Analysis of MHC Class II Antigen Processing by Quantitation of Peptides that Constitute Nested Sets

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3-36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.