty F.W. Efficient sequence analysis of the six gene products (7–74 kDa) from the by tandem mass Scholar, N.L. McLafferty F.W. protein characterization by tandem mass Chem. 1999; Scholar, F.W. mass 1999; Scholar, N.L. primary structure to from mass Biol. 2000; Scholar, B.J. N.L. and of protein identification in and the Biotechnol. 19: Scholar, B.J. N.L. complex of proteins for analysis by mass spectrometry.Anal. Chem. 2002; Scholar, Y. M. E. J. Begley T.P. McLafferty F.W. characterization of proteins kDa) by electron capture dissociation mass Chem. 2002; Scholar, Y. M. Begley T.P. McLafferty F.W. H. characterization of proteins from by electron capture dissociation mass 2003; 14: is not and the protein mixture is the Fourier transform MS McLafferty F.W. Fourier transform electrospray for tandem high mass spectrometry of Scholar) electrospray ionization J.B. Mann M. Electrospray ionization for mass spectrometry of Scholar, McLafferty F.W. J. D.F. Fourier transform mass spectrometry of by electrospray S. A. Scholar). to proteins is with a 5% mixture top-down characterization of mixture has been Y. M. E. J. Begley T.P. McLafferty F.W. characterization of proteins kDa) by electron capture dissociation mass Chem. 2002; Scholar, Y. M. Begley T.P. McLafferty F.W. H. characterization of proteins from by electron capture dissociation mass 2003; 14: Scholar). The resulting mass values for the proteins present in the high of the molecular ions of an protein, these are for dissociation Fourier transform mass dissociation technique that infrared multiphoton Scholar, McLafferty F.W. multiphoton dissociation of ions for Chem. Scholar, McLafferty F.W. of ions Fourier transform mass spectrometry.Anal. Chem. Scholar, N.L. McLafferty F.W. capture dissociation of protein A Chem. Scholar, McLafferty F.W. capture dissociation of proteins is and of high Chem. 1999; Scholar, N.L. McLafferty F.W. capture dissociation for characterization of protein Chem. 2000; Scholar, Y. McLafferty F.W. ion electron capture dissociation for mass sequencing of kDa) Chem. 2000; Scholar, Y. H. McLafferty F.W. electron capture dissociation for the characterization of proteins by mass spectrometry.Anal. Chem. 2003; and the resulting are matched against the DNA-predicted sequence to the protein. its that of the predicted signal peptide not the such discrepancies in the modifications sequence (11Kelleher N.L. Taylor S.V. Grannis D. Kinsland C. Chiu H.J. Begley T.P. McLafferty F.W. Efficient sequence analysis of the six gene products (7–74 kDa) from the by tandem mass Scholar, N.L. McLafferty F.W. protein characterization by tandem mass Chem. 1999; Scholar, F.W. mass 1999; Scholar, N.L. primary structure to from mass Biol. 2000; Scholar, B.J. N.L. and of protein identification in and the Biotechnol. 19: Scholar, B.J. N.L. complex of proteins for analysis by mass spectrometry.Anal. Chem. 2002; Scholar, Y. M. E. J. Begley T.P. McLafferty F.W. characterization of proteins kDa) by electron capture dissociation mass Chem. 2002; Scholar, Y. M. Begley T.P. McLafferty F.W. H. characterization of proteins from by electron capture dissociation mass 2003; 14: Scholar). approach is applied to (±1 values for 22 proteins and to and from the three proteomes thylakoid and of the chloroplast of A. thaliana identified previously by bottom-up MS (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar, 6Schubert M. Petersson U.A. Haas B.J. Funk C. Schroder W.P. Kieselbach T. Proteome map of the chloroplast lumen of Arabidopsis thaliana.J. Biol. Chem. 2002; 277: 8354-8365Google Scholar). A. was on a and a of were in The were in 7 and of 22 and the was for 3 The resulting in the were on a for 3000 to two The was in and in with 5 and in the with and and and a of of was three a and the thylakoid were by The was with with and of in 6 and to the thylakoid lumen proteins. The thylakoid were on with of for with for and for and the was with a to 5 The proteins were in with in a of and in 6 and to the thylakoid peripheral proteins. chloroplast were that they were in the and were on a and for The was with and for 3 The was in 5 and the and this was to the proteins. The were for and the was to to the protein lumen proteins were by on a with with as a were to of peripheral proteins were ion on a and with a of was on the with and as a The as a was in 6 and and the resulting and were by as for thylakoid with and were to the with was for for and for were on a protein trap with of and with of was a with a with the MS a of The resulting ions were the ion cell of a 6 FTMS McLafferty F.W. Fourier transform electrospray for tandem high mass spectrometry of Scholar). was achieved by ion electron capture dissociation Y. McLafferty F.W. ion electron capture dissociation for mass sequencing of kDa) Chem. 2000; infrared multiphoton dissociation McLafferty F.W. multiphoton dissociation of ions for Chem. Scholar) for ions the FTMS cell by ions in the cell stored waveform inverse Fourier transform of in Fourier transform ion mass spectrometry stored inverse Fourier transform Chem. Scholar) by collisionally activated dissociation for dissociation Fourier transform mass dissociation technique that infrared multiphoton Scholar, McLafferty F.W. of ions Fourier transform mass spectrometry.Anal. Chem. Scholar). sequence from the were used to the of A. thaliana H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; Scholar). the were the L. and developed for top-down FTMS by and B.J. N.L. complex of proteins for analysis by mass spectrometry.Anal. Chem. 2002; Scholar, Y. R. N.L. and for identification of proteins mass spectrometry.Anal. Chem. 2003; Scholar). of were made with the McLafferty F.W. and of high electrospray mass of 2000; Scholar). The mass of the and the is in The chloroplast proteome from A. thaliana was (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar) three those that the thylakoid peripheral the thylakoid lumen and the proteins. For the first two the molecular of the are in and were further by the of this is by of the of lumen of from of the thylakoid lumen mass of with of molecular ion Direct electrospray ionization of the protein mass such as that of the thylakoid peripheral For the is the molecular weight and the of of the the mass the of proteins of values and The of from the thylakoid lumen values of and whereas that of the proteins values of and values of and in three mass spectrometry primary sequence by MS and dissociation of molecular on of an amino by the mass in the resulting ions and such for residues provide a E. Roepstorff P. N.L. McLafferty F.W. Mann M. identification of proteins by tandem mass against sequence S. A. 1996; such as in be in the the was from the and to ions of which the ions were and fragmented to the sequence In the in 7 A protein characterization in lumen by and of the of the molecular ions from that the sequence cleavage from this full those from an with the DNA-predicted sequence of protein of two similar peripheral lumen of a to the of two for values by cleavage from the ECD, CAD, and of the ions from to the DNA-predicted sequences for and For the residues that are are the and that are unique are by a of posttranslational by three-stage mass of the purified of the molecular of ions of the in A. of of molecular ions also provide sequence the protein and these against DNA-predicted sequences and posttranslational modifications (11Kelleher N.L. Taylor S.V. Grannis D. Kinsland C. Chiu H.J. Begley T.P. McLafferty F.W. Efficient sequence analysis of the six gene products (7–74 kDa) from the by tandem mass Scholar, N.L. McLafferty F.W. protein characterization by tandem mass Chem. 1999; Scholar, F.W. mass 1999; Scholar, N.L. primary structure to from mass Biol. 2000; Scholar, B.J. N.L. and of protein identification in and the Biotechnol. 19: Scholar, B.J. N.L. complex of proteins for analysis by mass spectrometry.Anal. Chem. 2002; Scholar, Y. M. E. J. Begley T.P. McLafferty F.W. characterization of proteins kDa) by electron capture dissociation mass Chem. 2002; Scholar, Y. M. Begley T.P. McLafferty F.W. H. characterization of proteins from by electron capture dissociation mass 2003; 14: Scholar). The by the for dissociation Fourier transform mass dissociation technique that infrared multiphoton Scholar, McLafferty F.W. of ions Fourier transform mass spectrometry.Anal. Chem. Scholar) and McLafferty F.W. multiphoton dissociation of ions for Chem. Scholar) are to the from N.L. McLafferty F.W. capture dissociation of protein A Chem. Scholar, McLafferty F.W. capture dissociation of proteins is and of high Chem. 1999; Scholar, N.L. McLafferty F.W. capture dissociation for characterization of protein Chem. 2000; of the protein by ions from McLafferty F.W. sequencing of proteins by tandem mass S. A. 2000; Scholar). The bottom-up MS identification of the Arabidopsis proteins of the protein, and the top-down approach complex protein mixtures, and are for the of the protein of the For this the mass is For example, the values from the thylakoid peripheral proteins of and to and provide molecular weight However, with the of values in this the kDa) proteins by not in the For the thylakoid peripheral the analysis an protein in 5% of in Fourier transform ion mass spectrometry stored inverse Fourier transform Chem. Scholar) of ions from the cell isolated the in of these ions high mass products to mass of and Da are of and the DNA-predicted protein sequences H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; Scholar) for one with this sequence found only for H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; Scholar) with its sequence However, this protein the predicted of the signal peptide (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google the of (±1 this the in the were of to be fragmented by this of and be from the as the predicted from the of this ion be and The predicted of loss of the signal only of the sequence the protein is by a The signal peptide loss did not the two predicted mass of Da to the of the of these residues a predicted of in with the of the molecular ion showed a high for amino loss in MS2, the of this ion C. T. dissociation in of the N-terminal and J. 1997; Scholar) has this that further dissociation the identified the protein from those but the showed that the predicted sequence was with the sequence of the to the N-terminal signal peptide of the protein the of sequence and such top-down identification In from the thylakoid lumen only six protein molecular ions in and are of the isolated ions of a sequence of mass of and Da Of the predicted the only found in the was H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; Scholar) for with the residues sequence of but with the predicted of the signal peptide (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar). The of Da to the of the of the amino residues in the DNA predicted sequence signal peptide and Da), that the protein also these N-terminal amino In N-terminal ions and the predicted sequence and the of posttranslational the bottom-up methodology identified the DNA-predicted precursor protein, top-down fully the primary structure of the isolated protein. For the protein, of the ions and of the and ions sequence that not only a that matched the predicted protein H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; Scholar) but also a of ions fully this sequence posttranslational this is also by the with the predicted of are in which an (±1 an B.J. N.L. and of protein identification in and the Biotechnol. 19: Scholar, Y. M. Begley T.P. McLafferty F.W. H. characterization of proteins from by electron capture dissociation mass 2003; 14: Scholar). the bottom-up identification has been by but with far of the thylakoid peripheral to in the although the is that expected for a in and of the isolated and ions a sequence of that matched of two H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; Scholar) and these of are for However, the mass values of the and ions the sequence only matched those expected for also the technique of electron capture and and of the ions a of ions that be only from the protein that from proteins. However, ions were also found that be only by the protein, the of The bottom-up study found only one peptides with of the DNA-predicted precursors (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar). the of the bottom-up methodology to these highly similar proteins the FTMS the protein mixture was further and to and the was for the resulting peptide peptide mass of of which are to protein and to protein. by ion peptide mass of of which the only were two for protein. mass of of which matched the protein and matched the protein. in this bottom-up approach values that the of the protein, but with values as well as further and three characterization of these with and the of the be two predicted these values the A 3:1 of the the in In a of the of ions uniquely from the whereas with three a of peptides uniquely from MS the protein to be the of are and a for the protein used in the bottom-up Of these the two and of their isolated molecular ions sequence although these mass were from ion mass of this the DNA-predicted sequence identified two and H. P. H. Lemcke K. S. G. R. Arabidopsis thaliana an on the first plant 2002; of the of that is in the The bottom-up studies only identified (5Peltier J.B. Emanuelsson O. Kalume D.E. Ytterberg J. Friso G. Rudella A. Liberles D.A. Soderberg L. Roepstorff P. von Heijne G. van Wijk K.J. Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction.Plant Cell. 2002; 14: 211-236Google Scholar, 6Schubert M. Petersson U.A. Haas B.J. Funk C. Schroder W.P. Kieselbach T. Proteome map of the chloroplast lumen of Arabidopsis thaliana.J. Biol. Chem. 2002; 277: 8354-8365Google Scholar). However, for these the predicted values signal peptide loss are these discrepancies with a the complex H. of in ribulose-bisphosphate carboxylase/oxygenase. and of an Biol. Chem. Scholar) was isolated by with identification and and the was isolated by mass is by these two and dissociation of these ions with and ions cleavage of of in and of in of the N-terminal ion are Da the of these in proteins the +14-Da to the first of the with the predicted the +14-Da is in to the first two N-terminal residues by the ions and to the first six residues in by the The ions from the two precursors, as well as the N-terminal of are of the mass the of is by in of the ions the in that dissociation a of ions The of Da that the N-terminal has the further of and Da the of the is that these from only one of the two the in its first N-terminal ions be Da in mass The of these as well as the expected similar of the two highly that the N-terminal is the with N-methylation far on the posttranslational was recently found for the first time in also in with the N-terminal by electron ionization MS R. M. C. K. T. J. of the of in 1997; Scholar) of the first from has valuable sequence the ion from the cleavage that the For the present bottom-up and top-down the of the the for the first of the precursor proteins from a genome such as identified 97 whereas this study found 22 protein molecular