title: Proteinase K improves quantitative acylation studies.
abstract: Acetylation is a common PTM of proteins but is still challenging to analyze. Only few acetylome studies have been performed to tackle this issue. Yet, the detection of acetylated proteins in complex cell lysates remains to be improved. Here, we present a proteomic approach with proteinase K as a suitable protease to identify acetylated peptides quantitatively. We first optimized the digestion conditions using an artificial system of purified bovine histones to find the optimal protease. Subsequently, the capability of proteinase K was demonstrated in complex HEK293 cell lysates. Finally, SILAC in combination with MudPIT was used to show that quantification with proteinase K is possible. In this study, we identified a sheer number of 557 unique acetylated peptides originating from 633 acetylation sites.
authors.affiliation: Department of Analytical Chemistry, Biomolecular Mass Spectrometry, Institute of Chemistry and Biochemistry, Ruhr-Universität Bochum, Bochum, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fränzel
authors.forename: Frank
authors.initials: F
authors.lastname: Fischer
authors.forename: Clemens
authors.initials: C
authors.lastname: Steegborn
authors.forename: Dirk Andreas
authors.initials: DA
authors.lastname: Wolters
mesh_terms: D000107:Acetylation; D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019286:Endopeptidase K / Q000378:metabolism*; D057809:HEK293 Cells; D006657:Histones / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics; D037761:Sirtuins / Q000378:metabolism
chemical_list: D006657:Histones; D010455:Peptides; D019286:Endopeptidase K; D037761:Sirtuins
keywords: Acetylation; Digestion conditions; Proteinase K; Sirtuins; Technology