title: Getting intimate with trypsin, the leading protease in proteomics.
abstract: Nowadays, mass spectrometry-based proteomics is carried out primarily in a bottom-up fashion, with peptides obtained after proteolytic digest of a whole proteome lysate as the primary analytes instead of the proteins themselves. This experimental setup crucially relies on a protease to digest an abundant and complex protein mixture into a far more complex peptide mixture. Full knowledge of the working mechanism and specificity of the used proteases is therefore crucial, both for the digestion step itself as well as for the downstream identification and quantification of the (fragmentation) mass spectra acquired for the peptides in the mixture. Targeted protein analysis through selected reaction monitoring, a relative newcomer in the specific field of mass spectrometry-based proteomics, even requires a priori understanding of protease behavior for the proteins of interest. Because of the rapidly increasing popularity of proteomics as an analytical tool in the life sciences, there is now a renewed demand for detailed knowledge on trypsin, the workhorse protease in proteomics. This review addresses this need and provides an overview on the structure and working mechanism of trypsin, followed by a critical analysis of its cleavage behavior, typically simply accepted to occur exclusively yet consistently after Arg and Lys, unless they are followed by a Pro. In this context, shortcomings in our ability to understand and predict the behavior of trypsin will be highlighted, along with the downstream implications. Furthermore, an analysis is carried out on the inherent shortcomings of trypsin with regard to whole proteome analysis, and alternative approaches will be presented that can alleviate these issues. Finally, some reflections on the future of trypsin as the workhorse protease in mass spectrometry-based proteomics will be provided.
authors.affiliation: Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium; Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium.
authors.forename: Elien
authors.initials: E
authors.lastname: Vandermarliere
authors.forename: Michael
authors.initials: M
authors.lastname: Mueller
authors.forename: Lennart
authors.initials: L
authors.lastname: Martens
mesh_terms: D000818:Animals; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D011487:Protein Conformation; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010455:Peptides; D020543:Proteome; D014357:Trypsin
keywords: missed cleavage; peptides; protein cleavage; proteomics; trypsin