Protein analysis via MudPIT-based shot-gun proteomics

The extracted proteins were analyzed with MudPIT-based shot-gun proteomics. Briefly, 100 μg of protein was first digested by Trypsin (Mass Spectrometry Grade, Promega, WI, USA) at 37 °C for 24 h, with the ratio of 1:40 w/w. A Sep-Pak Plus C18 column (Waters Limited, ON, Canada) was applied to desalt the digested peptides, which were subsequently loaded into a customized biphasic capillary column. The column, which included 5 cm of C18 reverse-phase resin (C18-AQ, The Nest Group Inc, MA, USA) and 3 cm of strong cation exchange (SCX) resin PolySULFOETHYL A, (The Nest Group Inc, MA, USA), was used to provide 2-dimensional separation of peptides, as described by Washburn et al⁵². The Thermo Fisher Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific, San Jose, CA) was used for peptide detection. The mass spectrometer was set to record the peptides over a 300–1700 m/z range. The five most abundant ions were then subjected to the following tandem mass (MS/MS) analysis. The Xcalibur software (Thermo Fisher Scientific, San Jose, CA) was used for LC-MS system control and data collection.