The secret scalpel: Plastic surgery for wartime disguise

摘要

Proteolytic digestion is a time consuming and critical step in bottom-up proteomic analysis.Themost widely used protease, trypsin, has high specificity and generates peptides that are consideredto be ideally suited for bottom-up LC-MS technology. By exploiting key factors affectingenzymatic activity we obtained a simple, straightforward, and rapid in-solution digest protocolthat performed better than the conventional overnight digestionmethod in terms of amino acidcoverage of proteins, number of peptides generated, and peptide ion abundances. Prolongeddigestion time, such as overnight digestion, leads to decline in protein amino acid coverage andloss of tryptic peptides. This was found to be caused by complete digestion by trypsin leadingto an increased number of small peptides that are not LC-MS detectable. Slow-rate nontrypticdigestion of peptides is a contributing factor for loss of peptide ion intensities during extendeddigestion time. Our work demonstrates that for both qualitative and quantitative bottom-upproteomic studies it is beneficial to prevent trypsin digestion to go to completion by reducingtreatment time from the conventional several hours to a few minutes cleavage time.