Targeted Analyses of MHC-I Peptides Using TOMAHAQ

Advances in several key technologies, including major histocompatibility (MHC) peptidomics, have significantly enhanced our understanding of basic immune-regulatory mechanisms and the identification of T-cell receptor targets for the development of immune-therapeutics. Isolating and accurately quantifying MHC-bound peptides from cells and tissues enables the characterization of dynamic changes in the MHC peptidome due to cellular perturbations. However, the current multistep analytical process is challenging, and improvements in throughput, sensitivity, and reproducibility would enable rapid characterization of multiple conditions in parallel.In this chapter, we describe a robust, sensitive, and quantitative method for enriching peptides derived from MHC-I complexes from a variety of cell lines, including challenging adherent lines such as MC38, in a semiautomated fashion using reusable, dry-storage, customized antibody cartridges. This method allows a researcher, with minimal hands-on time, to perform up to 96 simultaneous enrichments reproducibly in a single day, achieving a quality level comparable to that of a manual workflow.TOMAHAQ (Triggered by Offset, Multiplexed, Accurate-mass, High-resolution, and Absolute Quantification) is a targeted mass spectrometry technique that combines sample multiplexing and high sensitivity 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. This unique combination of robust, semiautomated MHC-I peptide isolation and high-throughput multiplexed-targeted quantitation allows for both the routine analysis of over 4000 unique MHC-I peptides from 250 million cells using nontargeted methods, as well as quantitative sensitivity down to the low amol/μL level using TOMAHAQ targeted MS. The protocol and tips on how to execute are outlined below.