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Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ - PLoS ONE (2008)

Bottom Line: The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude.Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction.No evidence was found for suggested targeting via the secretory system.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.

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Tandem MS spectra of N-terminally acetylated peptides suggest presence of two isoforms of Cysteine Synthase, AT2G43750.1.(A) Tandem MS spectrum of doubly charged 25 aa-long, N-terminally acetylated AVSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read right-to-left, starting with the most massive y(20) ion. Singly charged b ions are indicated by red lines with corresponding aa residues shown top – peptide sequence should be read left-to-right, starting with the lightest b(4) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, b0(4), y++(23), and y++(24) are also indicated. (B) Tandem MS spectrum of doubly charged 24 aa-long, N-terminally acetylated VSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read from right-to-left, starting with the most massive y(20) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, y++(21), and y++(23) are also indicated.
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pone-0001994-g005: Tandem MS spectra of N-terminally acetylated peptides suggest presence of two isoforms of Cysteine Synthase, AT2G43750.1.(A) Tandem MS spectrum of doubly charged 25 aa-long, N-terminally acetylated AVSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read right-to-left, starting with the most massive y(20) ion. Singly charged b ions are indicated by red lines with corresponding aa residues shown top – peptide sequence should be read left-to-right, starting with the lightest b(4) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, b0(4), y++(23), and y++(24) are also indicated. (B) Tandem MS spectrum of doubly charged 24 aa-long, N-terminally acetylated VSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read from right-to-left, starting with the most massive y(20) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, y++(21), and y++(23) are also indicated.

Mentions: We identified 47 N-terminal acetylated nuclear-encoded proteins (Table S5). Acetylation leads to an increase in retention time (∼6 min under our on-line LC conditions) and if both the non-acetylated and acetylated peptides are present, they can be identified as two peptides with different retention times and a mass difference of 43.018 Da. An interesting example is shown for cysteine synthase (Figure 5A, B). In this case, two acetylated N-termini were identified that differ by one aa in length. Figure 5A shows the MS/MS spectrum for the longer doubly-charged N-terminal peptide, Acetyl-AVSIKPEAGVEGLNIADNAAQLIGK, and Figure 5B shows MS/MS spectrum for the shorter doubly-charged N-terminal peptide, Acetyl-VSIKPEAGVEGLNIADNAAQLIGK. Both spectra are of high quality, with respective ion scores of 100 and 79, and with ions supporting assignment of the acetylation of the N-terminal residue (as opposed to lysines) present well above the noise level. The longer peptide was also observed in the triply-charged state in both non-acetylated and N-terminally acetylated forms eluting 6.4 minutes apart (spectra not shown). This example demonstrates that the assignments of acetylated residues are not false positives. It also demonstrates that either cTP cleavage by SPP can occur at more than one precise position, and/or that after the initial cTP cleavage by SPP, additional processing occurs by chloroplast amino peptidases, followed by N-terminal acetylation.


Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.

Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ - PLoS ONE (2008)

Tandem MS spectra of N-terminally acetylated peptides suggest presence of two isoforms of Cysteine Synthase, AT2G43750.1.(A) Tandem MS spectrum of doubly charged 25 aa-long, N-terminally acetylated AVSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read right-to-left, starting with the most massive y(20) ion. Singly charged b ions are indicated by red lines with corresponding aa residues shown top – peptide sequence should be read left-to-right, starting with the lightest b(4) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, b0(4), y++(23), and y++(24) are also indicated. (B) Tandem MS spectrum of doubly charged 24 aa-long, N-terminally acetylated VSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read from right-to-left, starting with the most massive y(20) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, y++(21), and y++(23) are also indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2291561&req=5

pone-0001994-g005: Tandem MS spectra of N-terminally acetylated peptides suggest presence of two isoforms of Cysteine Synthase, AT2G43750.1.(A) Tandem MS spectrum of doubly charged 25 aa-long, N-terminally acetylated AVSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read right-to-left, starting with the most massive y(20) ion. Singly charged b ions are indicated by red lines with corresponding aa residues shown top – peptide sequence should be read left-to-right, starting with the lightest b(4) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, b0(4), y++(23), and y++(24) are also indicated. (B) Tandem MS spectrum of doubly charged 24 aa-long, N-terminally acetylated VSIKPEAGVEGLNIADNAAQLIGK peptide. Precursor ion is indicated with red asteric. Singly charged y ions are indicated by blue lines with corresponding aa residues shown on top - peptide sequence should be read from right-to-left, starting with the most massive y(20) ion. Ions, whose presence strengthen the assignment of the N-terminal acetylation, y++(21), and y++(23) are also indicated.
Mentions: We identified 47 N-terminal acetylated nuclear-encoded proteins (Table S5). Acetylation leads to an increase in retention time (∼6 min under our on-line LC conditions) and if both the non-acetylated and acetylated peptides are present, they can be identified as two peptides with different retention times and a mass difference of 43.018 Da. An interesting example is shown for cysteine synthase (Figure 5A, B). In this case, two acetylated N-termini were identified that differ by one aa in length. Figure 5A shows the MS/MS spectrum for the longer doubly-charged N-terminal peptide, Acetyl-AVSIKPEAGVEGLNIADNAAQLIGK, and Figure 5B shows MS/MS spectrum for the shorter doubly-charged N-terminal peptide, Acetyl-VSIKPEAGVEGLNIADNAAQLIGK. Both spectra are of high quality, with respective ion scores of 100 and 79, and with ions supporting assignment of the acetylation of the N-terminal residue (as opposed to lysines) present well above the noise level. The longer peptide was also observed in the triply-charged state in both non-acetylated and N-terminally acetylated forms eluting 6.4 minutes apart (spectra not shown). This example demonstrates that the assignments of acetylated residues are not false positives. It also demonstrates that either cTP cleavage by SPP can occur at more than one precise position, and/or that after the initial cTP cleavage by SPP, additional processing occurs by chloroplast amino peptidases, followed by N-terminal acetylation.

Bottom Line: The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude.Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction.No evidence was found for suggested targeting via the secretory system.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that 'spectral counting' can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models.

Show MeSH