*4.4. Peptide Sequence Analysis Based on LC-MS/MS*

The peptide sequence analysis used an Easy-nLC 1200 system coupled to a Q-Exactive quadrupole-Orbitrap mass spectrometry (Thermo Fisher Scientific, San Jose, CA, USA). One μL of the samples was injected with an autosampler into an Acclaim Pep Map RPLC C18 column (150 μm i.d. × 150 mm, C18, particle size: 1.9 μm, pore size: 100 Å, Dr. Maisch GmbH, Darmstadt, Hessen, Germany) with mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in the water, 80% acetonitrile. The flow rate was 600 nL/min, and the LC linear gradient ranged from 4% to 40% for 55 minutes and 10 minutes at 95% mobile phase B. Finally, the molecular mass, sequence, peak area (with respect to base peak), and relative peak area (peak area/total peak area) of the peptides were identified and calculated as previously described [81,91]. The conditions of the mass spectrometer were as follows: Resolution: 70,000, AGC target: 3e6, NCE/stepped NCE: 28. The samples were analyzed with a full-scan MS mode in the range of 100–1500 *m*/*z* to obtain the total ion chromatogram. The raw MS files were analyzed and searched against the target protein database based on the species of the samples using Byonic.
