Mass Spectrometry-based Peptide Identification

Each fraction was further subjected to a TripleTOF 5600 mass spectrometer system (AB SCIEX, USA) equipped with a nanoACQuity UPLC system (Waters, USA) according to previously described [2 (link)]. Briefly, the peptide sample from each SCX fraction was loaded onto a nanoACQuity UPLC BEH130 column (Waters, USA) packed with Symmetry C18 resin (Waters, USA). A Triple TOF 5600 platform was used for peptide identification. The ion spray voltage was set at 2.5 kV, the curtain gas was set at 30 psi, the nebulizer gas was set at 15 psi, and the interface heater temperature was set at 150 °C, respectively. For TOF–MS scans, the resolving power (RP) was greater than or equal to 30,000 FWHM. 250 ms was required for survey scans for the information dependent acquisition (IDA) analysis. 30 products were collected if the ion scans were more than 120 counts per second and with a 2+ to 5+ charge state. The Q2 transmission window was set at 100 Da for 100%. A sweeping collision energy setting of 35 ± 5 eV coupled with iTRAQ adjusted rolling collision energy was applied to all precursor ions for collision-induced dissociation. The parent ion dynamic exclusion was set to half of the peak time, and then the precursor was refreshed off the exclusion list.