title: Pitfalls in histone propionylation during bottom-up mass spectrometry analysis.
abstract: Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom-up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom-up MS on histones.
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Belgium.
authors.forename: Paulien
authors.initials: P
authors.lastname: Meert
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Belgium.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Govaert
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Belgium.
authors.forename: Ellen
authors.initials: E
authors.lastname: Scheerlinck
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Dhaenens
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Belgium.
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006657:Histones / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D011422:Propionates / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D006657:Histones; D011422:Propionates
keywords: Cell biology; Histone; MS-LC-MS; Method optimization; Propionylation