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.

Keywords: acetylation; data-independent acquisition; immunoaffinity enrichment; posttranslational modification; succinylation.

Figure 1

Workflow and reproducibility of one‐pot enrichment of PTM's. A) Antibodies are combined into a single tube and immunoaffinity enrichment of both modifications is performed in a single step, followed by DIA analysis. B) Table summary of time and resource requirements for one‐pot, serial, and single pulldowns. C) Boxplot of coefficients of variation of all acetylated or succinylated peptides identified following acetyl‐lysine, succinyl‐lysine, or one‐pot enrichments. The median coefficient of variation (CV) for modified peptide areas was 15.3%, 24.5%, and 23.4% for one‐pot, acetyl‐lysine, and succinyl‐lysine affinity enrichments, respectively. D) Correlation of acetylation site‐level quantification of two one‐pot pulldowns performed in parallel with two acetylation‐only pulldowns (*d, days; **h, hours). E) Correlation of succinylation site‐level quantification one‐pot enrichments to succinylation‐only pulldowns. Ac, acetylated; Su, succinylated. Ac_single‐PTM, acetyl‐lysine pulldown sites; Ac_onepot, acetylation sites from one‐pot pulldown; Su_single‐PTM, succinyl‐lysine pulldown sites; Su_onepot, succinylation sites from one‐pot pulldown.

Figure 2

Database searching for acetylation and succinylation together versus individually. A) Table summary of the total number of acetylated and succinylated peptides (and percentage of total peptide IDs containing either modification) identified from performing database searching of both acyl modifications combined or individually. B) MS/MS spectrum of a peptide containing both acetylation and succinylation on K209 and K211, respectively, on the protein 3‐ketoacyl‐CoA thiolase, mitochondrial (precursor ion selected at m/z 828.07). C) MS/MS spectrum of the same peptide succinylated on both corresponding lysines (precursor ion selected at m/z 847.40). Ac, acetylated; Su, succinylated. Peptide MS/MS spectra were taken from a DDA spectral library.

Figure 3

Crosstalk between lysine acetylation and succinylation. A) Venn diagram of acetylation and succinylation sites identified following one‐pot enrichment. B) Term enrichment analysis of protein complexes among indiscriminately modified acylation sites (sites that are both acetylated and succinylated). Black bars represent the percent of total subunits containing sites. The red line represents the significance of enrichment by Fischer exact test, and the dotted line is the significance threshold. C) Schematic of ATP synthase highlighting the subunits containing indiscriminately modified acylation sites in red. Ac, acetylated; Su, succinylated.