Mass spectrometry-based bottom-up proteomics is rapidly evolving and routinely applied in large biomedical studies. Proteases are a central component of every bottom-up proteomics experiment, digesting proteins into peptides. Trypsin has been the most widely applied protease in proteomics, due to its characteristics. With ever-larger cohort sizes and possible future clinical application of mass spectrometry-based proteomics, the technical impact of trypsin becomes increasingly relevant. To assess possible biases introduced by trypsin digestion, we evaluated the impact of eight commercially available trypsins in a variety of bottom-up proteomics experiments, and across a range of protease concentrations and storage times. To investigate the universal impact of these technical attributes, we included bulk HeLa-cell lysate, human plasma and single HEK293 cells, which were analyzed over a range of Selected Reaction Monitoring (SRM), Data-Independent Acquisition (DIA), and Data-Dependent Acquisition (DDA) instrument methods on three LC-MS instruments. Quantification methods employed encompassed both label-free approaches and absolute quantification utilizing spike-in heavy-labeled recombinant protein fragment standards. Based on this extensive dataset, we report variations between commercial trypsins, their source, as well as their concentration. Furthermore, we provide suggestions on the handling of trypsin in large scale studies.

The impact of eight trypsins was assessed in HeLa bulk cell lysate, human plasma and single HEK293 cells. To evaluate the impact on different experimental setups, we conducted data-dependent acquisition (QExactive HF), data-independent acquisition (QExactive HF, Orbitrap Eclipse Tribrid) and selected reaction monitoring (TSQ Altis). Furthermore, LFQ and absolute quantification were explored by incorporating heavy labeled standards for the absolute quantification of plasma proteins with SRM.

Pooled human plasma from five healthy individuals (DIA), Pooled human plasma from five healthy individuals spiked with heavy-labeled recombinant protein fragment standards (SRM), HeLa cell lysate (DDA), single HEK293 cells (DIA)