Top-Down Proteomic Characterization of Histone H3 Proteoforms in Disease

Zheng et al. performed top-down mass spectrometric proteomic analysis to characterize human histone H3 proteoforms in primary multiple myeloma cell lines.¹ Using malignant cells that overexpress histone methyltransferase MMSET, the researchers investigated driver mutations that contribute to modification of histones responsible for epigenetic control in disease.

Although researchers understand that specific proteoforms correlate well with disease and that mutations often contribute to pathogenesis, it is difficult to fully characterize histone modifications acetylation and methylation by traditional methods. This is because the modifications often associate together, and this close proximity interferes with antibody recognition, for example. Mass spectrometry offers more sensitivity, and top-down proteomics is a potentially useful tool for examining intact histones.

Zheng et al. grew multiple myeloma cell lines in culture, selecting primary lines that overexpress MMSET, a finding in 20% of clinical cases. As a comparison, they also grew KMS11 TKO cells as a low-MMSET control. After harvesting the cells, the researchers purified the nuclei and extracted the histones by traditional methods, fractionating the preparations to obtain H3.1 and H3.3 isolates. Following this, the team analyzed the fractions by mass spectrometry on an electron transfer dissociation–enabled Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific) operating in intact protein mode. They used the MS¹ spectra to define the histones’ proteoform peaks, noting that 14 Da is the mass difference for one methylation group. The team then used the MS² spectra for isolation and fragmentation of these clusters. They then conducted semi-quantitative analysis on the results, looking at all the theoretical fragment ion concentrations for the histone modifications.

The research team found that they achieved good separation for three histone H3 variants—H3.1, H3.2 and H3.3—from top-down proteomic analysis. MMSET-high cells showed elevated levels of H3.3 (+20%) and H3.2 (+10%), but reduced levels of H3.1 (−25%). Zheng et al. also saw evidence of combinatorial modifications with multiple proteoforms within an isobaric mix. In addition, they could discriminate between methylation and acetylation.

Using the MS² intensities, the team was able to determine the site of the modifications, and they could map both methylation and acetylation in a single sample according to ion type, charge and mass. The results showed a difference in modification patterns between MMSET-high and MMSET-low cells. Zheng et al. could see a maximum of seven methyl groups in MMSET-high cells compared to five in the MMSET-low cultures. They also saw that acetylation co-exists with up to five methyl groups in MMSET-high cells and three in MMSET-low cells.

The research team then chose two c-type fragment ions to visualize the modification patterns obtained from in vitro culture. They found that modification occurred in three key residues—K9, K27 and K36—and from this were able to unambiguously assign 16 proteoforms of H3.1 in the MMSET-low cells and 10 in the MMSET-high cells. From the analysis, Zheng et al. also found evidence of trivalent methylation in H3.1 and H3.3 proteoforms from MMSET-high cells.

Summarizing the results obtained from mass spectrometric characterization, Zheng et al. believe that the data show a clear hierarchy of acetylation with top-down analysis being a valuable and sensitive tool for determining histone proteoforms. With evidence of trivalent methylation as one of the modifications identified, the team suggest that the differences shown could influence disease through aberrant enzyme activity and abnormal chromatin effector recruitment.

Reference
1. Zheng, Y., et al. (2015) “Unabridged analysis of human histone H3 by differential top-down mass spectrometry reveals hypermethylated proteoforms from MMSET/NSD2 overexpression,” Molecular & Cellular Proteomics, 15(3) (pp.776–790), doi: 10.1074/mcp.M115.053819.

Post Author: Amanda Maxwell. Amanda is a freelance science writer and digital space explorer with a passionate curiosity for science and technology. She enjoys translating complex theories and subjects creatively into everyday language for all audiences. Equipped with a bachelor’s degree in veterinary medicine and a PhD in protein chemistry/small animal critical care nutrition, she brings clinical experience and practical research oversight into her writing.