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Quantitative Histone H3 Modification Characterization of Differentiating Hematopoetic Murine Erythroleukemia (MEL) Cells Using Parallel Reaction Monitoring Mass Spectrometry
Author(s) -
Hess Sonja,
Moradian Annie,
Raedle Matthias,
Sweredoski Michael
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.586.5
Subject(s) - acetylation , chemistry , histone , mass spectrometry , electron transfer dissociation , histone h3 , proteomics , methylation , tandem mass spectrometry , butyric acid , histone deacetylase , biochemistry , microbiology and biotechnology , chromatography , biology , gene
Murine Erythroleukemia cells differentiate into hematopoetic cells that produce either embryonic or adult hemoglobin, dependent on the chosen inducer (histone deacetylase inhibitor butyric acid vs. DMSO). The differentiation has, at least in part, been associated with changes in histone modifications that are not readily discernible by classic biochemical analyses such as antibodies or classical bottom‐up mass spectrometry. To establish a quantitative method for histone modifications, we developed a middle‐down proteomics workflow with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion. We then monitored the twenty most abundant combinations of methylation and acetylation of the N‐terminal tail of histone H3. The N‐terminal H3 tail was separated primarily by its acetylation states using weak cation exchange hydrophilic interaction chromatography. In addition, some separation can also be achieved by its methylation states. After extensive data evaluation, a minimum set of over 250 H3 proteoforms was identified. More importantly, quantitative acetylation and methylation maps were created. MEL cells treated with HDAC inhibitor butyric acid showed a bimodal distribution with one cluster showing significantly more acetylations and methylations than untreated samples and samples treated with DMSO. A second cluster emerged from the butryic acid treated samples showing significantly less acetylations and methylations than untreated and DMSO‐treated samples. Furthermore, we developed a fragment ion map to visualize specific differences between treated and untreated samples. Taken together the data presented here show that middle‐down proteomics with electron transfer dissociation using PRM is a novel, attractive mass spectrometry method for the effective analysis and quantification of histone tails. Support or Funding Information This project was supported by the Gordon and Betty Moore Foundation through grant GBM275, the Beckman Institute, HHMI (Fusion) and UO1 grant 4DNucleome.