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Comparative Study
. 2013 May 3;12(5):2233-44.
doi: 10.1021/pr400086y. Epub 2013 Mar 29.

Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei

Affiliations
Comparative Study

Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei

Michael D Urbaniak et al. J Proteome Res. .

Abstract

We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3-4 fold over-represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the T. brucei kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in T. brucei is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism.

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Figures

Figure 1
Figure 1
Growth of bloodstream form T. brucei in SILAC labeling media. Cells were grown in conventional HMI9-T medium or in the HMI11-SILAC medium developed in this paper. (A) Cumulative growth over 10 days (with subculturing every 2–3 days). (B) Growth to stationary phase. (C) Light microscopy of cells after 10 days in culture.
Figure 2
Figure 2
Phosphoproteomic workflow. Differentially labeled (heavy and light) bloodstream and procyclic form trypanosomes were lysed in SDS, and total proteins S-alkylated and digested with trypsin using the FASP method. Phosphopeptides were separated into eight fractions by SCX HPLC, enriched with TiO2, and analyzed by LC–MS/MS using MSA.
Figure 3
Figure 3
Correlation of label-swap biological replicates. (A) Changes in protein abundance. (B) Changes in phosphorylation site abundance. Replicates were trimmed at 2 σ prior to further analysis. Black squares <2 σ, gray square>2 σ.
Figure 4
Figure 4
Gene Ontology term enrichment for all observed phosphoproteins. Term-for-term enrichment, P < 0.01; white box, Biological process; light gray box, Cellular component; dark gray box, Molecular function. For clarity parental terms are omitted; the complete graph can be found in the Supporting Information (Figure S3).
Figure 5
Figure 5
Correlation of fold-changes in protein and fold-changes in phosphorylation sites. (A) Correlation of all phosphorylation sites. (B) Correlation of average fold-change in phosphorylation sites for each phosphoprotein, with bars indicating the upper and lower limits of the range.
Figure 6
Figure 6
Examples of the assignments of phosphorylation site location from tandem mass spectral data. (A) STE11 family protein kinase (Tb11.46.003) with pS357 and pS358 with MaxQuant localization score of 1.0 and Mascot delta score of 57.8. (B) CAMK family protein kinase Tb11.01.2290 with pS5 with MaxQuant localization score of 1.0 and Mascot delta score of 27.1, and pS8 with MaxQuant localization of 1.0 but which was not identified by Mascot. Data acquired on a LTQ Orbitrap Velos using MSA, * designates loss of phosphate (−98 Da), ** loss of two phosphates (−196 Da).
Figure 7
Figure 7
Differential phosphorylation of MAPK signaling components. Heatmap showing the change in abundance between the procyclic to bloodstream (Log2) at the protein and phosphorylation site level for the MAPK, STE7 and STE11 kinase quantified in this study. Red box, TxY motif; Gray shading, not observed. The Heatmap is generated with GENEE (http://www.broadinstitute.org/cancer/software/GENE-E/).
Figure 8
Figure 8
Distribution of intraprotein differentially regulated phosphorylation sites. Only proteins with more than one phosphorylation site are shown.

References

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