A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation

Abstract

Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.

Site specificity analysis of Piccolo NuA4-mediated acetylation for different histone complexes

“…The assays contained 0.02 to 0.8 µM Piccolo NuA4 (varied with substrate concentration) and 0.15 – 20 µM of (H3/H4) 2 , 0.15 – 15 µM of tetrasome, or 0.15 – 15 µM of NCP with saturating acetyl-CoA (300 µM). Sample preparation, including reaction quench, post-reaction propionylation, and tryptic digestion, were performed as in our previous published procedures [52,53]. …”

“…The UPLC and MS/MS settings, solvent gradient, and detailed mass transitions were reported in our previously published work [40,52,53] and Table 1. Retention time and specific mass transitions were both used to identify individual acetylated and/or propionylated peptides.…”

“…The areas under individual resolved peaks were integrated using Xcalibur software (version 2.1, Thermo). Relative quantitative analysis was used to determine the amount of acetylation on individual lysines [52,53], and the time course kinetics of Piccolo NuA4-mediated acetylation for individual lysines can be plotted. We also noted that the ionization efficiency of H4 tail peptide (K5-R17) is about 10-fold less than that of H3 K9-R17 peptide.…”

Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels

“…These samples were TCA-precipitated, acetone-washed, and prepared for mass spectrometry analysis as described previously (42). A Waters Acquity H-class ultra-performance liquid chromatography system coupled to a Thermo (Waltham, MA) TSQ Quantum Access triple-quadrupole (QqQ) mass spectrometer was used to quantify modified histones.…”

“…Selected reaction monitoring was used to monitor the elution of the acetylated and propionylated tryptic peptides. Samples were analyzed as described previously (42). Each acetylated and/or propionylated peak was identified by retention time and specific transitions.…”