A peptide-signal amplification strategy for the detection and validation of neoepitope presentation on cancer biopsies

Abstract

Targeting the right cancer-specific peptides presented by Human Leukocyte antigen (HLA) class I and II molecules on the tumor cell surface is a crucial step in cancer immunotherapy. Numerous approaches have been proposed to predict the presentation of potential neoepitopes that may be targeted through immune-based therapies. Often founded on patient specific somatic mutations, the routine validation of their actual appearance on the tumor cell surface is a significant barrier to realising personalized cancer immunotherapy. This can be attributed to the lack of robust and adaptable assays for antigen presentation that offer the required sensitivity to deal with the limited amounts of patient tumor tissue available. Rather than personalize individual assays we propose the use mass spectrometry to identify tumor neoepitopes from HLA-bound peptides directly isolated form the surface of tumor biopsies. We have developed a microscale HLA-peptide complex immunoprecipitation protocol combined with tandem mass tagging (TMT) to directly sequence HLA-bound peptides using mass spectrometry. Using this strategy, we identified HLA-bound peptides from as few as ~1000 cultured cells and from a small piece (~1 mg) of whole melanoma tumour tissue, encompassing epitopes derived from Melanoma-associated antigens and potential neoantigens.

Quantitative Consequences of Protein Carriers in Immunopeptidomics and Tyrosine Phosphorylation MS2 Analyses

“…1 , A and B ). Empty channels were not incorporated to more closely mirror the experimental design of a previously reported study ( 10 ). As a protein carrier, we utilized two samples (two channels) of 2.5 × 10 6 cells stimulated with 10 ng/ml interferon-gamma (IFN-γ) for 72 h. IFN-γ stimulation increases pMHCs levels approximately twofold, allowing for reduced cellular input requirements to generate approximately tenfold boost per sample.…”

“…used tenfold higher cellular input of the control sample as a protein carrier, and Chua et al. used a protein carrier that had been treated with pervanadate (PV) to halt tyrosine phosphatase activity and thereby increase tyrosine phosphorylation levels ( 5 , 10 , 11 ). While these initial results are encouraging, the quantitative impact of boosting in both approaches remains poorly understood.…”

Soluble HLA peptidome: A new resource for cancer biomarkers

“…The implementation of antibody-based IP enrichments of HLA-peptide complexes after cell lysis resulted in a 6-fold increase in peptide numbers compared to the earlier MAE approach ( 60 ). Similarly, significant advances in the sensitivity, selectivity and speed of mass spectrometric instrumentation have led in the past 10 years to an increase in peptide IDs that can be identified from decreasing amounts of starting material ( 61 ). Just a decade ago, 10 10 cells were required to identify approximately 3,000 peptides ( 62 ).…”

Immunopeptidomics for next-generation bacterial vaccine development

“…Immunopeptides are usually analyzed by LC-MS/MS in their native state, termed label-free; however, chemical derivatization and isotopic labeling were recently found to improve detectability of these short, nontryptic peptides by promoting positive charges on peptides, which are essential for successful peptide ionization during LC-MS/MS analysis [55][56][57]. Utilizing these techniques, efforts are ongoing to further reduce the required sample input by peptide amplification strategies [58]. MS fragmentation methods to infer immunopeptide sequence information include collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD), but also sophisticated techniques like EThcD combining electron transfer dissociation (ETD) with HCD to generate more diverse fragment ions and to improve identification rates and detection of post-translationally modified peptides [59,60].…”

“…Finally, even though immunopeptidomics typically requires large amounts of starting material, recent innovations have allowed downscaling to ca. 1E7 cells, and efforts are ongoing to downscale even further [38,53,55,58]. Together with more sensitive mass spectrometers, these developments will expand the number of infection models that are accessible to immunopeptidomics.…”