ABSTRACT: 20110318_GluC: Cell culture supernatants from Balb/c 3T3 mouse fibroblasts that had been incubated with the endoproteinase GluC (Staphylococcus aureus protease V8) as test protease for 1, 2, 4, 8, and 16 hours or buffer alone (0, 12, and 16 hours controls) were subjected to 8plex-iTRAQ-TAILS analysis. Labeling scheme: 113: 0h control, 114: 1h, 115: 2h, 116: 4h, 117: 8h, 118: 12h control, 119: 16h, 121: 16h control. 20130214_MMP10: Cell culture supernatants from matrix metalloproteinase (MMP) 10 deficient murine embryonic fibroblasts that had been incubated with recombinant human MMP10 as test protease for 1, 2, 4, 8, and 16 hours or buffer alone (0 and 16 hours controls) were subjected to 8plex-iTRAQ-TAILS analysis. Labeling scheme: 113: 0h control, 114: 1h, 115: 2h, 116: 4h, 117: 8h, 118: 12h, 119: 16h, 121: 16h control.MS data analysis: Mascot Distiller (Matrix Science) was used to extract peak lists from raw files and for merging of corresponding CID/HCD spectra pairs. Peak lists (mgf) were searched by Mascot v2.3 search engine against a mouse UniProtKB database (release 2012_03; 54232 entries), to which reversed decoy sequences as well as sequences for common contaminants and either for GluC or for human MMP10, respectively, had been added and with following parameters: semi-Arg-C for enzyme specificity allowing up to 2 missed cleavages; carbamidomethyl(C), iTRAQ(K) as fixed modifications; acetyl (N-term), iTRAQ(N-term), oxidation(M), and deamidation(NQ) as variable modifications; parent mass error at 10 ppm, fragment mass error at 0.8 Da. For GluC experiments an additional search was performed using the same parameters but with semi-GluC as enzyme and allowing up to 5 missed cleavages. The Trans-Proteomic Pipeline (TPP v4.6, rev 1, Build 201212051643)46 was used to secondary validate Mascot search results and to compile a single peptide list from all peptide fractions obtained from both the pre-pullout and the pullout samples. First, data were processed by PeptideProphet setting the ‘minimum peptide length’ to 1, using ‘accurate mass binning’ and omitting the ‘NTT model’. Next, iProphet was employed for additional validation and for combining PeptideProphet results from multiple searches, and only peptides with an iProphet probability of >=0.9 (corresponding to false discovery rate (decoy) of <1%) were included in subsequent analyses. For relative quantification iTRAQ reporter ion intensities were extracted from mgf files using a modified version of i-Tracker47 with a mass tolerance of 0.1 Da and purity corrections supplied by the iTRAQ manufacturer and assigned to filtered peptides.
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