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.

Lysine Succinylation and Lysine Malonylation in Histones

“…Peptide Sample Preparation and Affinity Enrichment-Chemical propionylation of histone extracts was performed using a procedure previously reported with slight modifications (17). Briefly, 3 mg of histone extracts were dissolved in 50 l of 100 mM ammonium bicarbonate buffer (pH 8.0) and added with 300 l of propionic anhydride in 300 l of methanol.…”

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

“…Traditional methods (bottom-up MS) of sequence determination and PTM site localization are not practical. Histone N-terminal regions are rich in lysine and arginine residues, and thus proteolysis using trypsin generates peptides that are too small or that are poorly retained on reversephase HPLC C18 resins for subsequent MS detection (11). With the advent of electron transfer dissociation (ETD) and more efficient electron capture dissociation fragmentation methods, which are better suited for larger, more highly charged peptides (12,13), several studies utilizing other endoproteases to generate longer peptides have emerged (14 -16).…”

Histone methyltransferase G9a contributes to H3K27 methylation in vivo

“…The reaction products were chemically propionylated [8] Figure S1). Moreover, they also contained robust signals for H3K27me1 and H3K27me2 ( Figure 1B).…”