Label-free measurement of histone lysine methyltransferases activity by MALDI-TOF mass spectrometry. - Université Pierre et Marie Curie Accéder directement au contenu
Article Dans Une Revue Analytical Biochemistry Année : 2014

Label-free measurement of histone lysine methyltransferases activity by MALDI-TOF mass spectrometry.

Résumé

: 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 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.

Domaines

Chimie organique
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Dates et versions

hal-00982533 , version 1 (24-04-2014)

Identifiants

Citer

Karine Guitot, Silvia Scarabelli, Thierry Drujon, Gérard Bolbach, Mehdi Amoura, et al.. Label-free measurement of histone lysine methyltransferases activity by MALDI-TOF mass spectrometry.. Analytical Biochemistry, 2014, 456, pp.25-31. ⟨10.1016/j.ab.2014.04.006⟩. ⟨hal-00982533⟩
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