蛋白質中特定離胺酸的乙醯化是一種轉譯後修飾，與生物調節相關，且由乙醯轉移酶催化產生。質譜儀分析方法被認為是一個適合鑑定蛋白質乙醯化的工具。在本研究中，我們提出一個分析策略，藉由追蹤穩定同位素標定乙醯基來決定蛋白質乙醯化的位置。同位素標定和無標定的乙醯基會使受質產生特定的質量差異。此外，乙醯化胜肽的訊號會與加入乙醯化反應中同位素標定和無標定的乙醯基比例成等比。因此，藉由這兩個條件可在質譜數據中認定出乙醯化胜肽的訊號，並且針對這些訊號做MS/MS分析，鑑定乙醯化位置。在受質乙醯化反應中加入乙醯轉移酶及等量的同位素標定乙醯輔酶A和無標定之乙醯輔酶A。待反應完成後，利用酵素將蛋白質水解，所得到的胜肽混和物由高效能液相層析儀分離收集，減少樣品的複雜度。由四極棒式/飛行時間質譜儀可精確地測量胜肽質量。接著選擇質量差異5 Da的訊號利用MS/MS分析，可鑑定出乙醯化位置。我們將此分析策略應用於組織蛋白H3胜肽(aa 1-20)和組織蛋白H2B蛋白質，得到組織蛋白H3胜肽(aa 1-20) 的乙醯化位置位於離胺酸18的位置上，且驗證組織蛋白H2B蛋白質的乙醯化位置位於離胺酸6的位置上。這個分析方法結合LC-MS和LC-MS/MS可能可應用於更快速和大量的乙醯化鑑定。

The acetylation of protein on specific lysine residues by acetyltransferase enzymes is a posttranslational modification of biologically relevant regulation. Mass spectrometry is an appropriate method for identifying protein acetylation. In this study, we proposed a strategy to determine the acetylation sites of proteins by tracing stable isotope-labeled acetyl groups. Labeled and unlabeled acetyl groups resulted in specific mass differences on substrates. Moreover, the responses of acetylated peptide signals were in proportion to the ratio of labeled to unlabeled acetyl groups added to the acetylation reaction. Therefore, acetylated peptides signals can be located and subjected to MS/MS analysis for the determination of acetylation sites. Acetylation reactions of substrates were performed in the presence of acetyltransferase and equal molar of isotopic labeled acetyl coenzyme A ([13C2-2-D3]acetyl-CoA) and unlabeled acetyl-CoA. After enzymatic digestion, the resulted peptide mixture was fractionated by high performance liquid chromatography to reduce sample complexity. Accurate mass measurement of peptides was achieved by a quadrupole/time-of-flight mass spectrometer. Signals with identical responses and 5-Da (or their multiples) mass differences were selected and subjected to MS/MS analyses for the identification of acetylation sites. We have demonstrated the applicability of this scheme for characterization of acetylation sites at Lys 18 of histone H3 peptide (aa 1-20) and Lys 6 of histone H2B protein. Our stratergy may be applied for rapid and large-scale determination of acetylation sites using LC-MS and LC-MS/MS.

Bestor, T. H. (1998) Gene silencing: methylation meets acetylation. Nature 393:311-312.

Borchers, C., Parker C.E., Deterding, L. J., Tomer, K. B. (1999) Priliminary comparison of precursor scans and liquid chromatography-tandem mass spectrometry on a hybrid quadrupole time-of flight mass spectrometer. J. Chromatogr. A 854:119-130.

Burns, K. L., Gelbaum, L. T., Sullards, M. C., Bostwick, D. E. (2005) Iso-coenzyme A. J. Biol. Chem. 280(17):16550-16558.

Bonenefant, D., Coulot, H., Towbin, H., Schindler, P. (2006) Characterization of Histone H2A and H2B variants and their post-translational modifications by mass spectrometry. Mol. Cell. Proteomics 5:541-552.

Clough, R. C., Barnum, S. R., Jaworski, J. G. (1989) Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate. Anal. Biochem. 176:82-84.

Dalluge, J. J., Liao, H., Gokarn, R., Jessen, H. (2005) Discovery of enzymatic activity using stable isotope metabolite labeling and liquid chromatography-mass spectrometry. Anal. Chem. 77:6737-6740.

Dormeyer, W., Dorr, A., Ott, M. Schnolzer, M. (2003) Acetylation of the HIV-1 tat protein: an in vitro study. Anal. Bioanal. Chem. 376:994-1005.

Dormeyer, W., Ott, M., Schnolzer, M. (2005) Probing lysine acetylation in proteins. Mol. Cell. Proteomics 4:1226-1238.

Kim, J. Y., Kim, K. W., Kwon, H. J., Lee, D. W., Yoo, J. S. (2002)Probing lysine acetylation with a modification-specific marker ion using high-performance liquid chromatography/electrospray-mass spectrometry with collision-induced dissociation. Anal. Chem. 74:5443-5449.
Kuo, M. H., Allis, C. D. (1998) Roles of histone acetyltransferases and deacetylases in gene regulation. BioEssays 20:615-626.

Nelson, D. L., Cox, M. M. (2000) Lehninger principles of biochemistry, 3rd edn, Worth, NY. 924-927.

Peterson, C. L., Laniel, M. A. (2004) Histones and histone modifications. Curr. Biol. 14:R546-R551.

Rajarhia, V. B., Priestley, N. D., Strohl, W. R. (1995) Efficient synthesis of radiolabeled propionyl-coenzyme A and acetyl-coenzyme A. Anal. Biochem. 224:159-162.

Roth, S. Y., Denu, J. M., Allis, C. D. (2001) Histone acetyltransferases. Annu. Rev. Biochem. 70:81-120.

Seo, J., Lee, K. J. (2004) Post-translational modifications and their biological functions: proteomic analysis and systematic approaches. J. Biochem. Mol. Biol. 37:35-44.

Skoog, D. A., Holler, F. J., Nieman, T. A. (1997) Principles of Instrumental analysis. 5th edn, Thomson learning Inc. 674-767.

Sterner, D. E., Berger, S. L. (2000) Acetylation of histones and transcription -related factors. Microbiol. Mol. Biol. Rev. 64:435-459.

Schweppe, R. E. H., Lewis, T. S., Resing, K. A., Ahn, N. G. (2003) The characterization of protein post-translational modifications by mass spectrometry. Acc. Chem, Res. 36:453-461.

Su, X., Zhang, L., Lucas, D. M., Davis, M. E., Knapp, A. R., Green-Church, K. B., Marcucci, G., Parthun, M. R., Byrd, J. C., Freitas, M. A. (2007) Histone H4 acetylation dynamics determined by stable isotope labeling with amino acids in cell culture and mass spectrometry. Anal. Biochem. 363:22-34.

Verdone, L., Agricola, E., Caserta, M., Mauro, E. D. (2006) Histone acetylation in gene regulation. Brief. Funct. Genomic Proteomic 5:209-221.

Zhang, K., Tang, H., Huang, L., Blankenship, J. W., Jones, P. R., Xiang, F., Yan, P. M., Burlingame, A. L. (2002) Identification of acetylation and methylation sites of Histone H3 from Chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry. Anal. Biochem. 306:259-269.

Zhang, K., Faiola, F., Martinez, E., (2005) Six lysine residues on c-Myc are direct substrates for acetylation by p300. Biochem. Biophys. Res. Commun. 336:274-280.

Zhang, K., Yau, P.M. (2004) Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: An application for determining lysine 9 acetylation and methylation of histone H3. Proteomic 4:1-10.

顏慶堂,鄭政峰. 民92. 高效率液相層析術基礎介紹. 科儀新知第十九卷第四期43-51.