title: The use of proteases complementary to trypsin to probe isoforms and modifications.
abstract: The wide diversity of proteins expressed in a cell or a tissue as a result of gene variants, RNA editing or PTMs results in several hundred thousand distinct functional proteins called proteoforms. The large-scale analysis of proteomes has been driven by bottom-up MS approaches. This allowed to identify and quantify large numbers of gene products and perform PTM profiling which yielded a significant number of biological discoveries. Trypsin is the gold standard enzyme for the production of peptides in bottom-up approaches. Several investigators argued recently that the near exclusive use of trypsin provided only a partial view of the proteome and hampered the discovery of new isoforms. The use of multiple proteases in a complementary fashion can increase sequence coverage providing more extensive PTM and sequence variant profiling. Here the various approaches to characterize proteoforms are discussed, including the use of alternative enzymes to trypsin in shotgun approaches to expand the observable sequence space by LC-MS/MS. The technical considerations associated with the use of alternative enzymes are discussed.
authors.affiliation: Luxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health, Strassen, Luxembourg.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Trevisiol
authors.affiliation: Luxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health, Strassen, Luxembourg.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ayoub
authors.affiliation: Luxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health, Strassen, Luxembourg.
authors.forename: Antoine
authors.initials: A
authors.lastname: Lesur
authors.affiliation: Luxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health, Strassen, Luxembourg.
authors.forename: Lina
authors.initials: L
authors.lastname: Ancheva
authors.affiliation: Luxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health, Strassen, Luxembourg.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gallien
authors.affiliation: Luxembourg Clinical Proteomics Center (LCP), Luxembourg Institute of Health, Strassen, Luxembourg.
authors.forename: Bruno
authors.initials: B
authors.lastname: Domon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D020033:Protein Isoforms / Q000032:analysis* / Q000235:genetics; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D020543:Proteome; D014357:Trypsin
keywords: Alternative proteases; Mass spectrometry; Proteoforms; Sequence coverage; Technology; Trypsin