Figure 2.
Proteasome-mediated cleavage patterns of 27-mer peptides encompassing 19 high affinity HLA-A*0201 binding PRAME peptides. 20S proteasomes isolated from an EBV-transformed B cell line were coincubated with 27-mer PRAME peptides at 37°C for the indicated time points. Digestion mixtures were analyzed by mass spectrometry as described in Materials and Methods. Major and minor cleavages sites at 1 h digestion are depicted. Notes: 1 all 19 high affinity binding peptides (IC50 < 6 μM) are listed and ranked according to their binding affinity for HLA-A*0201; 2 start aa position in PRAME of the HLA-A*0201 binding peptide. 3 IC50 (in μM) as determined in competition binding assay (see Table ); 4 start and end aa position in PRAME of 27-mer polypeptide encompassing the high affinity binding peptide; 5 aa sequence of 27-mer polypeptide (aa sequence of HLA-A*0201 binding peptide is printed in bold and shaded); 6 major (bold arrows) and minor (thin arrows) cleavage sites at 1 h incubation are depicted, classified according to the following definitions. Major site: fragments containing as COOH terminus the residue NH2-terminal from the cleavage are present for ≥5% at 1 h incubation. Minor site: fragments containing as COOH terminus the residue NH2-terminal from the cleavage are present for <5% at 1 h incubation. 7 Generation by digestion of fragments containing the correct COOH terminus of the HLA-A*0201 binding peptide. Generation at 1 h digestion or after a longer incubation period is separately indicated. Classification: (++) present for ≥5%, (+) present for <5%, (−) no fragments containing the correct COOH terminus were found. 8 Generation by digestion of fragments containing the intact HLA-A*0201 binding peptide and/or NH2-terminal elongated precursors of the peptide. Classification: (++) present for ≥5% at 1 h incubation, (+) present for <5% at 1 h or only detectable after 4 or 24 h, (−) no fragments containing the HLA-A*0201 binding peptide were found. 9 Epitope prediction based on digestion results. Classification: (+) most likely an epitope, (+/−) doubtful epitope, (−) not an epitope.