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.
- Authors:
-
- The Buck Institute for Research on Aging, Novato, CA (United States)
- The Buck Institute for Research on Aging, Novato, CA (United States); Amgen, South San Francisco, CA (United States)
- Publication Date:
- Research Org.:
- Univ. of Illinois at Urbana-Champaign, IL (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
- OSTI Identifier:
- 1465215
- Alternate Identifier(s):
- OSTI ID: 1465216; OSTI ID: 1545800
- Grant/Contract Number:
- SC0012443; OD016281
- Resource Type:
- Journal Article: Published Article
- Journal Name:
- Proteomics
- Additional Journal Information:
- Journal Volume: 18; Journal Issue: 17; Journal ID: ISSN 1615-9853
- Publisher:
- Wiley
- Country of Publication:
- United States
- Language:
- English
- Subject:
- acetylation; data‐independent acquisition; immunoaffinity enrichment; posttranslational modification; succinylation
Citation Formats
Basisty, Nathan, Meyer, Jesse G., Wei, Lei, Gibson, Bradford W., and Schilling, Birgit. Simultaneous Quantification of the Acetylome and Succinylome by ‘One-Pot’ Affinity Enrichment. United States: N. p., 2018.
Web. doi:10.1002/pmic.201800123.
Basisty, Nathan, Meyer, Jesse G., Wei, Lei, Gibson, Bradford W., & Schilling, Birgit. Simultaneous Quantification of the Acetylome and Succinylome by ‘One-Pot’ Affinity Enrichment. United States. doi:10.1002/pmic.201800123.
Basisty, Nathan, Meyer, Jesse G., Wei, Lei, Gibson, Bradford W., and Schilling, Birgit. Mon .
"Simultaneous Quantification of the Acetylome and Succinylome by ‘One-Pot’ Affinity Enrichment". United States. doi:10.1002/pmic.201800123.
@article{osti_1465215,
title = {Simultaneous Quantification of the Acetylome and Succinylome by ‘One-Pot’ Affinity Enrichment},
author = {Basisty, Nathan and Meyer, Jesse G. and Wei, Lei and Gibson, Bradford W. and Schilling, Birgit},
abstractNote = {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.},
doi = {10.1002/pmic.201800123},
journal = {Proteomics},
issn = {1615-9853},
number = 17,
volume = 18,
place = {United States},
year = {2018},
month = {7}
}
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