Quantification of Lysine Acetylation and Succinylation Stoichiometry in Proteins Using Mass Spectrometric Data-Independent Acquisitions (SWATH).
Jesse G MeyerAlexandria K D'SouzaDylan J SorensenMatthew J RardinAlan J WolfeBradford W GibsonBirgit SchillingPublished in: Journal of the American Society for Mass Spectrometry (2016)
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 Abstract ᅟ.
Keyphrases
- mass spectrometry
- amino acid
- histone deacetylase
- electronic health record
- big data
- single cell
- computed tomography
- high resolution
- liquid chromatography
- quantum dots
- multiple sclerosis
- gene expression
- magnetic resonance
- gas chromatography
- mesenchymal stem cells
- genome wide
- bone marrow
- high intensity
- nitric oxide
- fluorescent probe
- case control
- tandem mass spectrometry
- pet ct