Sensitive and quantitative detection of MHC-I displayed neoepitopes using a semi-automated workflow and TOMAHAQ mass spectrometry

Advances in several key technologies, including MHC peptidomics, has helped fuel our understanding of basic immune regulatory mechanisms and identify T cell receptor targets for the development of immunotherapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables characterization of dynamic changes in the ligandome due to cellular perturbations. This multi-step analytical process remains challenging, and throughput and reproducibility are paramount for rapidly characterizing multiple conditions in parallel. Here, we describe a robust and quantitative method whereby peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines, can be enriched in a semi-automated fashion on reusable, dry-storage, customized antibody cartridges. TOMAHAQ, a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity, was employed to characterize neoepitopes displayed on MHC-I by tumor cells and to quantitatively assess the influence of neoantigen expression and induced degradation on neoepitope presentation. Four treated samples were analyzed in technical triplicate (biological single replicate), from the same engineered cell line, MC38-idAdpgkG (which stands for inducible, degradable, Adpgk(R304M) neoantigen fused to turboGFP in the MC38 murine colon carcinoma cell line). The four treatments were as follows: mouse IFNg alone, mIFNg plus doxycycline, mIFNg plus doxycycline plus dTag13, and mIFNg plus dTag13 (see the treatment protocol section for full concentration and timing details).