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Mod Spec Service

quantitation of histone PTMs using mass spectrometry

Mod Spec is a mass spectrometry method that is used to measure the relative abundance of > 80 different histone modifications. Mod Spec is a great starting point to determine if epigenetic changes are occurring in response to disease or treatment. The large number of modifications monitored allows researchers to not only observe expected changes, but to discover unexpected changes, which can be even more informative.

Analyze histone modification changes in:

  • Response to epigenetic inhibitors
  • Normal vs diseased cells
  • Knock-out cells or animals
  • Inhibitor treated xenografts
  • Human biopsies

The Process

Submit cell pellets or tissue and Active Motif will perform histone extractions, sample preparation, and mass spectrometry using the Thermo Scientific™ TSQ Quantum Ultra™ Triple-Stage Quadrupole Mass Spectrometer. All samples are run in triplicate and data are presented in an easy to understand format. Data delivery typically occurs within 4 - 6 weeks of sample submission.

Sample Requirements

  • Cells required: 2 - 5 million
  • Tissue requirements: 25 - 100 mg

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Histone modifications detected by Mod Spec
H1.4: K25UN *H3R2UN: K4ME2 H3: Q55ME1 H3.3: K27AC
H1.4: K25AC *H3R2UN: K4ME3 H3: K56UN H3.3: K27M
H1.4: K25ME1 H3R2UN: Q5UN H3: K56AC H3.3: K27ME1
H1.4: K25ME2 H3R2UN: Q5ME1 H3: K56ME1 H3.3: K27ME2
H1.4: K25ME3 H3: K9UN H3: K64UN H3.3: K27ME3
H2A: K5UN H3: K9AC H3: K64AC H3.3: K36UN
H2A: K5AC H3: K9ME1 H3: K79UN H3.3: K36AC
H2A: K9UN H3: K9ME2 H3: K79AC H3.3: K36ME1
H2A: K9AC H3: K9ME3 H3: K79ME1 H3.3: K36ME2
H2A: K36UN H3: K14UN H3: K79ME2 H3.3: K36ME3
H2A: K36AC H3: K14AC H3: K79ME3 H4: K5UN
H2A1: K13UN H3: K18UN H3: K122UN H4: K5AC
H2A1: K13AC H3: K18AC H3: K122AC H4: K8UN
H2A1: K15UN H3: K18ME1 H3.1: K27UN H4: K8AC
H2A1: K15AC H3: Q19UN H3.1: K27AC H4: K12UN
H2A1: K15UB H3: Q19ME1 H3.1: K27ME1 H4: K12AC
H2A3: K13UN H3: K23UN H3.1: K27ME2 H4: K16UN
H2A3: K13AC H3: K23AC H3.1: K27ME3 H4: K16AC
H2A3: K15UN H3: K23ME1 H3.1: K36UN H4: K20UN
H2A3: K15AC H3: R42UN H3.1: K36AC H4: K20AC
H2A3: K15UB H3: R42ME2 H3.1: K36ME1 H4: K20ME1
*H3R2UN: K4UN H3: R49UN H3.1: K36ME2 H4: K20ME2
*H3R2UN: K4AC H3: R49ME2 H3.1: K36ME3 H4: K20ME3
*H3R2UN: K4ME1 H3: Q55UN H3.3: K27UN  


* H3R2me2 and H3K4me2/3 are mutually exclusive modifications. H3R2 methylation prevents H3K4 methylation. Therefore, H3K4 modifications  are reported only on the H3R2 unmodified peptide. For more information, see Nature. 2007 Oct 18; 449(7164):933-7.

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Mod Spec 25085 Request Quote
Mod Spec data from control HeLa cells and HeLa cells treated for 7 days with 0.5uM GSK-126
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.


EZH2 inhibition results in decreased H2K27me3 levels
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.
How does Mod Spec work?