Method for Differentially Quantifying Naturally Processed HLA-Restricted Peptides for Cancer, Autoimmune and Infectious Diseases Immunotherapy Development

Method for the identification and label-free quantification of MHC ligand peptides on primary tissue samples, comprising the steps of

• a) providing at least one diseased primary tissue sample and at least one sample of primary healthy tissue preferably corresponding to the diseased tissue,
• b) isolating MHC ligand peptides from said sample(s),
• c) performing an HPLC-MS analysis on said MHC ligand peptides,
• d) extracting the precursor ion signal intensity (area) for each signal, as derived from step c),
• e) identifying the sequences of said 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/sensitivity differences between replicate runs resulting in average quantities for each peptide per sample including error estimates,
• f) Assigning of 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 the allele-specific subgroups to account for different sample sizes or MHC expression levels:
• result is relative quantities comparable between samples,
• g) performing a data quality control based on peptide reproducibility for every sample, verifying that the total number of identified sequences and the 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 over-presentation of MHC ligand peptides,
• i) controlling the quality of identifications and area estimates, for example by visual inspection of the peptide identification results and of the area reproducibilites, or statistical analysis,
• j) comparing the values detected in said at least one diseased primary tissue sample with the value(s) obtained from said at least one primary healthy tissues, and
• k) quantifying said MHC ligand peptides.