PEP06 is a novel endostatin-Arg-Gly-Asp-Arg-Gly-Asp(RGDRGD)30-amino-acid polypeptide featuring a terminally fused RGDRGD hexapeptide at the N terminus.The active endostatin fragment of PEPO6 directly targets tumor cells and exerts an antitumoral effect.However,little is known about the kinetics and degradation products of PEP06 in vitro or in vivo.In this study,we investigated the in vitro metabolic stability of PEP06 after it was incubated with living cells obtained from animals of different species;we further identified the degradation characteristics of its cleavage products.PEP06 underwent rapid enzymatic degradation in multiple types of living cells,and the liver,kidney,and blood play important roles in the metabolism and clearance of the peptides resulting from the molecular degradation of PEP06.We identified metabolites of PEP06 using full-scan mass spectrometry(MS)and tandem MS(MS2),wherein 43 metabolites were characterized and identified as the degradation metabolites from the parent peptide,formed by successive losses of amino acids.The metabolites were C and N terminal truncated products of PEP06.The structures of 11 metabolites(M6,M7,M16,M17,M21,M25,M33,M34,M39,M40,and M42)were further confirmed by comparing the retention times of similar full MS spectrum and MS2 spectrum information with reference standards for the synthesized metabolites.We have demonstrated the metabolic stability of PEP06 in vitro and identified a series of potentially bioactive downstream metabolites of PEP06,which can support further drug research.