Method for differentially quantifying naturally processed HLA-restricted peptides for cancer, autoimmune and infectious diseases immunotherapy development

A method for identification and label-free quantification of at least one MHC ligand peptide on a primary tissue sample, comprising:

• a) providing at least one diseased primary tissue sample and at least one sample of primary healthy tissue optionally corresponding to diseased tissue,
• b) isolating one or more label-free MHC ligand peptides from said samples,
• c) performing an HPLC-MS analysis on said label-free MHC ligand peptides,
• d) extracting a precursor ion signal intensity area for each signal, as derived from c),
• e) identifying one or more sequences of said label-free MHC ligand peptides by fragment spectra clustering and database search, in order to group areas between different runs and samples, and normalizing areas within replicate runs to compensate technical performance and/or sensitivity differences between replicate runs resulting in average quantities for each peptide per sample including error estimates,
• f) Assigning said sequences to MHC alleles in order to generate allele-specific sequence subgroups for a comparison between different samples, and normalizing between different samples using allele-specific subgroups to account for different sample sizes or MHC expression levels wherein a result is relative quantities comparable between samples,
• g) performing a data quality control based on peptide reproducibility for every sample, verifying that a total number of identified sequences and a number of sequences with small variances in areas is as high as possible,
• h) calculating presentation profiles and scores in order to determine a potential overpresentation of MHC ligand peptides,
• i) controlling a quality of identifications and area estimates, optionally by visual inspection of peptide identification results and of area reproducibilities, or statistical analysis,
• j) comparing value detected in said at least one diseased primary tissue sample with the value obtained from said at least one primary healthy tissue, and
• k) quantifying said label-free MHC ligand peptide.