title: Extracting histones for the specific purpose of label-free MS.
abstract: Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885.
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Govaert
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: Van Steendam
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Ellen
authors.initials: E
authors.lastname: Scheerlinck
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Vossaert
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Paulien
authors.initials: P
authors.lastname: Meert
authors.affiliation: Department of Pharmaceutical Sciences, University of Eastern Piedmont, Novara, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Stella
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Sander
authors.initials: S
authors.lastname: Willems
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Laura
authors.initials: L
authors.lastname: De Clerck
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Maarten
authors.initials: M
authors.lastname: Dhaenens
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium.
authors.forename: Dieter
authors.initials: D
authors.lastname: Deforce
mesh_terms: D005591:Chemical Fractionation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D053595:Embryonic Stem Cells; D006657:Histones / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D057188:Workflow
chemical_list: D006657:Histones
keywords: Comparative study; Epigenetics; Extraction protocol; Histone; Label-free MS quantification; Technology