The search parameters were that 1] peptide mass tolerance (precursor ion) was set at 1.4 amu, 2] two internal cleavage sites were allowed for trypsin, and 3] group Scan was set at 7, minimum Group Count at 1, and minimum Ion Count at 15. Charge State and MSn level were set as Auto (peptide, 2.50; fragment ions, 0.00; ion series, B and Y).
with the following parameters: MW range 550 to 4000, group scan 1, minimum group count 1, minimum ion count 25, and an absolute threshold of 500.
The resulting MS/MS spectra (.raw files) were converted to.dta files using TurboSequest Bioworks version 3.1 (Thermo Fisher Scientific) (parameters = threshold cut-off 100, group scan default 100, minimum group count 1, minimum ion count 15, peptide tolerance 1.5).
MS/MS data were processed with in-house Bioworks (version 3.2, Thermo Electron Corporation, San Jose, CA) as described below: MS/MS spectra were extracted from the raw files by using SEQUEST with the following parameters; MW range: 250 4,000; threshold, 1,000; precursor mass tolerance: 2; group scan tolerance, 2; minimum group count, 1; precursor charge state, auto; MSn level, auto.
From raw files, MS/MS spectra were exported as dta (text format) files using the script extract_msn.exe (Thermo Fisher Scientific) under the following settings: peptide mass range: 500 3500; minimal total ion intensity threshold: 1000; minimal number of fragment ions: 15; precursor mass tolerance: 1.4 amu; group scan: 1; minimum group count: 1.
Raw mass spectra were converted to DTA peak lists using BioWorks Browser 3.2 (ThermoFinnigan) with the following parameter settings: peptide mass range 300 5000 Da, threshold 10, precursor mass ±1.4 Da, group scan 1, minimum group count 1, minimum ion count 15.
Our minimum group size is 20 and maximum is 35.
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