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

ABSTRACT: Posttranslational modification of lysine residues by Nepsilon-acylation is an important regulator of protein function. Many large-scale protein acylation studies have assessed relative changes of lysine acylation sites using mass spectrometry-based proteomics. While 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 over-estimate acetylation stoichiometries due to 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 from data-independent acquisitions (DIA). Specifically, we use SWATH acquisitions 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 by using deuterium labeled succinic anhydride. Proof of principle SWATH acquisition studies were first performed using BSA for both acetylation and succinylation occupancy measurements, followed by the analysis of more complex samples of E. coli cell lysates. While overall site occupancy was low (<1 percent), some proteins contained lysines with relatively high acetylation occupancy.

INSTRUMENT(S): TripleTOF 6600, TripleTOF 5600

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Escherichia Coli (ncbitaxon:562)

SUBMITTER: Brad Gibson Birgit Schilling 

PROVIDER: MSV000079779 | MassIVE | Thu May 26 20:04:00 BST 2016

SECONDARY ACCESSION(S): PXD004234

REPOSITORIES: MassIVE