TY - JOUR

T1 - Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application

AU - Woessmann, Jakob

AU - Petrosius, Valdemaras

AU - Üresin, Nil

AU - Kotol, David

AU - Aragon-Fernandez, Pedro

AU - Hober, Andreas

AU - auf dem Keller, Ulrich

AU - Edfors, Fredrik

AU - Schoof, Erwin M.

N1 - Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

U2 - 10.1021/acs.analchem.3c02543

DO - 10.1021/acs.analchem.3c02543

M3 - Journal article

C2 - 37639361

AN - SCOPUS:85171594219

VL - 95

SP - 13649

EP - 13658

JO - Industrial And Engineering Chemistry Analytical Edition

JF - Industrial And Engineering Chemistry Analytical Edition

SN - 0003-2700

IS - 36

ER -