Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics

Scott J. Walmsley, Paul A. Rudnick, Yuxue Liang, Qian Dong, Stephen E. Stein, Alexey I. Nesvizhskii

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55 Scopus citations


Trypsin is an endoprotease commonly used for sample preparation in proteomics experiments. Importantly, protein digestion is dependent on multiple factors, including the trypsin origin and digestion conditions. In-depth characterization of trypsin activity could lead to improved reliability of peptide detection and quantitation in both targeted and discovery proteomics studies. To this end, we assembled a data analysis pipeline and suite of visualization tools for quality control and comprehensive characterization of preanalytical variability in proteomics experiments. Using these tools, we evaluated six available proteomics-grade trypsins and their digestion of a single purified protein, human serum albumin (HSA). HSA was aliquoted and then digested for 2 or 18 h for each trypsin, and the resulting digests were desalted and analyzed in triplicate by reversed-phase liquid chromatography-tandem mass spectrometry. Peptides were identified and quantified using the NIST MSQC pipeline and a comprehensive HSA mass spectral library. We performed a statistical analysis of peptide abundances from different digests and further visualized the data using the principal component analysis and quantitative protein "sequence maps". While the performance of individual trypsins across repeat digests was reproducible, significant differences were observed depending on the origin of the trypsin (i.e., bovine vs porcine). Bovine trypsins produced a higher number of peptides containing missed cleavages, whereas porcine trypsins produced more semitryptic peptides. In addition, many cleavage sites showed variable digestion kinetics patterns, evident from the comparison of peptide abundances in 2 h vs 18 h digests. Overall, this work illustrates effects of an often neglected source of variability in proteomics experiments: the origin of the trypsin.

Original languageEnglish (US)
Pages (from-to)5666-5680
Number of pages15
JournalJournal of Proteome Research
Issue number12
StatePublished - Dec 6 2013
Externally publishedYes


  • digestion
  • endoprotease specificity
  • label-free quantification
  • mass spectrometry
  • missed cleavages
  • peptide abundance
  • proteomics
  • statistical analysis
  • trypsin
  • variability


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