title: Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics.
abstract: 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.
authors.affiliation: Department of Pathology, University of Michigan , 4237 Medical Science I, 1301 Catherine Road, Ann Arbor, Michigan 48109, United States.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Walmsley
authors.forename: Paul A
authors.initials: PA
authors.lastname: Rudnick
authors.forename: Yuxue
authors.initials: Y
authors.lastname: Liang
authors.forename: Qian
authors.initials: Q
authors.lastname: Dong
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Stein
authors.forename: Alexey I
authors.initials: AI
authors.lastname: Nesvizhskii
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D056148:Chromatography, Reverse-Phase; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D025341:Principal Component Analysis; D059748:Proteolysis; D040901:Proteomics / Q000592:standards*; D011786:Quality Control; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry*; D013045:Species Specificity; D013552:Swine; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D012709:Serum Albumin; D014357:Trypsin