title: Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC50 for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Guitot
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France.
authors.forename: Silvia
authors.initials: S
authors.lastname: Scarabelli
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Drujon
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine, IBPS / FR 3631, Plateforme de Spectrométrie de Masse et Protéomique, 7-9 Quai Saint Bernard, 75005 Paris, France.
authors.forename: Gérard
authors.initials: G
authors.lastname: Bolbach
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Amoura
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Burlina
authors.affiliation: Institute of Biochemistry, Faculty of Chemistry, University Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, LBM, 4, place Jussieu, F-75005 Paris, France; Ecole Normale Supérieure-PSL Research University, Département de Chimie, 24, rue Lhomond, 75005 Paris, France; CNRS, UMR 7203 LBM, F-75005 Paris, France. Electronic address: dominique.guianvarch@upmc.fr.
authors.forename: Dominique
authors.initials: D
authors.lastname: Guianvarc'h
mesh_terms: D000595:Amino Acid Sequence; D057075:Enzyme Assays / Q000191:economics / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods*; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D011495:Histone-Lysine N-Methyltransferase
keywords: ELISA; H3K4 methyltransferase; Histone lysine methyltransferase; Inhibitor; MALDI-TOF; Peptide methylation assay