In vitro/in vivo identification of zein degraded peptides using HPLC-MS/MS and their safety evaluation

Abstract

The identification of degraded products of implanted scaffolds is desirable to avoid regulatory concerns. In vivo identification of products produced by the degradation of natural protein-based scaffolds is complex and demands the establishment of a routine analytical method. In this study, we developed a method for the identification of peptides produced by the degradation of zein both in vitro and in vivo using HPLC-MS/MS. For in vitro experiments, zein was degraded enzymatically and analyzed produced peptides. In vitro study showed cytocompatibility of peptides present in the hydrolysate of zein with no induction of apoptosis and cell senescence. For in vivo experiment, zein gels were prepared and subcutaneously implanted in rats. Peptides produced by the degradation of zein were identified and some are selected as targeted (unique peptides) and two were synthesized as the main sequence of these peptides. Further, peptide analysis using HPLC-MS/MS of different organs after 2 and 8 weeks of implantation of zein gel in rats. It was found that zein-originated peptides were accumulated in different organs. QQHIIGGALF or long peptides with same fractions were identified as unique peptides. These peptides were also found in control rats with regular rat feed (having corn) which the degradation of implanted zein biomaterial food related peptides of non-toxic nature. Furthermore, H&E staining exhibited normal features. Overall, zein hydrolysate has cytocompatibility and did not found to induce organ toxicity and QQHIIGGALF can act as a standard peptide for determining zein degradation. The study also provides the feasibility of complex analysis of the identification of degradation products of protein-based scaffolds.