The identities of trypsinized proteins were analyzed on a LC/MS/MS mass spectrometry, TripleTOF 5600+ System (SCIEX, Ontario). Nano-flow LC Ultra system (Eksigent, Dublin, CA) packed with 3μm Luna C18 (Phenomenex, Torrance, CA) was inline installed. Samples were injected via a 300μm×5mm PepMap100 trap-column (ThermoFisher). Data acquisition of mass spectrometer were performed following settings: 250 ms survey MS spectra (m/z 380–1500) followed by up to 12 MS/MS measurements on the most intense parent ions (200 counts/sec threshold, +2 - +5 charge state, m/z single charged 400–1500 mass range for MS/MS, 100 ms each, high sensitivity mode).
Raw spectra files were converted into Mascot Generic File format (MGF) for peptide/protein identification by X!Tandem search algorithm (http://hs2.proteome.ca/tandem/thegpm_tandem_a.html). The X!Tandem search parameters were set at 20 ppm and 50 ppm mass tolerance for parent and fragment ions respectively, and two miss cleavage sites allowed [22 (link)]. The criterion of candidate molecule cut-off was: at least two matched peptides with Log(e) <-3, less than a thousandth probability of mismatching in database retrieval.
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