Otof processing software

The reconstituted peptides were used for shotgun proteomics experiments for the identification of the endogenous peptides. The peptides were separated using micro-LC (Dionex, Thermo UltiMate 3000 HPLC System, USA) through analytical column (Supelco, Ascentis® Express C18, 25cm × 4.6mm, 2.7 um) coupled with ESI source (Bruker Daltonics, Germany) spray in Maxis-HD qTOF (Bruker, Germany) mass spectrometer. The acquisition parameters were adapted from our previous reports with slight modifications 66,67. The elution was performed with a flow rate of 150 uL/min in a continuous gradient of 5-75% acetonitrile over 135 min. In the solvent system; Solvent A was 100% water with 0.1% formic acid, and solvent B was 100% acetonitrile with 0.1% formic acid. Data were acquired in the data-dependent mode in mass spectrometer operated in automatically switching between MS and MS/MS acquisition. The precursor ion MS spectra scan range of 200-2200 (m/z) was used in the Q-TOF with resolution R = 75, 000. The six most abundant precursor ions were searched for detection of different masses during acquisition and selected for fragmentation using collision-induced dissociation (CID) with a fixed cycle time of 3 sec along with 2 min of release for exclusion filter (otof processing software, Bruker Daltonics).