ANALYSIS OF THE DEGRADATION MECHANISMS OF MHC CLASS I-PRESENTED TUMORANTIGENIC PEPTIDES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY - APPLICATION TO THE DESIGN OF PEPTIDASE-RESISTANT ANALOGS

Peptide vaccines based on the use of MHC class I restricted epitopes a re currently assayed for anti-tumor and anti-viral immunotherapy. With the aim of designing minimally modified, peptidase-resistant analogs, we developed a rational approach based on a detailed understanding of the degradation mechanism of peptides in serum. Degradation of murine tumor antigen P198 and human tumor antigen MAGE-3.A1 was followed by on line high performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). This method provided high precision and sensitivity for rapid and direct analysis of degradation fragments in a complex mixture and, very importantly, precise identification of transient degradation fragments present at low concentrations. The de sign of structurally modified analogs, and the analysis of their degra dation by on-line HPLC/ESI-MS, allowed us to demonstrate the efficienc y of local modifications in the protection of a given peptide bond tow ards a specific peptidase activity. (C) 1998 John Wiley & Sons, Ltd.