Peptide Analysis on Orbitrap Analyzer

The peptides were analyzed on an Orbitrap analyzer (Q-Exactive, ThermoFisher, San Jose, CA, USA) outfitted with a nanospray source and EASY-nLC nano-LC system (ThermoFisher, San Jose, CA, USA). a 75 μm × 50 cm PepMax RSLC EASY-Spray column filled with 2 μM C18 beads (ThermoFisher, SanJose, CA, USA) was used to load the peptide mixture at a pressure of 800 Bar. Peptides were then subjected to a stepwise gradient elution over 240 min at a rate of 250 nL/min (0–4% Acetonitrile containing 0.1% Formic Acid over 2 min; 4–28% Acetonitrile containing 0.1% Formic Acid over 226 min, 28–95% Acetonitrile containing 0.1% Formic Acid over 2 min, constant 95% Acetonitrile containing 0.1% Formic Acid for 10 min). In the Q-Exactive mass spectrometer (ThermoFisher, San Jose, CA, USA), one MS full scan (525–1600 m/z) was performed with an automatic gain control (AGC) target of 1 × 10⁶ maximum ion injection time of 120 ms and a resolution of 35,000 with subsequent 15 data-dependent MS/MS scans with a resolution of 35,000, an AGC target of 1 × 10⁶ , maximum ion time of 120 ms, and one microscan. The intensity threshold required to trigger a MS/MS scan was at an underfill ratio of 0.2%. In the higher energy collision dissociation (HCD) trap, normalized collision energy of 30 V was used for the fragmentation. The dynamic exclusion was applied with an exclusion period of 40 s [17 (link)].