2.6. Peptide Sequencing Using LC-MS/MS

The peptides were identified by capillary high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis using the Thermo Fisher Scientific™ HPLC (Ultimate 3000) coupled to an Orbitrap Elite™ Hybrid Ion Trap–Orbitrap Mass Spectrometer (Thermo Fisher Scientific (China) Co., Ltd., Shanghai, China) according to previously reported methods [29]. The mass spectrometer was operated in the positive mode with the capillary voltage of 3.6 kV and the source temperature of 100 °C. The scanning m/z range was 200–1500 in the MS mode and 50–1500 in the MS/MS mode. The MS/MS data were processed using Thermo Xcalibur software (Thermo Fisher Scientific, San Jose, CA, USA) in combination with manual de novo sequencing. The spectra of hydrolysates were processed to MS (mass spectrum) and MS/MS, respectively, by Bruker Daltonics Data Analysis 4.0 (Bruker Daltonics Inc., Billerica, MA, USA). Analysis results were confirmed using protein database from National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/protein, accessed on 17 September 2018). Validated by Peptide/Protein MS Utility program (http://prospector.ucsf.edu/, accessed on 20 September 2018). Peptides were synthesized using the method of Fmoc solid-phase (GL Biochem Ltd., Beijing, China). The purity of all peptides used in the study was greater than 98% determined by HPLC.