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Title: Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)

Abstract

Post-translational modification of lysine residues by N ε-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinicmore » anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.« less

Authors:
 [1];  [1];  [1];  [2];  [3];  [4]; ORCiD logo [1]
  1. Buck Inst. for Research on Aging, Novato, CA (United States)
  2. Amgen Inc., South San Francisco, CA (United States)
  3. Loyola Univ. Chicago, IL (United States). Dept. of Microbiology and Immunology. Stritch School of Medicine. Health Sciences Division
  4. Buck Inst. for Research on Aging, Novato, CA (United States); Univ. of California, San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry
Publication Date:
Research Org.:
Buck Inst. for Research on Aging, Novato, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Inst. of Health (NIH) (United States)
OSTI Identifier:
1315863
Alternate Identifier(s):
OSTI ID: 1425373
Grant/Contract Number:  
SC0012443; R24DK085610; T32G000266; 1S10 OD016281
Resource Type:
Journal Article: Published Article
Journal Name:
Journal of the American Society for Mass Spectrometry
Additional Journal Information:
Journal Volume: 27; Journal Issue: 11; Journal ID: ISSN 1044-0305
Publisher:
American Society for Mass Spectrometry
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; stoichiometry; acetylation; succinylation; SWATH; data-independent acquisition; mass spectrometry; skyline

Citation Formats

Meyer, Jesse G., D’Souza, Alexandria K., Sorensen, Dylan J., Rardin, Matthew J., Wolfe, Alan J., Gibson, Bradford W., and Schilling, Birgit. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH). United States: N. p., 2016. Web. doi:10.1007/s13361-016-1476-z.
Meyer, Jesse G., D’Souza, Alexandria K., Sorensen, Dylan J., Rardin, Matthew J., Wolfe, Alan J., Gibson, Bradford W., & Schilling, Birgit. Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH). United States. doi:10.1007/s13361-016-1476-z.
Meyer, Jesse G., D’Souza, Alexandria K., Sorensen, Dylan J., Rardin, Matthew J., Wolfe, Alan J., Gibson, Bradford W., and Schilling, Birgit. Fri . "Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)". United States. doi:10.1007/s13361-016-1476-z.
@article{osti_1315863,
title = {Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)},
author = {Meyer, Jesse G. and D’Souza, Alexandria K. and Sorensen, Dylan J. and Rardin, Matthew J. and Wolfe, Alan J. and Gibson, Bradford W. and Schilling, Birgit},
abstractNote = {Post-translational modification of lysine residues by Nε-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites after antibody enrichment using mass spectrometry-based proteomics. Although relative acylation fold-changes are important, this does not reveal site occupancy, or stoichiometry, of individual modification sites, which is critical to understand functional consequences. Recently, methods for determining lysine acetylation stoichiometry have been proposed based on ratiometric analysis of endogenous levels to those introduced after quantitative per-acetylation of proteins using stable isotope-labeled acetic anhydride. However, in our hands, we find that these methods can overestimate acetylation stoichiometries because of signal interferences when endogenous levels of acylation are very low, which is especially problematic when using MS1 scans for quantification. In this study, we sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA). Specifically, we use SWATH acquisition to comprehensively collect both precursor and fragment ion intensity data. The use of fragment ions for stoichiometry quantification not only reduces interferences but also allows for determination of site-level stoichiometry from peptides with multiple lysine residues. We also demonstrate the novel extension of this method to measurements of succinylation stoichiometry using deuterium-labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using bovine serum albumin for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. Although overall site occupancy was low (<1%), some proteins contained lysines with relatively high acetylation occupancy.},
doi = {10.1007/s13361-016-1476-z},
journal = {Journal of the American Society for Mass Spectrometry},
issn = {1044-0305},
number = 11,
volume = 27,
place = {United States},
year = {2016},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1007/s13361-016-1476-z

Citation Metrics:
Cited by: 6 works
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