基于UPLC-Q-Exactive-MS的菲牛蛭酶解物肽段鉴定与分子特性表征

目的 基于超高效液相色谱-四极杆-静电场轨道阱高分辨质谱（UPLC-Q-Exactive-MS）技术对菲牛蛭酶解物中肽段进行鉴定与表征。方法 采用胰蛋白酶对菲牛蛭进行酶解，利用UPLC-Q-Exactive-MS技术结合Maxquant软件对菲牛蛭酶解物进行分析并对其肽段进行序列鉴定，同时采用生物信息学平台对这些肽段进行生物活性与不良反应预测以及相对分子质量、等电点（pI）、净电荷、亲水性氨基酸比例、不稳定指数等基本分子特性的预测。结果 利用UPLC-Q-Exactive-MS技术联合Maxquant软件在菲牛蛭酶解物中共鉴定出32条肽段，其中肽段AGFAGDDAPR的各项肽段分子特性符合目前已知抗血栓肽的共性特征，鉴定分数为103.83，可信度高；活性概率为0.56，相对分子质量976.01，肽链长度为10；平均亲水性为0.5，亲水性氨基酸残基比例为30%，水溶性良好；pI值为4.21，净电荷为-1；不稳定性指数为20.72，在水中稳定性强；具有抗血栓活性的可能性较大。同时6条肽段SSGETSSIIRR、AGFAGDDAPR、SSGETSSIIR、DSYVGDEAQSKR、GARRER、SIEDQVKR被预测具有抗血管生成活性且无溶血现象，但仍需进一步的合成与活性验证。结论 以菲牛蛭为例，提供了一种快速从酶解物中鉴定动物药肽段序列的方法，以期为动物药的肽类成分的研究提供思路。

Peptide identification and molecular characterization of enzymatic hydrolysates from Poecilobdella manillensis based on UPLC-Q-Exactive-MS

Objective In view of the difficulties in the separation and identification of protein and peptides of Poecilobdella manillensis leading to the ambiguity of peptides components of it, a method for the identification and characterization of polypeptides in enzymatic hydrolysate of Poecilobdella manillensis was developed. Methods Poecilobdella manillensis was hydrolyzed by trypsin, and the polypeptides were identified by UPLC-Q-Exactive-MS technology and Maxquant software from the hydrolysate. Meanwhile, some bioinformatics platforms were used to predict the biological activity and side effects of these peptides, as well as molecular weight, isoelectric point, net charge, proportion of hydrophilic amino acids, instability index and other basic molecular properties. Results A total of 32 peptides were identified by UPLC-Q-Exactive-MS technology combined with Maxquant software. Among them, the molecular properties of AGFAGDDAPR accorded with the common characteristics of antithrombotic peptides which were known to researchers. The identification score was 103.83, with high reliability. The activity probability was 0.56, the molecular weight was 976.01, and the peptide chain length was 10. The average hydrophilicity was 0.5, hydrophilic amino acid residue proportion was 30%, good water solubility; The isoelectric point was 4.21 and the net charge was -1. Instability index was 20.72, strong stability in water, so it would be possible to have antithrombotic activity. Six peptides (SSGETSSIIRR, AGFAGDDAPR, SSGETSSIIR, DSYVGDEAQSKR, GARRER, and SIEDQVKR) were predicted to have antiangiogenic activity and no hemolysis,but further synthesis and activity verification experiments were needed. Conclusion This study takes Poecilobdella manillensis as an example to provide a method for rapid identification of the peptide sequence in enzymatic hydrolysates of animal drugs and provide ideas for the study of peptide components of animal drugs.