Abstract
Post-translational modification of lysine residues by NƐ-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. Graphical ᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-016-1476-z) contains supplementary material, which is available to authorized users.
Highlights
Post-translational modification of lysine residues by N acetylation can modulate protein activities, conformation, and protein–protein interactions [1, 2]
We sought to improve the accuracy of determining acylation stoichiometry using data-independent acquisition (DIA)
We first benchmarked this new acylation occupancy workflow with experiments using bovine serum albumin (BSA), and we investigated protein lysates from E. coli under different growth conditions where we had previously observed large fold-changes in acetylation levels [23], but where data on the site occupancy of these changes was not known
Summary
Post-translational modification of lysine residues by N acetylation can modulate protein activities, conformation, and protein–protein interactions [1, 2]. To address the challenges inherent in accurate quantification of lysine acylation stoichiometry, we have developed a method that applies a variation of the stable isotope labeling method from Baeza et al [15], followed by SWATH acquisition that collects both precursor and multiple fragment ion abundances [42] We first benchmarked this new acylation occupancy workflow with experiments using (acetylated) bovine serum albumin (BSA), and we investigated protein lysates from E. coli under different growth conditions where we had previously observed large fold-changes in acetylation levels [23], but where data on the site occupancy of these changes was not. All raw files are uploaded at to the Center for Computational Mass Spectrometry, MassIVE (massive.ucsd.edu), and can be accessed using the MassIVE ID number: MSV000079779 (ProteomeXchange Accession PXD004234)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Journal of the American Society for Mass Spectrometry
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
