Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application
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Assessing the Role of Trypsin in Quantitative Plasma and Single-Cell Proteomics toward Clinical Application. / Woessmann, Jakob; Petrosius, Valdemaras; Üresin, Nil; Kotol, David; Aragon-Fernandez, Pedro; Hober, Andreas; auf dem Keller, Ulrich; Edfors, Fredrik; Schoof, Erwin M.
I: Analytical Chemistry, Bind 95, Nr. 36, 2023, s. 13649-13658.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 -
ID: 368250170