Isotopic Labeling and Quantitative Proteomics of Acetylation on Histones and Beyond

Abstract

Lysine acetylation is an important post-translational 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 for not only 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 then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.

Stable Isotopes for Tracing Cardiac Metabolism in Diseases

“…In combination with other tracers it is possible to determine the overall utilization (uptake and metabolization) of glucose. To gain insight into the systems-wide response during disease development, it is also necessary to assess how metabolic alterations are linked to protein dynamics and posttranslation modifications ( 83 , 84 ). Stable isotope tracers enable measuring posttranslational modifications of proteins.…”

Current Methods of Post-Translational Modification Analysis and Their Applications in Blood Cancers

“…However, other residues such as histidine, proline, and glutamine may also be subject to methylation. Methylation is catalyzed by lysine or arginine methyltransferases and reversed by demethylases [ 149 ] Research on acetylation and methylation is often focused on histone modifications which requires specific sample preparation approaches, as described above [ 22 ].…”

“…Once again, acetylated proteins are enriched prior to LC–MS/MS. Immunoaffinity purification of acetylated peptides using specific antibodies is the most common and effective method of enrichment [ 22 , 156 , 157 , 158 ]. Combined fractional diagonal chromatography (COFRADIC) is also a popular technique that specifically enriches N-terminal acetylated proteins through the derivatization of primary amines [ 159 , 160 ].…”

“…Subsequently, this technique has been adapted to facilitate the labelling of specific PTMs. For example, 13 C-glucose and D 3 -acetate, 13 CD 3 -methionine; and γ-18 O 4 -labeled ATP are added to cell culture media to label acetylation, methylation, and phosphorylation, respectively [20][21][22]. Label-free quantitation (LFQ) is a commonly used method of quantitation which refers to the use of peak intensity analysis and spectral counting for quantitation [23].…”

Stable Isotope Tracing In Vivo Reveals A Metabolic Bridge Linking The Microbiota To Host Histone Acetylation

“…Histones were isolated from the nuclei of frozen cell pellets by acid extraction and derivatized with propionic anhydride similarly as described previously (Lund et al, 2019). Briefly, cell pellets were resuspended in nuclear isolation buffer (NIB) containing 15 mM Tris pH 7.5, 15 mM NaCl, 60 mM KCl, 5 mM MgCl 2 , 1 mM CaCl 2 , and 250 mM sucrose supplemented with 1 mM DTT, 10 mM sodium butyrate, 500 µM AEBSF, 5 nM microcystin, and 0.2% NP-40 Alternative.…”