Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression.
Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such "trivalent hypermethylation" (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry can now decode crosstalk involving up to three modified sites.
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208;
Yupeng
Y
Zheng
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208;
Luca
L
Fornelli
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208;
Philip D
PD
Compton
§Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134;
Seema
S
Sharma
§Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134;
Jesse
J
Canterbury
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208;
Christopher
C
Mullen
§Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134;
Vlad
V
Zabrouskov
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208;
Ryan T
RT
Fellers
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208;
Paul M
PM
Thomas
¶Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611.
Jonathan D
JD
Licht
§Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, California 95134;
Michael W
MW
Senko
From the ‡Departments of Chemistry and Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, 2145 N. Sheridan Road, Evanston, Illinois 60208; n-kelleher@northwestern.edu.
Neil L
NL
Kelleher
D045744:Cell Line, Tumor; D044127:Epigenesis, Genetic; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D009101:Multiple Myeloma / Q000235:genetics* / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics*; D015854:Up-Regulation
D006657:Histones; D020543:Proteome; D012097:Repressor Proteins; D011495:Histone-Lysine N-Methyltransferase; C419586:NSD2 protein, human; D008239:Lysine