Computational protocol: Site-specific mapping and quantification of protein S-sulfenylation in cells

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Protocol publication

[…] Raw data files were analyzed by using TagRecon algorithm (Version 1.4.47) against a decoy protein database consisting of forward and reversed human RefSeq database (Version 20130621). Precursor ion mass tolerance was 0.01 Da, and fragmentation tolerance was 0.1 Da for the database search. The maximum number of modifications allowed per peptide was three, as was the maximum number of missed cleavages allowed. Methionine oxidation (15.9949 Da) and cysteine modifications either by iodoacetamide (carbamidomethyl, 57.0214 Da) or by S-sulfenylcysteine tagged with DYn-2-triazohexanoic acid group (DTH, , 333.1689 Da) were searched as dynamic modifications. For quantitative S-sulfenylome analysis, the heavy DTH modification on cysteine (DTH-d6, 339.2065) was also included as dynamic modification during database searching. The maximum Q value of peptide-spectrum matches (PSMs) was adjusted to achieve either a peptide or a protein false discovery rate (FDR) no greater than 5% using IDPicker software (Version 3.0.529) . Because identifications based on one peptide per protein were permitted, a streamlined manual validation approach was used to establish peptide identification and site localization (). First, the modified peptides whose matched spectra did not contain the major three diagnostic fragment ions (m/z 368.16, 366.15, and 336.19) were excluded from further consideration. MS/MS spectra of identified S-sulfenylated peptides then were annotated automatically with the SeeMS tool embedded in IDPicker software. The mass errors of the annotated peaks were required to be less than 20 ppm. All the automatically annotated spectra were manually evaluated according a previously published protocol for verification of automated peptide identification. For identified peptides containing more than one cysteine residue, at least three DTH modification-specific fragment ions were required for unambiguous site localization. PSMs with ambiguously localized sites identified with proper peak annotation also were retained in the final dataset. […]

Pipeline specifications

Software tools TagRecon, IDPicker
Application MS-based untargeted proteomics
Organisms Homo sapiens
Chemicals Cysteine, Hydrogen Peroxide