Mod Spec

Figure 1: Mod Spec^® data from control HeLa cells and HeLa cells treated for 7 days with 0.5uM GSK-126.(Click image to enlarge)

GSK126 is an inhibitor that blocks the methytransferase activity of EZH2, resulting in global decreases in H3K27me2 and H3K27me3. A) A selection of H3 modifications are shown from Active Motif’s Mod Spec^® assay that confirm significant decreases in H3K27 methylation with concomitant increases in acetylated and unmodified H3K27 (highlighted in yellow). Changes at H3K36 were also detected (highlighted in red). B) A selection of H4 modifications do not show significant changes in response to GSK126 treatment.

Figure 2: Mod Spec^® data from control HEK293 cells and HEK293 cells treated for 6 hours with 5 mM sodium butyrate (NaB).

NaB is a general HDAC inhibitor and treatment is expected to increase histone acetylation levels. A) Mod Spec^® data for a selection of acetylated and unmodified histone sites is shown. Acetylation is increased at all sites after treatment. Unmodified peptide is displayed for some sites and shows a concomitant decrease after NaB treatment. B) Methylation at various histone residues is not affected by NaB treatment.

How does Mod Spec^® work?

1. Histones are extracted from samples.
2. Derivatization of histones using propionic anhydride.
   • Mass spectrometry of proteins requires digestion of the proteins into small peptides before being injected into the mass spectrometer. However, if peptides are too small they can not be separated in the LC step and if too big they are too complex to analyze. The most common method of digestion is with trypsin, an enzyme that cleaves proteins at lysine and arginine residues. This presents a problem when analyzing histones since histone tails are rich in lysine, resulting in digested peptides that are too small. Propionylation, using propionic anhydride, blocks cleavage at lysines while allowing digestion at arginines to occur. The resulting peptides are of the appropriate size for mass spec analysis.
3. Histones are digested with trypsin.
4. Samples are analyzed on a Triple-Stage Quadrupole Mass Spectrometer.
   • Peptides of the appropriate molecular weight are selected.
   • Peptides are further fragmented in the collision chamber.
   • Peptides fragments of interest are selected and analyzed by mass spectrometry.
5. Fragments are analyzed and graphed to show changes between samples.

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