Using Mass Spectrometry to Characterize the Complex Posttranslational Modifications of Histones

As one of the major molecular mechanisms that mediate epigenetics, histone proteins undergo diverse posttranslational modifications (PTMs), most notably acetylation and methylation. It is now well known that different PTMs can co‐occur on proteins that may act sequentially or in combination and constitute a ‘histone code’. Mass spectrometry is the method of choice for studying protein PTMs. Conventional mass spectrometry strategy relies on protein digest analysis using chromatographic separation of peptides and tandem mass spectrometry with collisional induced dissociation (CID) for fragmentation analysis. Although usually sensitive enough to determine even low stoichiometry modifications, it is difficult to determine the co‐occurrence of different modifications on the same protein species using this analytical approach. However, new fragmentation mechanisms such as electron capture dissociation (ECD) are capable of preserving labile PTMs and allow characterization of PTMs at the intact protein level. Through our study of histone modifications from several different samples, this presentation will show the power of ECD for protein modification analysis. Specific PTM patterns will be compared among different histone preparations to further the understanding of how epigenetic regulation can be modulated through histone modification. Financial support was provided by NCRR 01614(ALB), NIH R01 GM63959 (CDA) and NSF CHE‐99‐09502 (AGM).