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Chemical derivatization of histones for facilitated analysis by mass spectrometry


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.

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Figure 1: Peptides generated from the trypsin digestion of propionylated histone H3.2.
Figure 2: MS/MS spectrum of the [M+2H]2+ precursor ion from the digest of propionylated histone H3.2 taken on an LTQ-FT mass spectrometer.
Figure 3: Differential expression mass spectrometry analysis of histone PTMs.

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Funding from the National Institutes of Health (GM 37537) to D.F.H. and the Ford Foundation to B.A.G is gratefully acknowledged.

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Correspondence to Donald F Hunt.

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Garcia, B., Mollah, S., Ueberheide, B. et al. Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat Protoc 2, 933–938 (2007).

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