Identification of Antioxidant Peptide Sequences in Tilia Tuan Honey by UPLC-Q-Exactive Quadrupole-Electrostatic Field Orbitrap High Resolution Mass Spectrometry

Tilia tuan honey has many biological activities, and the source of biological activities is inseparable from the protein and peptides in tilia tuan honey. Antioxidant activity is one of the many biological activities of tilia tuan honey. In this paper, the composition and structure of trypsinolysis peptides in tilia tuan honey were identified by ultrahigh performance liquid chromatographyquadrupole/electrostatic field orbitrap high resolution mass spectrometry (UPLCQExactive), and the relationship between trypsinolysis peptides in tilia tuan honey and antioxidant activity was discussed. Tilia tuan honey protein was precipitated by 20%trichloroacetic acid and lyophilized. The lyophilized protein was dissolved in 100 mmol/L NH4HCO3 buffer solution. Dithiothreotol was added to reduce protein, iodoacetamide was added to alkylate protein, and 500 mmol/L dithiothreotol solution was added to the final protein solution to stop the alkylation reaction, then the protein was hydrolyzed by trypsin. The enzymatic peptides was enriched and purified by C18 solid phase extraction column. Gradient elution was carried out with 0.1% formic acid (A) and acetonitrile (B) as mobile phase. The enzymatic hydrolyzed tilia tuan honey peptides was identified by Full MS/ddMS2 mode of high resolution mass spectrometry (QExactive). The results of mass spectrometry were analyzed by MaxQuant software, and compared on Uniprot by Blast. A total of 52 peptides in tilia tuan honey were identified, 7115% of which were derived from major royal jelly proteins (MRJPs), and the identified peptides belonged to 10 proteins, of which 6 (60%) were MRJP family proteins. Reproducibility analysis of tilia tuan honey peptides mass spectrometry results, more than two repetitive mass spectrometry can achieve reliable identification of tilia tuan honey peptides (871%). According to the structureactivity relationship of honey peptides reported in the literature, 11 peptides with antioxidant activity were screened out. Four different antioxidant activities were tested after chemical synthesis of 11 peptides. The results showed that the antioxidant activities of these 11 peptides were significantly different, and 1 g/L VIYEWK showed strong 2,2′azinobis(3ethylbenzthiazoline6sulphonate) (ABTS) free radical scavenging activity (338±06%), 2,2diphenyl1picryhydrazyl (DPPH) free radical scavenging activity (96.5±0.0%), and showed a certain reducing power(0010±0001) and Fe2+ chelating activity (40±02%). The method can quickly and effectively identify the structure of tilia tuan honey peptides, provide a basis for revealing the relationship between tilia tuan honey peptides structure and antioxidant activity. But the results of antioxidant activity in vitro can not fully explain the antioxidant activity of peptides. Therefore, it is necessary to study the antioxidant activity of peptides in vivo and the antioxidant activity of peptides is the result of the interaction of various amino acids. The modification of amino acids of antioxidant peptides can be considered to determine the specific amino acids that play an antioxidant role.