Method for differential quantification of naturally processed HLA-restricted peptides that are intended for the development of cancer immunotherapies and against autoimmune and infectious diseases

摘要

Method for the identification and quantification without labeling of at least one MHC peptide ligand in one or more primary tissue samples from at least one mammal, wherein said method comprises: a) Isolation of at least one MHC peptide ligand a starting from at least a first healthy tissue sample and a second diseased tissue sample corresponding to said primary diseased tissue, b) Carrying out an analysis by HPLC-MS of said MHC peptide ligands in order to generate a signal of such peptides c) Determination of the signal area of the precursor ion for each of said MHC peptide ligand signals d) identification of the sequences of each of the said MHC peptide ligands, by grouping the fragmentation spectra and / or database search e) Determination of an average amount of the MHC peptide ligand by grouping the areas obtained in different series of analyzes is repeated and the normalization of said areas within each of the analysis series, in order to create an average value for each sample f) Determination of relative amounts of the MHC peptide ligand that are comparable between the samples, which comprises f1) Assignment of the MHC peptide ligand to a subset of allele-specific sequences for comparison between different samples by identifying at least one MHC allele to which the MHC peptide ligand is predicted to bind, and f2) Normalization between different samples by allele specific subgroups to account for different sample sizes and / or MHC expression levels; and f3) Execution of a quality control of the data based on the reproducibility of the peptide in each sample g) Determination of the over-presentation of the MHC peptide ligand by calculating a presentation profile and / or a presentation score corresponding to the MHC peptide ligand from said diseased tissue sample and from said healthy tissue sample based on said average and relative amounts of MHC peptide ligand, and h) Unlabeled quantification of MHC peptide ligand.