期刊
ANALYTICAL CHEMISTRY
卷 80, 期 16, 页码 6178-6189出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac800440r
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资金
- National Science Council [NSC-96-2628-M006-005]
- National Cheng Kung University Project of Promoting Academic Excellence & Developing World Class Research Centers from Ministry of Education of Taiwan
Acetylation of proteins on specific lysine residues by acetyltransferase enzymes is-a post-translational modification for biologically relevant regulation. In this study, we proposed a strategy to determine the in vitro acetylation sites of proteins by tracing isotope-labeled acetyl groups using mass spectrometry. Isotope-labeled and unlabeled acetyl groups transferred onto the substrates in vitro result in a specific mass difference that can be measured by MS analysis and utilized for localization of potential acetylated peptide signals. The identification of acetylation site is facilitated by conducting MS/MS experiments on those selected signals. Acetylation reactions of substrates were performed in the presence of acetyltransferase and equal molar of isotope-labeled acetyl coenzyme A ([C-13(2)-2-D-3]-acetyl-CoA) and unlabeled acetyl-CoA. After enzymatic digestion, the resulting peptide mixture was fractionated by off-line, reversed-phase high-pressure liquid chromatography and the accurate mass measurement of peptides was achieved by a quadrupole/time-of-flight mass spectrometer. Signals with 5-Da (or their multiples) mass differences and equal responses were selected out by program computation. Those potential acetylated peptide signals were subjected to MS/MS analyses for determination of acetylation sites. We have used histone H3 peptide (aa 1-20), histone H2B peptide (aa 1-21), histone H2A, and histone H2B proteins as the model compounds to demonstrate the applicability of this analytical scheme for the characterization of in vitro acetylation sites.
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