目的 基于超高效液相色譜-四極桿-靜電場軌道阱高分辨質(zhì)譜（UPLC-Q-Exactive-MS）技術(shù)對菲牛蛭酶解物中肽段進(jìn)行鑒定與表征。方法 采用胰蛋白酶對菲牛蛭進(jìn)行酶解，利用UPLC-Q-Exactive-MS技術(shù)結(jié)合Maxquant軟件對菲牛蛭酶解物進(jìn)行分析并對其肽段進(jìn)行序列鑒定，同時采用生物信息學(xué)平臺對這些肽段進(jìn)行生物活性與不良反應(yīng)預(yù)測以及相對分子質(zhì)量、等電點(diǎn)（pI）、凈電荷、親水性氨基酸比例、不穩(wěn)定指數(shù)等基本分子特性的預(yù)測。結(jié)果 利用UPLC-Q-Exactive-MS技術(shù)聯(lián)合Maxquant軟件在菲牛蛭酶解物中共鑒定出32條肽段，其中肽段AGFAGDDAPR的各項肽段分子特性符合目前已知抗血栓肽的共性特征，鑒定分?jǐn)?shù)為103.83，可信度高；活性概率為0.56，相對分子質(zhì)量976.01，肽鏈長度為10；平均親水性為0.5，親水性氨基酸殘基比例為30%，水溶性良好；pI值為4.21，凈電荷為-1；不穩(wěn)定性指數(shù)為20.72，在水中穩(wěn)定性強(qiáng)；具有抗血栓活性的可能性較大。同時6條肽段SSGETSSIIRR、AGFAGDDAPR、SSGETSSIIR、DSYVGDEAQSKR、GARRER、SIEDQVKR被預(yù)測具有抗血管生成活性且無溶血現(xiàn)象，但仍需進(jìn)一步的合成與活性驗證。結(jié)論 以菲牛蛭為例，提供了一種快速從酶解物中鑒定動物藥肽段序列的方法，以期為動物藥的肽類成分的研究提供思路。

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.

R917

國家自然科學(xué)基金資助項目（82173955）；北京市自然科學(xué)基金資助項目（7172130）