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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.


Related in: MedlinePlus

Workflow of determination of occupancy (Raqu or Risf) using AQUIP. A known quantity of 15N-coded recombinant proteins, including both the target phosphorylated isoform and all of its cognates of different types of PTMs, are highly purified from 15N-labeled transgenic plant (either the control or the treated plants) and divided into six aliquots and resolved on SDS-PAGE gel, followed by in-gel trypsin digestion. The 14N-coded synthetic phosphopeptide and its non-phosphorylated cognate are mixed together with a known concentration and spiked into 6 aliquots of 15N-coded peptide samples. After the oxidization of methionine residues on the peptides and LC-MS/MS analysis, two standard curves are built using a method between the ion intensity ratios of peptide P and NP standards and the molar ratios of peptide standards. The molar amount of phosphorylated peptide (Paqu) and its non-phosphorylated cognate (NPaqu), which are derived from the highly purified and 15N-coded protein isoforms, is determined according to these two standard curves separately. The percentage of target-phosphorylated isoform among all isoforms in the total cellular proteins is defined as the site-specific phosphorylation occupancy Risf, which is equivalent to Raqu when the ratio of P and NP peptide amount is in concern (Li et al., 2012). Error values indicate standard deviations. The Risf obtained from either the control or the treated plants is used to calculate the relative abundance of the phosphorylation event and subject to statistical assessment, as shown at the bottom of the figure. ***Means significant difference.
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Figure 2: Workflow of determination of occupancy (Raqu or Risf) using AQUIP. A known quantity of 15N-coded recombinant proteins, including both the target phosphorylated isoform and all of its cognates of different types of PTMs, are highly purified from 15N-labeled transgenic plant (either the control or the treated plants) and divided into six aliquots and resolved on SDS-PAGE gel, followed by in-gel trypsin digestion. The 14N-coded synthetic phosphopeptide and its non-phosphorylated cognate are mixed together with a known concentration and spiked into 6 aliquots of 15N-coded peptide samples. After the oxidization of methionine residues on the peptides and LC-MS/MS analysis, two standard curves are built using a method between the ion intensity ratios of peptide P and NP standards and the molar ratios of peptide standards. The molar amount of phosphorylated peptide (Paqu) and its non-phosphorylated cognate (NPaqu), which are derived from the highly purified and 15N-coded protein isoforms, is determined according to these two standard curves separately. The percentage of target-phosphorylated isoform among all isoforms in the total cellular proteins is defined as the site-specific phosphorylation occupancy Risf, which is equivalent to Raqu when the ratio of P and NP peptide amount is in concern (Li et al., 2012). Error values indicate standard deviations. The Risf obtained from either the control or the treated plants is used to calculate the relative abundance of the phosphorylation event and subject to statistical assessment, as shown at the bottom of the figure. ***Means significant difference.

Mentions: Once phosphosites are identified from SILIA-based phosphoproteomics and confirmed with in vitro kinase assays, an in planta validation and measurement of these phosphosites and signal-specific phosphorylation level will be performed using a transgenic approach. A transgene encoding the target protein will be transferred into plant to measure the alteration of a particular phosphorylation in response to various treatments, cell types, or disease models (Havlis and Shevchenko, 2004; Steen et al., 2008). The term occupancy (Raqu or Risf) defines the ratio of the amount of a protein PTM isomer (or PTM peptide) over the total amount of the corresponding protein (or the corresponding PTM site peptide; Li et al., 2012). A signal-induced alteration in occupancy on a phosphosite usually measures the change in phosphorylation level elicited by both internal and external signals. It is therefore that Raqu or Risf are used to describe signal-specific phosphorylation change. To determine Raqu (or Risf) of a phosphosite, 14N-coded peptide standards are established (Figure 2) against the 15N-coded recombinant protein expressed in the transgenic plant. One is the synthetic phosphopeptide that contains targeted phosphosite, whereas the other is its unmodified cognate. A series of synthetic peptide samples with a wide range of concentrations are then made, in the meantime, an aliquot of known amount of highly purified recombinant protein expressed in transgenic plant is resolved on SDS-PAGE and undergo in-gel tryptic digestion. Once the peptides are produced from the 15N-coded recombinant protein, 14N-coded standard phosphopeptides (both phosphorylated and non-phosphorylated) of various amounts are added into aliquots of the recombinant protein-derived peptides and analyzed using MS. The resulting data is used to establish two individual standard curves to calculate the molar amount of phosphopeptide Paqu and its non-phosphorylated cognate NPaqu. The Raqu is determined using Eq. 1:


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

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

Workflow of determination of occupancy (Raqu or Risf) using AQUIP. A known quantity of 15N-coded recombinant proteins, including both the target phosphorylated isoform and all of its cognates of different types of PTMs, are highly purified from 15N-labeled transgenic plant (either the control or the treated plants) and divided into six aliquots and resolved on SDS-PAGE gel, followed by in-gel trypsin digestion. The 14N-coded synthetic phosphopeptide and its non-phosphorylated cognate are mixed together with a known concentration and spiked into 6 aliquots of 15N-coded peptide samples. After the oxidization of methionine residues on the peptides and LC-MS/MS analysis, two standard curves are built using a method between the ion intensity ratios of peptide P and NP standards and the molar ratios of peptide standards. The molar amount of phosphorylated peptide (Paqu) and its non-phosphorylated cognate (NPaqu), which are derived from the highly purified and 15N-coded protein isoforms, is determined according to these two standard curves separately. The percentage of target-phosphorylated isoform among all isoforms in the total cellular proteins is defined as the site-specific phosphorylation occupancy Risf, which is equivalent to Raqu when the ratio of P and NP peptide amount is in concern (Li et al., 2012). Error values indicate standard deviations. The Risf obtained from either the control or the treated plants is used to calculate the relative abundance of the phosphorylation event and subject to statistical assessment, as shown at the bottom of the figure. ***Means significant difference.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Workflow of determination of occupancy (Raqu or Risf) using AQUIP. A known quantity of 15N-coded recombinant proteins, including both the target phosphorylated isoform and all of its cognates of different types of PTMs, are highly purified from 15N-labeled transgenic plant (either the control or the treated plants) and divided into six aliquots and resolved on SDS-PAGE gel, followed by in-gel trypsin digestion. The 14N-coded synthetic phosphopeptide and its non-phosphorylated cognate are mixed together with a known concentration and spiked into 6 aliquots of 15N-coded peptide samples. After the oxidization of methionine residues on the peptides and LC-MS/MS analysis, two standard curves are built using a method between the ion intensity ratios of peptide P and NP standards and the molar ratios of peptide standards. The molar amount of phosphorylated peptide (Paqu) and its non-phosphorylated cognate (NPaqu), which are derived from the highly purified and 15N-coded protein isoforms, is determined according to these two standard curves separately. The percentage of target-phosphorylated isoform among all isoforms in the total cellular proteins is defined as the site-specific phosphorylation occupancy Risf, which is equivalent to Raqu when the ratio of P and NP peptide amount is in concern (Li et al., 2012). Error values indicate standard deviations. The Risf obtained from either the control or the treated plants is used to calculate the relative abundance of the phosphorylation event and subject to statistical assessment, as shown at the bottom of the figure. ***Means significant difference.
Mentions: Once phosphosites are identified from SILIA-based phosphoproteomics and confirmed with in vitro kinase assays, an in planta validation and measurement of these phosphosites and signal-specific phosphorylation level will be performed using a transgenic approach. A transgene encoding the target protein will be transferred into plant to measure the alteration of a particular phosphorylation in response to various treatments, cell types, or disease models (Havlis and Shevchenko, 2004; Steen et al., 2008). The term occupancy (Raqu or Risf) defines the ratio of the amount of a protein PTM isomer (or PTM peptide) over the total amount of the corresponding protein (or the corresponding PTM site peptide; Li et al., 2012). A signal-induced alteration in occupancy on a phosphosite usually measures the change in phosphorylation level elicited by both internal and external signals. It is therefore that Raqu or Risf are used to describe signal-specific phosphorylation change. To determine Raqu (or Risf) of a phosphosite, 14N-coded peptide standards are established (Figure 2) against the 15N-coded recombinant protein expressed in the transgenic plant. One is the synthetic phosphopeptide that contains targeted phosphosite, whereas the other is its unmodified cognate. A series of synthetic peptide samples with a wide range of concentrations are then made, in the meantime, an aliquot of known amount of highly purified recombinant protein expressed in transgenic plant is resolved on SDS-PAGE and undergo in-gel tryptic digestion. Once the peptides are produced from the 15N-coded recombinant protein, 14N-coded standard phosphopeptides (both phosphorylated and non-phosphorylated) of various amounts are added into aliquots of the recombinant protein-derived peptides and analyzed using MS. The resulting data is used to establish two individual standard curves to calculate the molar amount of phosphopeptide Paqu and its non-phosphorylated cognate NPaqu. The Raqu is determined using Eq. 1:

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.


Related in: MedlinePlus