High-Throughput, Quantitative Analysis of Peptide-Exchanged MHCI Complexes by Native Mass Spectrometry.

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis─precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc