In vitro digestion of synthetic peptides with human proteasomes. (A) Digestion of the peptide TEL-AML1-319. Predominant cleavage products and cleavage sites are shown. Synthetic peptides were incubated until 50% of the peptide substrate was turned over by purified LCL (lymphoblastoid cell lines) 20S proteasomes, which mainly expresses immunoproteasomes. Peptide fragments are detected by LC-MS/MS (reversed phase liquid chromatography coupled online with tandem mass spectrometry) using a normal triple method. Arrows of various thickness are proportional to the relative cleavage intensity. Note the large number of destructive cleavages within the epitope (in red) and the complete absence of a potentially functional N-terminal cleavage. (B) Generation of the LLO-296-304 CD8⁺ T-cell epitope by digestion of synthetic polypeptide LLO-291 derived from bacterial listeriolysin O. The experiment was performed as described in panel A. The dominant relevant cleavage products and the MHC class I ligand Val₂₉₆-Leu₃₀₄ (in red) as well as the N-terminally elongated epitope precursor peptide thereof (in green) are shown. Arrows indicate major and minor cleavage sites. Note the strong cleavage behind the C-terminal Leu₃₀₄ residue, resulting in the predominant generation of the epitope. Cleavage within the epitope does not effect the overall predominance of Val₂₉₆-Leu₃₀₄ peptide generation. Numbers designate amino acid residue positions in the corresponding proteins (TEL-AML1 and listeriolysin O).