Computational protocol: Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation

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Protocol publication

[…] All raw MS spectra were processed with MaxQuant software suite (version 1.5.1.0) (Cox et al., ) and default settings. Identified peaks were searched against the target-decoy databases of Synechocystis sp. PCC 6803 from Cyanobase (http://genome.microbedb.jp/cyanobase) and Uniprot (http://www.uniprot.org), containing 3672 and 3507 protein sequences and 245 common contaminants, with the following database search criteria: Trypsin was defined as cleaving enzyme and up to two missed cleavages were allowed. Carbamido-methylation of cysteines was set as a fixed and methionine oxidation, protein N-termini acetylation and S/T/Y phosphorylation were set as variable modifications. Light-, medium-heavy- and heavy- dimethylation labeling on peptide N-termini and lysine residues was defined for samples from the quantitative experiments. The initial mass tolerance of precursor ions was limited to 6 ppm and 0.5 ppm for fragment ions. False discovery rates (FDRs) of peptides and proteins were set to 1%, respectively. Quantification of dimethylation labeled peptides required at least two ratio counts. Peptides were only allowed with a posterior error probability (PEP) <1% at the peptide- and <5% at the phosphopeptide level. Phosphopeptide MS/MS spectra were further manually filtered and validated with stringent acceptance criteria: Phosphorylation site localization with ≥ 75% localization probability was manually validated as well as comprehensive coverage of b- and y-ion series for CID and HCD spectra as well as a low noise to signal ratio (Supplementary MS Spectra). To exclude quantitation bias of phosphopeptide ratios due to fluctuations of corresponding proteins, phosphopeptide ratios were normalized by division with corresponding protein ratios. Significance analysis of regulated phosphorylation events was performed with Perseus software (version 1.5.0.31), normalized phosphopeptide ratios were log2 transformed and plotted against the respective log10 transformed phosphopeptide intensities. Significantly regulated phosphorylation events were identified by significance A analysis with a p-value of 0.05. […]

Pipeline specifications

Software tools MaxQuant, Perseus
Databases CyanoBase MicrobeDB
Application MS-based untargeted proteomics
Organisms Synechocystis sp. PCC 6803
Chemicals Carbon, Nitrogen