Why less is more when generating tryptic peptides in bottom-up proteomics.

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