Proteomics quality and standard: from a regulatory perspective

Cultured mouse lymphoma cells were lysed with a buffer containing 50 mM Tris-HCl (pH 8.0) and 8 M Urea with or without a protease inhibitor cocktail (Sigma). The lysates were desalted and digested with sequencing-grade modified trypsin (Promega). Different amounts of peptides were analyzed using nanoflow RPLC-MS/MS on a linear ion trap mass spectrometer (LTQ XL, Thermo Electron, San Jose, CA). The raw MS/MS data were searched using SEQUEST running under BioWorks (Rev. 3.3.1 SP1) (Thermo Electron, San Jose, CA) against a mouse IPI proteome database (Version 3.62) downloaded from the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk). For the SEQUEST analysis, the peptide mass tolerance was set as 2.0 Da and the fragment ion tolerance was 1.0 Da. A tryptic enzyme restriction with a maximum of two internal missed cleavage sites was used. The Xcorr versus charge state values were filtered at Xcorr ≥ 1.7 for [M+H]1+ ions, ≥ 2.5 for [M+2H]2+ ions, ≥ 3.2 for [M+3H]3+ ions, and P ≤ 0.01 and ΔCn ≥ 0.08 were applied for identification of all fully tryptic peptides. The peptide ion chromatogram was extracted using a minimum intensity threshold of 100, mass tolerance of 2.0 amu and smoothing point of 5, and the area of the extracted ion chromatographic (XIC) peak was integrated and calculated using the PepQuan module in Bioworks (Thermo Electron, San Jose, CA).