Heavy Methyl-SILAC Labeling Coupled with Liquid Chromatography and High-Resolution Mass Spectrometry to Study the Dynamics of Site-Specific Histone Methylation
Histone lysine and arginine methylation involved in gene activation and silencing is dynamically regulated. However, partly limited to the research technologies previously available, the dynamics of global histone methylation on a site-specific basis have not been fully pursued. Heavy methyl-SILAC (Stable Isotope Labeling of Amino Acids in Cell Culture) labeling provides a remarkable signpost to distinguish the preexisting and newly generated methyl marks on histones. Using this technology coupled with quantitative LC-MS analysis make it possible to monitor changes in the dynamics of histone site-specific methylation. In this chapter, we comprehensively describe the experimental strategy to determine the dynamics of multiple histone methylated residues including SILAC labeling, histone extraction/purification and mass spectrometry analysis.
Key wordsHistone Methylation Dynamics Heavy methionine SILAC Quantitation Mass spectrometry
B.A.G. gratefully acknowledges funding from a National Science Foundation grant (CBET-0941143), an Agilent Thought Leader award, an NSF Early Faculty CAREER award, and an NIH Innovator award (DP2OD007447) from the Office Of The Director, NIH.
- 21.Pedrioli PG, Eng JK, Hubley R, Vogelzang M, Deutsch EW, Raught B, Pratt B, Nilsson E, Angeletti RH, Apweiler R, Cheung K, Costello CE, Hermjakob H, Huang S, Julian RK, Kapp E, McComb ME, Oliver SG, Omenn G, Paton NW, Simpson R, Smith R, Taylor CF, Zhu W, Aebersold R (2004) A common open representation of mass spectrometry data and its application to proteomics research. Nat Biotechnol 22:1459–1466PubMedCrossRefGoogle Scholar