Abstract
Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Chemical regents used in this study purchased from Sigma-Aldrich: Propionic anhydride and phenyl isocyanate (Fluka brand), perchloric acid, hydroxylamine (50% wt%), phenyl 13C6 isocyanate (Aldrich brand), 1 M Triethylammonium bicarbonate buffer solution (Sigma brand)
Tors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5
The field of Epigenetics has become important in drug discovery as many diseases have been linked to aberrations in chromatin and changes of histone post-translational modifications (PTMs)1 [1, 2]
Summary
Materials—Chemical regents used in this study purchased from Sigma-Aldrich: Propionic anhydride and phenyl isocyanate (Fluka brand), perchloric acid, hydroxylamine (50% wt%), phenyl 13C6 isocyanate (Aldrich brand), 1 M Triethylammonium bicarbonate buffer solution (Sigma brand). Five-l aliquots containing nominally 1 pmol/l of each synthetic peptide sample were injected into a 200 l per minute solvent stream (0.1% v/v formic acid, 20% v/v acetonitrile in water) directed to the ion source (Turbo Ion Spray) of an Applied Biosystems 4000 QTRAP mass spectrometer This instrument was operated in selected ion monitoring mode, with 125 ms dwell times at each peptide’s [MϩH]ϩ and [Mϩ2H]2ϩ mass to charge ratio at unit resolution. Orbitrap LC-MS/MS of Endogenous Histone Peptides—Stage-tip desalted histone peptides were resuspended with HPLC solvent A (0.1% v/v formic acid, 2% v/v acetonitrile in HPLC grade water) to final concentrations of 200 to 500 ng/l total histones in order that ϳ500 ng was analyzed by capillary LC-MS/MS using a hybrid linear ion trap/Orbitrap mass spectrometer (Orbitrap-Elite; Thermo Fisher Scientific, Waltham, MA). Once peaks’ relative abundances for each mark are calculated, these are used to measure mark-by-mark changes, for examplein a two-sample comparison (chemical labeled or SILAC), among samples with a common internal standard (chemical labeled or super-SILAC), or among samples without internal standard (label free)
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