Controlling nonspecific trypsin cleavages in LC-MS/MS-based shotgun proteomics using optimized experimental conditions.

摘要: Trypsin has traditionally been used for enzymatic digestion during sample preparation in shotgun proteomics. The stringent specificity of trypsin is essential accurate protein identification and quantification. But nonspecific cleavages are often observed LC-MS/MS-based To explore the extent cleavages, a series biological systems including standard mixture, Saccharomyces cerevisiae, human serum, cancer cell lines mouse brain were examined. We found that commonly occurred various digested samples with high frequency. control these we optimized fundamental parameters homogenates. These included denaturing agents storage time, type, enzyme-to-substrate ratio, as well concentration digestion. experimental conditions significantly decreased ratio partially tryptic peptides total identifications from 28.4% to 2.8%. Furthermore, protocol was applied study N-glycoproteomics, proportions enriched mixtures also sharply reduced. Our work demonstrates importance controlling both proteomics glycoproteomics provides better understanding standardization routine treatment.