title: Purification of Antioxidant Peptides by High Resolution Mass Spectrometry from Simulated Gastrointestinal Digestion Hydrolysates of Alaska Pollock (Theragra chalcogramma) Skin Collagen.
abstract: In this study, the stable collagen hydrolysate was prepared by alcalase hydrolysis and twice simulated gastrointestinal digestion from Alaska pollock skin. The characteristics of hydrolysates and antioxidant activities in vitro, including 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS•+) scavenging activity, ferric-reducing antioxidant power (FRAP) and hydroxyl radical (OH·) scavenging activity, were determined. After twice simulated gastrointestinal digestion of skin collagen (SGI-2), the degree of hydrolysis (DH) reached 26.17%. The main molecular weight fractions of SGI-2 were 1026.26 and 640.53 Da, accounting for 59.49% and 18.34%, respectively. Amino acid composition analysis showed that SGI-2 had high content of total hydrophobic amino acid (307.98/1000). With the simulated gastrointestinal digestion progressing, the antioxidant activities increased significantly (p< 0.05). SGI-2 was further purified by gel filtration chromatography, ion exchange chromatography and high performance liquid chromatography, and the A1a3c-pfraction with high hydroxyl radical scavenging activity (IC50 = 7.63 μg/mL) was obtained. The molecular weights and amino acid sequences of key peptides of A1a3c-pwere analyzed using high resolution mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) combined with de novo software and UniProt of MaxQuant software. Four peptides were identified from A1a3c-p, including YGCC (444.1137 Da) and DSSCSG (554.1642 Da) identified by de novo software and NNAEYYK (900.3978 Da) and PAGNVR (612.3344 Da) identified by UniProt of MaxQuant software. The molecular weights and amino acid sequences of four peptides were in accordance with the features of antioxidant peptides. The results indicated that different peptides were identified by different data analysis software according to spectrometry mass data. Considering the complexity of LC-ESI-LTQ-Orbitrap-MS, it was necessary to use the different methods to identify the key peptides from protein hydrolysates.
authors.affiliation: Yunnan Institute of Food Safety, Kunming University of Science and Technology, No. 727 South Jingming Road, Kunming 650500, Yunnan, China. kmlpsun@163.com.
authors.forename: Liping
authors.initials: L
authors.lastname: Sun
authors.affiliation: Yunnan Institute of Food Safety, Kunming University of Science and Technology, No. 727 South Jingming Road, Kunming 650500, Yunnan, China. changweidan08023@163.com.
authors.forename: Weidan
authors.initials: W
authors.lastname: Chang
authors.affiliation: Yunnan Institute of Food Safety, Kunming University of Science and Technology, No. 727 South Jingming Road, Kunming 650500, Yunnan, China. maqingyu0323@163.com.
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Ma
authors.affiliation: Yunnan Institute of Food Safety, Kunming University of Science and Technology, No. 727 South Jingming Road, Kunming 650500, Yunnan, China. ylzhuang@kmust.edu.cn.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Zhuang
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000494:pharmacology*; D052160:Benzothiazoles / Q000737:chemistry; D003094:Collagen / Q000737:chemistry; D029941:Fish Proteins; D016166:Free Radical Scavengers / Q000737:chemistry / Q000494:pharmacology; D048388:Gadiformes / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000737:chemistry*; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011492:Protein Hydrolysates / Q000737:chemistry / Q000494:pharmacology*; D012867:Skin / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013451:Sulfonic Acids / Q000737:chemistry
chemical_list: D000596:Amino Acids; D000975:Antioxidants; D052160:Benzothiazoles; D029941:Fish Proteins; D016166:Free Radical Scavengers; D010455:Peptides; D011492:Protein Hydrolysates; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D003094:Collagen
keywords: Alaska pollock skin collagen; UniProt of MaxQuant; antioxidant peptide; de novo software; peptide purification; simulated gastrointestinal digestions