Dataset Information

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.

SUBMITTER: Basisty N

PROVIDER: S-EPMC6175148 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Simultaneous Quantification of the Acetylome and Succinylome by 'One-Pot' Affinity Enrichment.

Basisty Nathan N Meyer Jesse G JG Wei Lei L Gibson Bradford W BW Schilling Birgit B

Proteomics 20180819 17

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-f ...[more]

PMID: 30035354

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Project description:Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA) increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is common in B. subtilis, just as it is in Escherichia coli. Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

Dataset Information

Simultaneous Quantification of the Acetylome and Succinylome by 'One-Pot' Affinity Enrichment.

Publications

Simultaneous Quantification of the Acetylome and Succinylome by 'One-Pot' Affinity Enrichment.

Similar Datasets

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