Quantitative analysis of chaotropic solvent effects on the trypsin digestion efficiency of human plasma

摘要

No single digestion condition is 100percent efficient for all 45 proteins tested. The efficiency of trypsin digestion widely varies protein by protein with complete digestion is attainable in <2 h for many proteins. Urea denaturation is incomplete and results in reduced signal intensity. SDS denaturation is most efficient at denaturation, but MS incompatible. DOC denaturation is efficient and MS compatible (precipitates during acidification). Solvent denaturation with TFE and MeOH quite effective, but not present during reduction & alkylation. A combined DOC/solvent denaturation to improve reduction reduces digestion efficiency. Some proteins digest slowly regardless of protocol used. Standardized protocols are critical for reproducible quantitation.