Limits...
Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

Franchin C, Cesaro L, Pinna LA, Arrigoni G, Salvi M - PLoS ONE (2014)

Bottom Line: Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2.A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS.Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy; Proteomics Center of Padova University, Padova, Italy.

ABSTRACT
Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

Show MeSH

Related in: MedlinePlus

Logarithmic distribution of quantification values.A. Distribution of Log2 ratios relative to all phosphopeptides identified in this study. B. Distribution of Log2 ratios relative to all non-phosphopeptides.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206460&req=5

pone-0111018-g002: Logarithmic distribution of quantification values.A. Distribution of Log2 ratios relative to all phosphopeptides identified in this study. B. Distribution of Log2 ratios relative to all non-phosphopeptides.

Mentions: Figure 2 shows the logarithmic distribution of dimethyl label ratios for phosphorylated and non-phosphorylated peptides. In particular, panel A shows the distribution of Log2 ratios relative to phosphorylated peptides, where it is evident that, except for few cases, the very large majority of identified phosphopeptides is present almost exclusively in the sample treated with recombinant PLK2 (the maximum ratio was set at 100, as specified in the methods section). To assess a threshold above which we could consider the fold change as significant, we plotted Log2 ratios for all quantified non-phosphorylated peptides (panel B). As it is possible to see, the Log2 ratio for these peptides never exceeds the value of 1 (dashed line), equivalent to a PLK2-treated/control of 2. Hence this was chosen as the threshold above which the differences between PLK2-treated samples and untreated samples were considered as significant.


Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

Franchin C, Cesaro L, Pinna LA, Arrigoni G, Salvi M - PLoS ONE (2014)

Logarithmic distribution of quantification values.A. Distribution of Log2 ratios relative to all phosphopeptides identified in this study. B. Distribution of Log2 ratios relative to all non-phosphopeptides.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206460&req=5

pone-0111018-g002: Logarithmic distribution of quantification values.A. Distribution of Log2 ratios relative to all phosphopeptides identified in this study. B. Distribution of Log2 ratios relative to all non-phosphopeptides.
Mentions: Figure 2 shows the logarithmic distribution of dimethyl label ratios for phosphorylated and non-phosphorylated peptides. In particular, panel A shows the distribution of Log2 ratios relative to phosphorylated peptides, where it is evident that, except for few cases, the very large majority of identified phosphopeptides is present almost exclusively in the sample treated with recombinant PLK2 (the maximum ratio was set at 100, as specified in the methods section). To assess a threshold above which we could consider the fold change as significant, we plotted Log2 ratios for all quantified non-phosphorylated peptides (panel B). As it is possible to see, the Log2 ratio for these peptides never exceeds the value of 1 (dashed line), equivalent to a PLK2-treated/control of 2. Hence this was chosen as the threshold above which the differences between PLK2-treated samples and untreated samples were considered as significant.

Bottom Line: Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2.A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS.Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy; Proteomics Center of Padova University, Padova, Italy.

ABSTRACT
Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

Show MeSH
Related in: MedlinePlus