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, A., Boichenko, A. P., van den Bosch, T., Hermans, J., Dekker, F., Andrisano, V. & Bischoff, R., May-2016, In : Analytical and Bioanalytical Chemistry. 408, 13, p. 3547-3553 7 p.

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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 epsilon-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(6)- (heavy) or d(0)- (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) a parts per thousand yenaEuro parts per thousand 0.94), precision (RSD a parts per thousand currency signaEuro parts per thousand 10 %), and accuracy (a parts per thousand currency sign27 %) 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.

Original languageEnglish
Pages (from-to)3547-3553
Number of pages7
JournalAnalytical and Bioanalytical Chemistry
Issue number13
Publication statusPublished - May-2016


  • Histone acetylation, Tandem mass spectrometry, Multiple reaction monitoring (MRM), Post-translation modification (PTM), Histone deacetylase (HDAC) inhibitors, MASS-SPECTROMETRY, DEACETYLASE INHIBITORS, ALZHEIMERS-DISEASE, PROPIONYLATION, MS

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