Simultaneous Quantification of the Acetylome and Succinylome by ‘One‐Pot’ Affinity Enrichment

Abstract

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi‐PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label‐free proteomic workflow—“one‐pot” PTM enrichment—that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label‐free, data‐independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one‐pot method are identified and quantified and peak areas are shown to be highly correlated between the one‐pot and traditional single‐PTM enrichments. The ‘one‐pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single‐PTM enrichments, the one‐pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.

Quantitative Method for Assessing the Role of Lysine & Arginine Post-Translational Modifications in Nonalcoholic Steatohepatitis

“…Acetyl and Succinyl Enrichment Sample Preparation: Mitochondrial extracts and total protein lysate were prepared from mouse tissue as previously described. 28 Briefly, crude mitochondrial fractions were isolated from the livers of SIRT5-/-(C57BL/6) mice by differential centrifugation. Total protein lysate was also isolated from the livers of WT (C57BL/6) mice.…”

“…Immunoaffinity enrichments of modified peptides were done using the PTMScan® Acetyl-Lysine Motif [Ac-K] Kit (#13416) and PTMScan® Succinyl-Lysine Motif [Succ-K] Kit (#13764) (Cell Signaling Technology, Danvers, MA), using the 'one-pot' affinity enrichment previously described. 28 For one-pot enrichments, 1 mg peptide digests were incubated in a tube containing equal parts (~62.5 µg immobilized antibody) of both the succinyl-and acetyl-lysine antibodies. TripleTOF 6600 mass spectrometer (SCIEX, Redwood City, CA).…”

“…35 This gave us an assay library of 808 methyl Arg and Lys peptides. We further supplemented the library with DDA acquisitions of a 'one-pot' acetyl-Lys and succinyl-Lys immunoaffinity enrichment of mitochondrial extract and whole liver lysate isolated from WT mouse liver as described in Basisty et al 28 which provided us with 2428 succinylated peptides and 2547 acetylated peptides.…”

“…In addition to methylation, lysine residues can be modified by various other PTM's, including acetylation and succinylation. [25][26][27][28][29] To understand the extent of lysine post translational modification, we expanded our DIA assay library to include succinylated and acetylated peptides, thus providing an opportunity to quantify methylated, acetylated, succinylated, and unmodified peptides from the same sample and acquisition run. This not only allows for classical cellular protein quantification, but also provides the opportunity to quantify peptidoforms containing Lys methylation, succinylation, acetylation, and Arg methylation simultaneously from a single DIA acquisition.…”

“…Traditional DDA and DIA based PTM-immunoprecipitation enrichment approaches rely on antibody enrichment for the modified peptide and by nature are unable to quantify unmodified peptides in the same acquisition or enrich at the single modified protein which has limited scope. 4,28 In our method, we are able to quantify both modified and unmodified peptides of seven different PTMs using DIA acquisitions with small precursor mass window requiring only five percent of starting material necessary to perform PTM immunoprecipitations. The important distinction with using small precursor mass windows is that we are able to physically trap each modified peptidoform away from other modified peptidoforms in addition to the unmodified peptide, so that all mono-methylated peptides are in a different precursor mass window than the unmodified peptidoform as well as the di-the tri-methylated peptidoforms.…”