Photosynthesis-related phosphopeptides in Synechocystis 6803
Martina Angeleri, Dorota Muth-Pawlak, Eva-Mari Aro, and Natalia Battchikova
- Organism: Synechocystis sp. PCC 6803
- Instrument: TSQ Vantage
O-phosphorylation has been shown in photosynthesis-related proteins in a cyanobacterium Synechocystis sp. strain PCC 6803 (thereafter Synechocystis 6803) suggesting that phosphorylation of S,T and Y residues might be important in photosynthesis-related processes. Investigation of biological roles of these phosphorylation events requires confident knowledge of the phosphorylated sites and prospects for their individual assessment. We performed phosphoproteomic analysis of Synechocystis 6803 using TiO2 enrichment of the phosphopeptides followed by LC-MS/MS and discovered 367 phosphorylation sites in 190 proteins participating in various cellular functions. Further, we focused on the large group of phosphoproteins which are involved in light harvesting, photosynthesis-driven electron flow, photoprotection and CO2 fixation. The SRM approach was applied to verify/improve assignments of phosphorylation sites in these proteins and to investigate possibilities for analysis of phosphopeptide isomers. The SRM assays were designed for peptides comprising 45 phosphorylation sites. The assays contain peptide iRT values and Q1/Q3 transitions comprising those discriminating between phosphopeptide isoforms. The majority of investigated phosphopeptides and phosphorylated isoforms could be individually assessed with the SRM technique. The assays could be potentially used in future quantitative studies to evaluate an extent of phosphorylation in photosynthesis-related proteins in Synechocystis 6803 cells challenged with various environmental stresses.
SRM assays were designed for assessment of phosphorylation sites in Synechocystis proteins involved in light harvesting, photosynthesis-driven electron flow, photoprotection and CO2 fixation. The spectra library was obtained by LC-MS/MS analysis (QExactive)of pre-fractionated TiO2-enriched tryptic peptides. Phosphopeptide isoforms were differentiated with specific transitions pinpointing the phosphorylation site.
When necessary, synthetic “heavy” peptides were applied to distinguish phosphopeptide isoforms.
Synechocystis cells were broken by French press in the denaturing buffer containing inhibitors of proteases, kinases and phosphatases. Proteins were purified with the organic extraction method described in Wessel and Flügger (1984). After TCEP reduction, IAA alkylation and trypsing digestion, the peptides were enriched with TiO2 resin and desalted. For SRM analysis, samples were supplemented with iRT and "heavy" peptides
Created on 12/1/16, 10:33 AM