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EIO Team Develops Workflow for Mass Spec-Based Histone PTM Analysis in Clinical Samples

NEW YORK(GenomeWeb) – A team led by researchers at the European Institute of Oncology has developed a workflow for mass spec-based analysis of histone post-translational modification patterns in formalin-fixed parrafin embedded clinical samples.

Using the technique, which they detailed in a paper published this week in Molecular & Cellular Proteomics, they analyzed a series of breast cancer samples identifying what they characterized as significant changes in histone H3 methylation patterns in luminal A-like and triple negative breast cancer subtypes.

The results provide a proof of concept for the approach, which, they wrote, could enable "retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with FFPE archives."

One of the most common storage formats for clinical samples, FFPE tissue has presented challenges in proteomic analysis due to the large amount of protein cross-linking caused by the fixing process. In recent years, though, researchers have developed new extraction methods that have made such samples more amenable to proteomic approaches.

Building on these methods, the EIO team developed an extraction approach specifically targeted at histones capable of collecting these proteins from FFPE tissue with yield and purity sufficient for downstream mass spec analysis. They validated the method by comparing FFPE and frozen tissue from mice, identifying in this way a set of histone H3 peptides whose modification profiles were not as affected by long-term storage as FFPE tissue.

Using a Thermo Fisher Scientific Q Exactive instrument and super-SILAC labeling, they generated quantitative profiles of 24 different modified histone H3 peptides from FFPE human breast cancer samples from the four main breast cancer subtypes — luminal A, luminal B, HER2-positive, and triple negative.

Via non-supervised clustering they were able to define four main groups based on their histone PTM profiles — one containing mainly luminal-A tumors, one containing primarily triple-negative samples with one HER2-positive sample, and two containing a mix of luminal B, HER2-positive, and triple-negative samples.

While these groupings did not, by and large, recapitulate established breast cancer subtypes, the researchers did in some cases identify histone PTM profiles linked to these traditional subtypes. For instance, they found that increased trimethylation of histone H3 at lysine 27 was associated with luminal-A tumors, an association, the authors noted, that had been previously reported in immunohistochemistry studies.

Additionally, they found significant differences between the PTM profiles of luminal-A and triple-negative samples.

More generally, the researchers noted, the paper demonstrates the feasibility of using mass spec for histone PTM analysis in actual clinical samples, something which has previously been largely the domain of antibody assays. Mass spec analysis, they wrote, has several potential advantages, including the possibility of better accuracy and the ability to develop highly multiplexed assays to a number of modifications including those for which high-quality antibodies don't currently exist.