超高效液相色谱-四极杆/静电场轨道阱高分辨质谱联用鉴定椴树蜂蜜中的抗氧化活性肽

应用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱联用法(UPLC-Q-Exactive)鉴定椴树蜂蜜蛋白经胰蛋白酶水解后获得的肽类物质的组成和结构,并探讨椴树蜂蜜酶解多肽与其抗氧化活性的关系。椴树蜂蜜经三氯乙酸溶液沉淀,所得蛋白用胰蛋白酶酶解,酶解肽段经C18固相萃取柱富集净化后,采用0.1%甲酸(A)-乙腈(B)作为流动相进行梯度洗脱,以高分辨质谱(Q-Exactive)的Full MS/ddMS2模式对其进行鉴定。采用MaxQuant软件分析质谱结果,所得肽段在Uniprot上进行Blast序列对比,共鉴定出52种椴树蜂蜜胰蛋白酶解多肽,其中71.15%来自蜂王浆主要蛋白(MRJPs),鉴定出的肽段归属于10种蛋白质,其中6种(60%)属于MRJP家族蛋白。对椴树蜂蜜酶解多肽质谱鉴定结果进行重复性分析,2次以上重复质谱分析可实现对椴树蜂蜜多肽(87.1%)的可靠鉴定。结合文献报道的蜂蜜多肽抗氧化活性的构效关系,筛选出11条可能具有抗氧化活性的多肽。化学合成11条多肽后,进行4种不同的抗氧化活性实验。结果表明,这11条多肽的抗氧化活性存在较大差异,其中1 g/L VIYEWK多肽显示出较强的ABTS自由基清除活性(33.8%±0.6%)、DPPH自由基清除活性(96.5%±0.0%),并表现出一定的还原力(0.010±0.001)和Fe2+螯合活性(4.0%±0.2%)。该方法能快速有效鉴定椴树蜂蜜多肽结构,可为揭示椴树蜂蜜多肽结构与抗氧化活性的关系提供依据。

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.