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

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 high performance liquid chromatography with tandem mass spectrometry (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 few were selected as targeted (unique peptides) and two peptides were synthesized as the reference sequence of these peptides. Further, peptide analysis using HPLC-MS/MS of different organs was performed 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 peptides with same fractions were identified as unique peptides. These peptides were also found in control rats with regular rat feed, which means the degradation of implanted zein biomaterial produced food related peptides of non-toxic nature. Furthermore, hemotoxylin and eosin (H&E) staining exhibited normal features. Overall, zein degraded products showed cytocompatibility and did not induce organ toxicity, and QQHIIGGALF can act as a standard peptide for tracing and determining zein degradation. The study also provides the feasibility of complex analysis on identification and quantification of degradation products of protein-based scaffolds.