Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach.

D'Urzo, Annalisa; Boichenko, Alexander; Bosch, Thea; Hermans, Jos; Dekker, Frank; Andrisano, Vincenza; Bischoff, Rainer

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 d- (heavy) or d- (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 ≥ 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.

SPECTRUM analysis; QUALITATIVE chemical analysis; RADIATION; ACOUSTIC spectroscopy; ALPHA ray spectrometry

Analytical & Bioanalytical Chemistry, 2016, Vol 408, Issue 13, p3547

1618-2642

Academic Journal

10.1007/s00216-016-9431-1