The major histocompatibility complex (MHC) is a region of highly polymorphic genes encoding for glycoproteins (MHC molecules) that form part of the cell-mediated branch of the acquired immune system. In the cytosol, cellular self and foreign (non-self) proteins are constantly being degraded; it is the peptides generated that are presented, non-covalently bound to MHC molecules, on the surface of cells for inspection by cytotoxic T-lymphocytes (CTLs). Nonrecognition of the presented peptide ultimately leads to cell destruction.
Characterizing the factors associated with nonrecognition is an attractive proposition for anyone interested in generating tools for targeted cell destruction. In the field of oncology the obvious application then is the targeted destruction of cancerous cells. To enable the molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Most frequently an immunoprecipitation is used to isolate the target complex. The peptide elution is performed under conditions minimizing protein contamination and finally peptide analysis is accomplished by mass spectrometry.
Here we present a case study of our recent work optimizing and performing a workflow for the analysis of peptides associated with Class I and Class II MHC molecules. The goal of the assay optimization was to minimize the amount of antibody required for the assay, to minimize the amount of biological material needed from which the complex is isolated and to achieve the optimum sensitivity towards the peptides of interest.
Citation Format: Michael Ford, Richard Jones, David Allen, Ravi Amunugama, Paul Del Rizzo, Michael Pisano, James Mobley, Paul Domanski, Bill Ho, Daniel Bochar, Melissa Holt. Mass spectrometric characterization of peptides associated with molecules of the major histocompatibility complex [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B80.