Comprehensive Analysis of Protein Digestion Using Six Trypsins Reveals the Origin of Trypsin As a Significant Source of Variability in Proteomics

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.