Robust trypsin

Selected spots were automatically cut form gels, treated and, washed twice with Milli-Q water for 30 min in 50% methyl alcohol (Guangzhou Chemical Reagent Factory, Guangzhou, China) until destained. The spots were dissolved in 100 mM NH₄HCO₃ (Sigma-Aldrich Co. LLC, MO, USA) and extracted with 50% acetonitrile (ACN; Sigma-Aldrich Co. LLC., MO, USA). The spots were then digested with 1 μg/μL robust trypsin (Promega Corporation, WI, USA) for 30 min. Coverage solution (10% ACN, Milli-Q water, 50 mM NH₄HCO₃) was then added, following incubation at 16 h. Following digestion, the generated peptides were extracted with an appropriate solvent that was 90% ACN and 2.5% trifluoroacetic acid (TFA; Promega Corporation, WI, USA) for 30 min. Finally, the peptides were vacuum dried.

Selected spots were automatically cut form gels, washed twice with Milli-Q water for 30 min, and destained in 50% methyl alcohol (Guangzhou Chemical Reagent Factory, Guangzhou, China). The spots were dissolved in 100 mM NH₄HCO₃ (Sigma-Aldrich Co. LLC, MO, USA) and the proteins were extracted with 50% acetonitrile (ACN; Sigma-Aldrich Co. LLC., MO, USA). The proteins were then digested with 1 μg/μL robust trypsin (Promega Corporation, WI, USA) for 30 min. Subsequently, coverage solution (10% ACN, Milli-Q water, and 50 mM NH₄HCO₃) was added, and the samples were incubated further for 16 h. Following digestion, the generated peptides were extracted with a solvent that consisted of 90% ACN and 2.5% trifluoroacetic acid (TFA; Promega Corporation, WI, USA) for 30 min. Finally, the peptides were vacuum-dried.