With the rapid pace of liquid biopsy assay development, it can be a challenge to keep up with the latest and greatest early cancer detection methods. Although most methods are focused on assaying the association of cell-free DNA methylation profiles with cancer, a new method homes in on circulating nucleosomes and their histone post-translational modifications (PTMs).
In order to take the road less traveled, Marielle Herzog’s team (Volition, Belgium) developed Nu.Q Capture-MS, an innovative blood-based method, which uses less than 1 ml of plasma, to discover histone PTMs on circulating nucleosomes. Mass spectrometry (MS) is commonly used to discover histone PTMs in cells and tissues but is not effective in blood due to high protein abundance, which may conceal less abundant circulating nucleosomes and histone PTMs. Until now, detection of circulating histone PTMs in blood was mostly feasible only by immunoassay, provided you already knew which modifications you were looking for. Nu.Q Capture-MS is the first method that can enrich circulating nucleosomes in blood samples, allowing for histone PTMs to be detected and quantified by MS. Here’s how Nu.Q Capture-MS works:
- First, 900 μl of plasma collected with K2-EDTA tubes is incubated with an anti-histone H3.1 antibody coated on magnetic beads for quantitative immunoprecipitation of circulating nucleosomes
- Spiking in defined quantities of recombinant H3.1 nucleosome into nucleosome-free plasma demonstrates that the immunoprecipitation is quantitative
- The pull-down efficacy is 82.2% ± 3.7% (n = 6), which was confirmed by Coomassie blue staining and anti-histone H3 Western blot analysis in immunoprecipitated material and by verifying the depletion of nucleosomes in plasma samples using the ELISA method following immunoprecipitation
- The circulating nucleosomes immunoprecipitated with the Nu.Q Capture protocol are then analyzed by liquid chromatography with tandem MS (LC-MS/MS) to identify histone PTMs
- Analysis of native plasma samples not subjected to the Nu.Q Capture protocol confirms that 95% of the detectable peptides are from common blood proteins whereas histone peptides are barely detectable
- Enrichment of circulating nucleosomes with the Nu.Q Capture protocol enables a large increase in histone peptide detection compared to native plasma, with a sequence coverage of over 60% and a 52% reduction of blood peptides
- Importantly, all core histone proteins are detectable, indicating that pull-down of H3.1 is effective to isolate entire circulating nucleosome
The team then applied the Nu.Q Capture-MS protocol in a pilot study to investigate the circulating nucleosome histone PTM signatures of colorectal cancer (CRC) (9 CRC patients and 9 healthy donors). 13 histone PTMs were distinctive of CRC out of the 54 proteoforms identified and quantified. H3K9 and H3K27 methylation, H3 acetylation, and notably highlighted for the first time, H2A1R3 citrullination are particularly upregulated in CRC plasma samples. These results indicate that the Nu.Q Capture-MS method has great promise in catching early cancerous clues.
Get that new new in Scientific reports, March 2021