Strategy for determination of in vitro protein acetylation sites by using isotope-labeled acetyl coenzyme a and liquid chromatography-mass spectrometry

Hsin Yi Wu, Fu Yung Huang, Yu Chen Chang, Meng Chun Hsien, Pao Chi Liao

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

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 ([ 13C2-2-D3]-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.

Original languageEnglish
Pages (from-to)6178-6189
Number of pages12
JournalAnalytical chemistry
Volume80
Issue number16
DOIs
Publication statusPublished - 2008 Aug 15

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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