Chemical derivatization of histones for facilitated analysis by mass spectrometry

Benjamin A. Garcia, Sahana Mollah, Beatrix M. Ueberheide, Scott A. Busby, Tara L. Muratore, Jeffrey Shabanowitz, Donald F. Hunt

Research output: Contribution to journalArticlepeer-review

291 Scopus citations


Histone post-translational modifications have been recently intensely studied owing to their role in regulating gene expression. Here, we describe protocols for the characterization of histone modifications in both qualitative and semiquantitative manners using chemical derivatization and tandem mass spectrometry. In these procedures, extracted histones are first derivatized using propionic anhydride to neutralize charge and block lysine residues, and are subsequently digested using trypsin, which, under these conditions, cleaves only the arginine residues. The generated peptides can be easily analyzed using online LC-electrospray ionization-tandem mass spectrometry to identify the modification site. In addition, a stable isotope-labeling step can be included to modify carboxylic acid groups allowing for relative quantification of histone modifications. This methodology has the advantage of producing a small number of predicted peptides from highly modified proteins. The protocol should take approximately 15-19 h to complete, including all chemical reactions, enzymatic digestion and mass spectrometry experiments.

Original languageEnglish
Pages (from-to)933-938
Number of pages6
JournalNature Protocols
Issue number4
StatePublished - Apr 2007


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