Fig. 5

The proposed IA-MS workflow for in vitro measurements of pMHC-specific TCRs. (A) Immunopeptidome workflows facilitate the identification of MHC-specific peptides through the isolation of antigen-presenting cells, affinity enrichment of peptide-MHC (pMHC) complexes, dissociation of class I (9–10 aa) or class II (13–25 aa) MHC peptides, and de novo sequencing of MHC peptides by LC-MS. (B) Recombinant MHC I or MHC II proteins representing the patient-specific Human Leukocyte Antigen (HLA) variants are conjugated to magnetic particle-bound streptavidin tetramers and incubated with the synthetic MHC I or MHC II peptides previously identified with immunopeptidome workflows. Numerous pMHC complexes need to be prepared. (C) CD8 + cytotoxic T lymphocytes (MHC I) and CD4 + helper T lymphocytes (MHC II) are isolated from the patient’s blood and lysed to release soluble TCRs (α-β1 or α-β2). (D) T lymphocyte lysates are incubated with the individual pMHC complexes, and endogenous pMHC-specific TCRs are enriched and covalently cross-linked to pMHCs to retain low-affinity interactions. Following trypsin digestion, unique peptides of α constant (TRAC_HUMAN), β1 constant (TRBC1_HUMAN), and β2 constant (TRBC2_HUMAN) chains are quantified by LC-SRM. Corresponding heavy isotope labeled peptide internal standards (IS) facilitate accurate relative or absolute quantification