Immunopeptidomics is a bioanalytical method by which presented peptides are extracted from biological samples and analysed by LC-MS, giving researchers a qualitative and quantitative snapshot of the immunopeptidome. BIOGNOSYS is a proteomics and immunopeptidomics CRO that creates platforms for accessing smaller inputs on tissue biopsies.
Industry opinion suggests that one of the biggest limitations in immunopeptidomics is the amount of material needed to extract the HLA receptors, and the peptides associated with them. Therefore, the main goal is to make a sensitive assay for immunopeptidomics discovery and reduce the inputs required. Dan Redfern, Business Development Manager at BIOGNOSYS, explained how to address the challenge above by using BIOGNOSYS’ immunopeptidome profiling platform to optimise the workflow for HLA peptidomics.
Firstly, enriching the HLA antibodies is key. By using mass spectrometry, implementing a data-independent acquisition (DIA), and developing a target platform that uses parallel reaction monitoring, Redfern developed a sensitive assay for HLA peptidomics, capable of optimising workflows to work with minimal inputs. To increase the amount of immunopeptidome covered in the studies, in-house software programs such as Spectronaut and SpectroMine were applied.
Redfern wanted to build on this research by testing other inputs and investigating coverage levels, so he conducted various studies for HLA Class 1 and Class 2 peptidomics. Cell line data for class 1 demonstrated that between 20 and 25 million cells appeared to be optimal cell count and that beyond this point the number of peptide IDs began to plateau at approximately 11,000 and gains were minimal. Class 2 followed a very similar pattern. Regarding tissue samples, a sample between 10 and 15mg was effective for discovery studies.
Additionally, Redfern investigated how we can expand immunopeptidomics beyond pre-clinical studies into a clinical setting, specifically when there is no tissue available. In this case, PBMCs (peripheral blood mononuclear cells) are used as a surrogate matrix. The results showed that although coverage is reduced in comparison to tissue samples, PBMCs still provide useful data on immunopeptidome modulation.
BIOGNOSYS is collaborating with Greywolf Therapeutics to investigate the effects of ERAP inhibition on the HLA-I immunopeptidome. The experiment involved selecting 30 new antigens to quantify and developing a targeted assay to work out a rough copy number per cell in these peptide targets. The initial experiment examined different cancer cell lines and ran the panel of 30 peptides to explore how copy numbers per cell varied in the presence of an ERAP1 inhibitor and without one. The results showed that ERAP1 inhibition led to an increase in longer HLA-I peptides, revealing novel immune targets.
