Multiplexed relative and absolute quantitative immunpeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition [I]

Major histocompatibility complex (MHC) class I peptides play a critical role in immune cell recognition. Cancer cells modulate surface MHC levels in response to therapy, thereby affecting antitumor immunity. However, understanding the peptide repertoire response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking robust normalization controls. We describe a novel approach that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging for quantitation of peptide repertoires using low sample input. HipMHCs improve quantitative accuracy by normalizing for variation across analyses, and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens. Application of this platform to profile the immunopeptidome response to CDK4/6 inhibition and Interferon gamma, known modulators of antigen presentation, uncovered treatment-specific alterations that connect the intracellular response to extracellular immune presentation. This method quantifies repertoire changes that can inform targeted and combination immunotherapy design. 3 replicates each of DMSO-treated and Palbociclib-treated SKMEL5 cells