Quantification of Site-Specific Protein Lysine Acetylation and Succinylation Stoichiometry using Data-Independent Acquisitions (DIA) Mass Spectrometry --Protocol Example
Lei Wei, Jesse G. Meyer, Birgit Schilling "Quantification of Site-Specific Protein Lysine Acetylation and Succinylation Stoichiometry using Data-Independent Acquisitions (DIA) Mass Spectrometry" Journal of Visualized Experiments (JoVE), 2017, under revision.
- Organism: Bovine
- Instrument: TripleTOF 5600
Post-translational modification (PTM) of protein lysine residues by NƐ-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. Here, we describe an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, our workflow offers unbiased, precise and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.
Chemical Treatments to Quantitatively Acetylate and Succinylate Proteins - Acylation Stoichiometry
Stoichiometry workflow. (a) First, protein samples were incubated three times with either succinic anhydride-d4 (or alternatively acetic anhydride-d6) to chemically acylated unmodified lysine residues. Second, samples were digested with endoproteinase Glu-C, followed by optional offline fractionation of the proteolytic peptides by basic reversed-phase chromatography. Finally, peptides were analyzed by LC-MS in SWATH acquisition mode. These workflows will either determine succinylation stoichiometry (using succinic anhydride-d4) or acetylation stoichiometry (using acetic anhydride-d6).
The protein samples used here in the representative results section are bovine serum albumin (BSA), quantitatively succinylated BSA and mixtures thereof yielding BSA samples with defined succinylation occupancy at 0%, 1%, 10%, 50%, and 100%, respectively. This protocol has also been performed using protein lysates from E. coli and mouse liver. The protocol can also be applied to other cell or tissue lysates.
Created on 11/20/17 10:13 AM