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Quantitation, networking, and function of protein phosphorylation in plant cell.

Zhu L, Li N - Front Plant Sci (2013)

Bottom Line: One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure.Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA.The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, The Hong Kong University of Science and Technology Hong Kong, China.

ABSTRACT
Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a (15)N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs.

No MeSH data available.


Workflow of in vitro assay for signal-regulated phosphorylation. Upper left panel shows two alignments of conserved sequences of phosphorylation motif generated with CLUSTAL W, which was reported in a previous study (Li et al., 2009). In this case, 9–21 amino acid-long oligopeptide sequences deduced from the primary sequence of an aluminum-induced protein covering the entire phosphorylation motif was employed to search against the non-redundant protein sequence database (organism: Arabidopsis thaliana, taxid: 3702). Candidates with a homology of more than 55.5% are considered to contain the phosphorylation motif. The most conserved candidates are aligned to obtain the motif sequence. The phosphorylation site Ser(S) is marked with a black asterisk and TAIR IDs of the genes are shown on the left. To validate such a prediction, peptides including those amino acid sequences flanking the predicted phosphosite motif are synthesized and used as substrates for in vitro kinase assay. The control or treated means the plant without or with particular treatment of interest, respectively. The quantitation is performed at MS2 spectra by directly comparing the reporter ion intensity. Plant extracts from the control or the treated plants are used separately. Peptides assayed are reciprocally labeled with iTRAQ reagents. Three sets of reciprocal labelings are required before a statistical analysis is performed.
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Figure 1: Workflow of in vitro assay for signal-regulated phosphorylation. Upper left panel shows two alignments of conserved sequences of phosphorylation motif generated with CLUSTAL W, which was reported in a previous study (Li et al., 2009). In this case, 9–21 amino acid-long oligopeptide sequences deduced from the primary sequence of an aluminum-induced protein covering the entire phosphorylation motif was employed to search against the non-redundant protein sequence database (organism: Arabidopsis thaliana, taxid: 3702). Candidates with a homology of more than 55.5% are considered to contain the phosphorylation motif. The most conserved candidates are aligned to obtain the motif sequence. The phosphorylation site Ser(S) is marked with a black asterisk and TAIR IDs of the genes are shown on the left. To validate such a prediction, peptides including those amino acid sequences flanking the predicted phosphosite motif are synthesized and used as substrates for in vitro kinase assay. The control or treated means the plant without or with particular treatment of interest, respectively. The quantitation is performed at MS2 spectra by directly comparing the reporter ion intensity. Plant extracts from the control or the treated plants are used separately. Peptides assayed are reciprocally labeled with iTRAQ reagents. Three sets of reciprocal labelings are required before a statistical analysis is performed.

Mentions: To place the newly identified phosphosites in the context of a cellular process or a specific signal regulation, a general approach has been developed using quantitative proteomics in combination with kinase extracts of both the treated and mutant plants (Figure 1; Li et al., 2009, 2012; Zhu et al., 2012). The in vitro kinase assay is then performed using the synthetic peptides mixed with plant kinase extracts in the presence of phosphatase inhibitors. The chemical labeling quantitative proteomic protocol, such as iTRAQ, is then applied to investigate the differentially regulated phosphorylation events. A routine iTRAQ-based in vitro phosphosite quantitation requires at least two sets of forward and reciprocal experiments. Results on MS-derived and bioinformatics-derived synthetic peptides can provide information on the differential regulation of phosphosites in response to the internal and external stimuli.


Quantitation, networking, and function of protein phosphorylation in plant cell.

Zhu L, Li N - Front Plant Sci (2013)

Workflow of in vitro assay for signal-regulated phosphorylation. Upper left panel shows two alignments of conserved sequences of phosphorylation motif generated with CLUSTAL W, which was reported in a previous study (Li et al., 2009). In this case, 9–21 amino acid-long oligopeptide sequences deduced from the primary sequence of an aluminum-induced protein covering the entire phosphorylation motif was employed to search against the non-redundant protein sequence database (organism: Arabidopsis thaliana, taxid: 3702). Candidates with a homology of more than 55.5% are considered to contain the phosphorylation motif. The most conserved candidates are aligned to obtain the motif sequence. The phosphorylation site Ser(S) is marked with a black asterisk and TAIR IDs of the genes are shown on the left. To validate such a prediction, peptides including those amino acid sequences flanking the predicted phosphosite motif are synthesized and used as substrates for in vitro kinase assay. The control or treated means the plant without or with particular treatment of interest, respectively. The quantitation is performed at MS2 spectra by directly comparing the reporter ion intensity. Plant extracts from the control or the treated plants are used separately. Peptides assayed are reciprocally labeled with iTRAQ reagents. Three sets of reciprocal labelings are required before a statistical analysis is performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539650&req=5

Figure 1: Workflow of in vitro assay for signal-regulated phosphorylation. Upper left panel shows two alignments of conserved sequences of phosphorylation motif generated with CLUSTAL W, which was reported in a previous study (Li et al., 2009). In this case, 9–21 amino acid-long oligopeptide sequences deduced from the primary sequence of an aluminum-induced protein covering the entire phosphorylation motif was employed to search against the non-redundant protein sequence database (organism: Arabidopsis thaliana, taxid: 3702). Candidates with a homology of more than 55.5% are considered to contain the phosphorylation motif. The most conserved candidates are aligned to obtain the motif sequence. The phosphorylation site Ser(S) is marked with a black asterisk and TAIR IDs of the genes are shown on the left. To validate such a prediction, peptides including those amino acid sequences flanking the predicted phosphosite motif are synthesized and used as substrates for in vitro kinase assay. The control or treated means the plant without or with particular treatment of interest, respectively. The quantitation is performed at MS2 spectra by directly comparing the reporter ion intensity. Plant extracts from the control or the treated plants are used separately. Peptides assayed are reciprocally labeled with iTRAQ reagents. Three sets of reciprocal labelings are required before a statistical analysis is performed.
Mentions: To place the newly identified phosphosites in the context of a cellular process or a specific signal regulation, a general approach has been developed using quantitative proteomics in combination with kinase extracts of both the treated and mutant plants (Figure 1; Li et al., 2009, 2012; Zhu et al., 2012). The in vitro kinase assay is then performed using the synthetic peptides mixed with plant kinase extracts in the presence of phosphatase inhibitors. The chemical labeling quantitative proteomic protocol, such as iTRAQ, is then applied to investigate the differentially regulated phosphorylation events. A routine iTRAQ-based in vitro phosphosite quantitation requires at least two sets of forward and reciprocal experiments. Results on MS-derived and bioinformatics-derived synthetic peptides can provide information on the differential regulation of phosphosites in response to the internal and external stimuli.

Bottom Line: One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure.Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA.The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science, The Hong Kong University of Science and Technology Hong Kong, China.

ABSTRACT
Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a (15)N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs.

No MeSH data available.