Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH)
- Buck Institute for Research on Aging, 94945, Novato, CA, USA
- Amgen, 94080, South San Francisco, CA, USA
- Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, 60153, Maywood, IL, USA
- Buck Institute for Research on Aging, 94945, Novato, CA, USA, Department of Pharmaceutical Chemistry, University of California, 94143, San Francisco, CA, USA
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.
- Research Organization:
- Buck Inst. for Research on Aging, Novato, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Inst. of Health (NIH) (United States)
- Grant/Contract Number:
- SC0012443; R24DK085610; T32G000266; 1S10 OD016281
- OSTI ID:
- 1315863
- Alternate ID(s):
- OSTI ID: 1425373
- Journal Information:
- Journal of the American Society for Mass Spectrometry, Journal Name: Journal of the American Society for Mass Spectrometry Vol. 27 Journal Issue: 11; ISSN 1044-0305
- Publisher:
- American Chemical SocietyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
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