The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis

摘要: The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60 °C), reduction and alkylation, digestion time could be reduced profoundly compared to conventional conditions (ratio 1:40, overnight) without compromising method sensitivity or repeatability. procedure was obtained a systematic evaluation level quality using set three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) cytochrome C (CytC). All peptides monitored were produced at similar higher levels 45 min 1:1, overnight (exception: CytC modified trypsin, required up 4 h (at 1:1 ratio) order achieve this). Peptide decay due chymotryptic activity observed longer times, but circumvented times <4 h. accelerated protocol (w/w), min) applied realistic samples containing biomarker hCG demonstrate its applicability.