PBMC#2 was typed positive for HLA-A02, -A03, -B35, -B39, and is shown here as a representative sample. (A) The stand-alone software package of the HLA binding prediction algorithm NetMHC 3.4 was used to predict the binding affinity of all identified peptides to HLA-A02, -A03, -B35 and -B39 (four peptides are shown for simplicity). For each peptide, an annotation score was calculated by dividing the second lowest IC50 value (second best predicted allele) by the lowest IC50 value (best predicted allele). Peptides with a score ≥3 were annotated to the HLA allele predicted to bind best. Peptides with a score below 3 were considered as non-annotated. Non-annotated peptides were curated in the output files in Figure 2—source data 2 and correspond to 1) non-HLA peptides/contaminants, 2) peptides predicted to strongly bind more than one HLA allele (supertype peptides), 3) peptides predicted to bind HLA-C alleles, 4) exceptional HLA peptides with no known binding motifs. Annotation scores of all eluted peptides are shown in Figure 2—source data 2. Additional information is provided in Supplementary file 1. (B) Curves showing the distribution of the predicted HLA binding affinities for all HLA-A03-annotated peptides with a score ≥3. Overall, 91% of all HLA-A03-annotated peptides are predicted to have a binding affinity below 500 nM for the HLA-A03 molecule (see also Figure 2—figure supplement 4 and Figure 2—figure supplement 5). The same peptides are predicted to be non-binders for the other alleles – i.e., HLA-A02, -B35 and -B39. (C) Heat map visualization following clustering of predicted HLA binding affinity values. The white box highlights HLA-A03-annotated peptides. The four peptides in the table in (a) are indicated by arrows and their respective predicted binding affinity for the HLA-A03 molecule is indicated in parenthesis.