Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application

Mass spectrometry-based bottom-up proteomics is rapidly
evolving
and routinely applied in large-scale 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. The
quantification methods employed encompassed both label-free approaches
and absolute quantification utilizing spike-in heavy-labeled recombinant
protein fragment standards. Based on this extensive data set, we report
variations between commercial trypsins, their source, and their concentration.
Furthermore, we provide suggestions on the handling of trypsin in
large-scale studies.