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

• Published in: Analytical and bioanalytical chemistry Vol. 408; no. 13; pp. 3547 - 3553
• Main Authors: D’Urzo, Annalisa, Boichenko, Alexander P., van den Bosch, Thea, Hermans, Jos, Dekker, Frank, Andrisano, Vincenza, Bischoff, Rainer
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-05-2016; Springer; Springer Nature B.V
• Subjects: Acetic acid; Acetylation; Alzheimer's disease; Amino Acid Sequence; Analytical Chemistry; Anhydrides; Animals; Biochemistry; Cell Line; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography, High Pressure Liquid - methods; Chymotrypsin; Disease; Enzymes; Food Science; High performance liquid chromatography; Histone Deacetylase Inhibitors - pharmacology; Histones; Histones - chemistry; Inhibitors; Labeling; Laboratory Medicine; Liquid chromatography; Lysine; Lysine - chemistry; Mass spectrometry; Methods; Mice; Monitoring/Environmental Analysis; Observations; Peptides; Physiological aspects; Proteins; Proteomics; Reproducibility of Results; Research Paper; Scientific imaging; Tandem Mass Spectrometry - methods; Trypsin; Netherlands; Tandem mass spectrometry; Histone deacetylase (HDAC) inhibitors; Histone acetylation; Multiple reaction monitoring (MRM); Post-translation modification (PTM)
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-016-9431-1

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
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
 ≥ 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.