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New Mass Spec Workflow Enables Low-Abundance Spatial Analysis of Histone PTMs

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NEW YORK — A team led by researchers at the European Institute of Oncology (IEO) has developed a mass spectrometry-based workflow for analyzing histone post-translational modifications in low-abundance tissue samples.

The method, detailed in a paper published last month in Clinical Epigenetics, could enable better measurement of histone PTMs in samples including cancer biopsies as well as analysis of tissue and tumor heterogeneity, said Tiziana Bonaldi, group leader in the department of experimental oncology at the IEO and senior author on the study.

Alterations in histone PTMs have been linked to a variety of diseases, including cancer where researchers are exploring whether various histone PTM changes could be useful as biomarkers for detection, diagnosis, or prognosis of the disease. Additionally, histone deacetylase inhibitors, which target the activity of enzymes involved in the acetylation of histones, have become a hot area within cancer drug development.

Antibody-based approaches are most commonly used for the analysis of histone PTMs in clinical samples, but Bonaldi said that mass spec-based approaches could enable more detailed and comprehensive histone PTM profiling.

Using mass spec "we can profile several modifications at a time, and we can get a much better view of the combinatorial aspects" of histone PTM patterns, the so-called "histone code," she said.

A major challenge has been developing mass spec methods capable of analyzing histone PTMs in the low abundance samples that are typically available in clinical settings, Bonaldi said, noting that reducing the sample requirements for this kind of work has been a major focus of her lab.

In previous research, Bonaldi and her colleagues developed workflows for analyzing histone PTMs in formalin-fixed paraffin-embedded samples and frozen tissue in samples as small as 500,000 cells. That, however, was still well beyond the number of cells available in many clinical samples, she said.

In the Clinical Epigenetics work, the researchers further refined their approach, developing a workflow that can measure the most common histone PTMs in samples as small as 1,000 cells.

The method relies on an in-gel digestion process that works to eliminate from the clinical samples contaminants that would hinder mass spec analysis while also separating the target histones from the other proteins in the samples. Additionally, the researchers used a two-step derivatization process adapted to the in-gel digestion that the authors noted improves mass spec detection of "short and hydrophilic peptides" such as "the histone H3 3-8 peptide" and of "low-abundance acetylations."

Applying the approach to serial dilutions of peptides from MDA-MB-436 cells, the researchers found they could quantify 85 modified histone peptides from 3 µg of sample and 31 modified peptides from 5 ng of sample. In laser microdissected mouse pancreatic tissue samples, the researchers were able to quantify more than 30 modified histone peptides in samples as small as 1,000 cells.

In addition to enabling analysis of small clinical samples, the ability to look at such small cell populations opens the door to studies of histone PTM heterogeneity within tissues, Bonaldi said.

The method, which the researchers named PRO-PIC, offers "the capability within a tumor section to profile histone modifications from different tumor areas in order to be able to start to correlate the spatial differences and the morphological differences that are typically visualized by pathologists, with the corresponding changes in the epigenetics," she said. "There is now the possibility to dissect epigenetic heterogeneity in the tissue so that you can make a sort of geographical mapping of histone marks within a biopsy section."

In their recent study, Bonaldi and her colleagues used MALDI mass spec imaging to analyze lipid heterogeneity in patient breast cancer tissue samples of between 700 and 4,500 cells in size. They subdivided the cancer tissue into two regions based on the different lipid profiles identified within them and then used the PRO-PIC approach to analyze the histone PTMs present within these different regions, finding that the levels of one peptide form — the tetra-acetylated histone H4 4-17 peptide — differed between the two regions.

"We wanted to prove the applicability of the [workflow] in combination with MALDI imaging," Bonaldi said, adding that "the next step would be to check whether there is a mechanistic correlation between the two — whether, for instance, we can correlate changes in histone modification with, for instance, the activity of specific enzymes or pathways."

With interest growing in single-cell proteomics, a number of workflows and products for sample preparation and mass spec analysis of extremely small samples have recently emerged. In June, Bruker introduced its Bruker timsTOF SCP, a version of the company's timsTOF system optimized for high-sensitivity analyses of very small sample sizes, down to single cells.

Bonaldi, whose team used Thermo Fisher Scientific Q Exactive instruments for the research, said that she had not looked into the new Bruker platform and its potential for her lab's histone work. She said, however, that as a follow-up to the Clinical Epigenetics study she hoped to look at what complementary information imaging mass cytometry could provide "using a panel of antibodies raised against both histone PTMs whose levels emerged [as] different among distinct tumor areas in our study and other markers, for instance of [tumor infiltrating lymphocytes]."

Currently, the researchers are applying the technique to study several questions, including how epigenetic heterogeneity in triple-negative breast cancer patients might be linked to chemotherapy response and how interactions between tumor infiltration lymphocytes and tumor cells influence epigenetic patterns in the tumor and tumor microenvironment.

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