Strategy for determination of in vitro protein acetylation sites by using isotope-labeled acetyl coenzyme A and liquid chromatography-mass spectrometry.
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 ([(13)C2-2-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.
Wu
Hsin-Yi
HY
Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Huang
Fu-Yung
FY
Chang
Yu-Chen
YC
Hsieh
Meng-Chun
MC
Liao
Pao-Chi
PC
D000105:Acetyl Coenzyme A / Q000737:chemistry*; D000107:Acetylation; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006657:Histones / Q000032:analysis; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D006657:Histones; D010446:Peptide Fragments; D000105:Acetyl Coenzyme A; D051548:Histone Acetyltransferases