Computational protocol: A THEMIS:SHP1 complex promotes T-cell survival

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Protocol publication

[…] Data were converted to.mzXML format using MSconvert (Proteowizard) and uploaded into the Central Proteomics Facility Pipeline (CPFP) (Trudgian et al, ) for analysis. Enzyme was set to trypsin allowing for up to 2 missed cleavages. Carbamidomethyl cysteine was set as a fixed modification and oxidation (methionine), deamidation (NQ), acetylation (Protein-N) and phosphotyrosine as variable modifications. Mass tolerances for MS and MS/MS peak identifications were 20 ppm and 0.1 Da, respectively. InterProphet probability (IP Prob) is derived by the combination of results from multiple search engines within CPFP and improves coverage and confidence over use of a single search engine. Label-free quantitation was performed using the SINQ (Spectral index quantitation) tool within CPFP (Trudgian et al, ). Only proteins which were >20-fold abundant over the biotin-blocked control sample were considered for further analysis using the CRAPOME contaminant repository (Mellacheruvu et al, ). Finally, proteins found in less than 5% of all Strep-Tag background pull-down experiments listed within CRAPOME were considered as true interactors. […]

Pipeline specifications

Software tools CPFP, iProphet
Application MS-based untargeted proteomics