title: Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach.
abstract: We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R(2) ≥ 0.94), precision (RSD ≤ 10%), and accuracy (≤27%) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)-K8 and K5-K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role.
authors.affiliation: Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.|Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso di Augusto, 237-47921, Rimini, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: D'Urzo
authors.affiliation: Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Boichenko
authors.affiliation: Department of Pharmaceutical Gene Modulation, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
authors.forename: Thea
authors.initials: T
authors.lastname: van den Bosch
authors.affiliation: Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
authors.forename: Jos
authors.initials: J
authors.lastname: Hermans
authors.affiliation: Department of Pharmaceutical Gene Modulation, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands.
authors.forename: Frank
authors.initials: F
authors.lastname: Dekker
authors.affiliation: Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, Corso di Augusto, 237-47921, Rimini, Italy.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Andrisano
authors.affiliation: Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands. r.p.h.bischoff@rug.nl.
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D051379:Mice; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D008239:Lysine
keywords: Histone acetylation; Histone deacetylase (HDAC) inhibitors; Multiple reaction monitoring (MRM); Post-translation modification (PTM); Tandem mass spectrometry