Analysis of the degradation mechanisms of MHC class I‐presented tumor antigenic 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 are 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 design of structurally modified analogs, and the analysis of their degradation by on‐line HPLC/ESI‐MS, allowed us to demonstrate the efficiency of local modifications in the protection of a given peptide bond towards a specific peptidase activity. © 1998 John Wiley & Sons, Ltd.