Identification of peptide superagonists for a self-K-ras-reactive CD4(+) Tcell clone using combinatorial peptide libraries and mass spectrometry

The proliferative responses of a human CD4(+) T cell clone 29.15.2, reactiv e with a self-K-ras-derived peptide ((3)EYKLVVVGAG GVGKSALT(20)), were test ed using a set of X9 combinatorial peptide libraries containing the flankin g residues (EYKLVXXXXXXXXXSALT, where X indicates random amino acids). Cert ain peptide libraries, such as EYKLVXXXXXXMXX SALT and EYKLVXXXXXXXHXSALT, stimulated a marked proliferation of 29.15.2, However, no combinations of s ubstitutions tested, such as EYKLVXXXXXXMHXSALT, exhibited additive effects . We subsequently synthesized peptides with degenerate sequences (a mixture of 480 species), where each position is composed of the wad-type (wt) resi due or of amino acids that induced the proliferation of 29.15.2, in positio nal scanning. Interestingly, one fraction of degenerate peptides, separated by reverse-phase HPLC, stimulated much higher proliferation than did the w t; in addition, the retention time of this fraction was distinct from that of the wt. Mass spectrometry analysis of this fraction and flanking fractio ns identified five peptide species that exhibit strong signals in a manner that parallels the antigenic activity. Finally, 17 candidate peptide sequen ces were deduced from mass spectrometry and hydrophobicity scoring results, of which two peptides (EYKLVVVGAGGMLKSALT and EYKLVVVGAGGMIKSALT) did indu ce 52- and 61-fold stronger proliferation, respectively, compared with the wt, These findings indicate that: 1) synthetic peptides that carry "the bes t" residue substitution at each position of combinatorial peptide libraries do not always exhibit superagonism, and 2) such a drawback can be overcome with the use of mass spectrometry, This approach provides new perspectives for the accurate and efficient identification of peptide superagonists.