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

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
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  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:
Grant/Contract Number:
SC0012443; R24DK085610; T32G000266; 1S10 OD016281
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
American Society for Mass Spectrometry
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)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; stoichiometry; acetylation; succinylation; SWATH; data-independent acquisition; mass spectrometry; skyline
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1425373