Separation And Analysis Of Peptide In Functional Food

In this paper, based on affinity chromatography separating proteins peptides biologicaland dual polarization interferometry, mass spectrometry identification of peptides wasanalyzed, and then the binding characteristic and kinetics between collagen peptides to FNwas further researched. Separation and analysis method of peptides were established.Using FN affinity chromatography, collagen peptides were separated from gelatin. Thesequence of collagen peptides which could bind to FN were identified by HPLC-MS/MS. Themolecular weight and hydroxylation modification of collagen peptides were analyzed theeffect on the FN-binding activity by MALDI-TOF MS and DPI. All of peptides which couldcombine with FN contained the sequence GPAG or GPPG. They were the FN-binding coresequence. The molecular weight of peptides was mainly in the1000-2000Da. The FN bindingregion of collagen was identified. The lower molecular weight of peptides was more easilyabsorbed by human body, providing a new basis for the development of new collagen activepeptides. The binding activity of collagen peptides with different molecular weight range toFN fixed on the chip was determinated by DPI. The results showed that the peptides of themolecular weight less than1kDa were difficult to bind FN, and the molecular weightbetween2and30kDa combined with FN more easily. This was explained that the modestmolecular weight range made the structure of peptides exposed the more FN binding sites.Therefore, the molecular weight range played an important role in the interaction collagen-FN.Four collagen peptides were synthesized, including two hydroxylation and twonon-hydroxylation modification respectively. Hydroxylation and non-hydroxylationmodification samples mixed balanced were researched the hydroxylation modification effecton FN-binding activity by MALDI-TOF MS. The four synthesized peptides could interactwith FN, however, the strength of combination was decided by the position and number ofhydroxylation modification in the sequence.Based on gel filtration chromatography, amino acid composition analysis combinedwith liquid chromatography-mass spectrometry, the protein peptides of tortoise-shell glue andantler glue were analyzed. We initially established the chromatographic method for theanalysis of the protein and peptides in the functional food. Due to the similarity amino acidcomposition between tortoise-shell glue, antler glue and type I collagen, collagen in thetortoise-shell glue and antler glue was type I. Gel filtration chromatography simply andrapidly analyzed the molecular weight range in the extraction solution of tortoise-shell glue and antler glue. Except the sugars, almost all of the protein could be precipitated by TCA.And the extraction solution was fully hydrolyzed by trypsin. Based on HPLC-MS coupledwith the full database searching of collagen sequence, the results showed that collagensequence of antler glue was similar to that of its similar material species. Collagen sequenceof tortoise-shell glue was similar to that of amphibian, and some of them were similar to thesequence of type I collagen in the reptiles. The results showed clearly that similar specieshave the similar collagen sequence. The results could contribute to the quality control oftortoise-shell glue. This provided theory for further research of collagen sequence intortoise-shell glue and antler glue.In order to verify the applicability of the chromatographic method for the analysis ofglycoproteins, another functional food-edible bird’s nest was analyzed. Edible bird’s nestcontained lots of glycoproteins. The glycosylation inhomogeneity for glycoprotein oftenresults in wide range of molecular weight and the difficulty for protein separation andcharacterization. In this paper, proteins in the edible bird’s nest were extracted using multipleextractions, and then digested by PNgase F and trypsin. The digest mixture was separatedwith HPLC, and peptides were identified based on MS/MS data searching. The results showedthat mass spectrometry identification method was an effective method of peptides analysis.The results indicated that the extracted proteins was amount to79.7%of total protein in theedible bird’s next. After the extracted proteins were digested by two enzymes, the digestionwas fully degradated by gel filtration chromatography. The amino acid analysis indicated thatthe content of Asp(including Asn), Ser and Thr were high, and there wasn’t almost Met. Theamino acid composition was extremely similar to that of sialoglycoprotein. This furtherindicated that the extracted of edible bird’s nest contained a lot of sialoglycoproteins. Due tonon-existent Hyp, the edible bird’s nest didn’t contain collagen. More than20species ofpeptides in the digested mixture were identified. The sequences of these peptides showedsimilarity with some human protein, especially was similar to the protein sequence ofapodidae. This provided the technical reference for glycoprotein analysis.