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. Overall design: 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).