Computational protocol: Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe*

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Protocol publication

[…] The MS data were analyzed with the MaxQuant Software Package version 1.3.0.5 () and searched using the Andromeda peptide search engine (). The MS/MS spectra were searched against a database containing the proteome sequence of the S. pombe downloaded from PomBase (03.01.2013). The database contained 5145 entries from the S. pombe proteome, 248 frequently observed contaminants (downloaded from http://www.maxquant.org/contaminants.zip), as well as the reverse version of all sequences. Mass tolerance was set to 6 ppm at the MS and 0.5 Da at the MS/MS level. A minimal length of seven amino acids and full tryptic enzyme specificity were required. Two enzyme missed cleavages were allowed. Oxidation of methionine and protein N-terminal acetylation were defined as variable modifications and carbamidomethylation of cysteines was used as fixed modification. The false discovery rate was set to 1% on the peptide and protein level. In quantitative measurements, SILAC amino acids Lys4, Lys8, Arg6, and/or Arg10 were defined as fixed modifications. Significantly regulated protein groups were defined as those with calculated “Significance B” lower than 0.01. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Organisms Schizosaccharomyces pombe
Chemicals Ammonia, Quaternary Ammonium Compounds, Guanidine