Isotopic Labeling and Quantitative Proteomics of Acetylation on Histones and Beyond.

Isotopic Labeling and Quantitative Proteomics of Acetylation on Histones and Beyond. Methods Mol Biol. 2019;1977:43-70 Authors: Lund PJ, Kori Y, Zhao X, Sidoli S, Yuan ZF, Garcia BA Abstract Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, 13C-glucose and D3-acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and ...
Source: Molecular Medicine - Category: Molecular Biology Authors: Tags: Methods Mol Biol Source Type: research