Chemical derivatization of histones for facilitated analysis by mass spectrometry

Abstract

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.

Figure 1

Peptides generated from the trypsin digestion of propionylated histone H3.2. As all lysine residues are blocked by endogenous modifications or chemically by conversion to propionyl amides, cleavage occurs only C-terminal to arginine residues (similar results can be obtained on other histones).

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. This peptide was determined to span residues 18–26 and was found to contain an acetylation (ac) modification on K23. Note propionyl amide groups (pr) on the unmodified K18 residue and also on the N-terminus. b and y type ions are labeled. Inset: mass spectrum of the precursor parent ion differentiates this peptide as being acetylated (+1.23 p.p.m.) and not trimethylated (−30.66 p.p.m.), as the experimental mass of the peptide is consistent with an acetylation mark.

Figure 3

Differential expression mass spectrometry analysis of histone PTMs. (a) An example of methyl ester stable isotope labeling of histone H3 samples. Summed full MS spectrum spanning the 551–555 m/z region from the comparative analysis of histone H3 variants, H3.1 (labeled D₀-methanol, light) and H3.2 (labeled D₃-methanol, heavy). Doublet peaks at 551.8212 and 553.3304 m/z both correspond to the peptide pr-YRPGTVALR with one stable isotope label incorporated at the C-terminus (pr = propionyl group). The observed ion ratio is roughly 1:1, demonstrating equal loading of the two samples as this peptide is not modified and only one form exists. (b) Summed full MS spectrum spanning the 828–833 m/z region from the same comparative analysis between histone H3 variants, H3.1 (labeled D₀-methanol, light) and H3.2 (labeled D₃-methanol, heavy). Doublet peaks at 829.4917 and 831.0007 m/z both corresponding to the peptide pr-K_me3SAPATGGVK_prK_prPHR with the incorporation of one stable isotope label at the C-terminus (pr = propionyl group). This peptide with a trimethylation modification at K27 from the H3.2 sample was found in twofold abundance compared to the H3.1 sample.