Analysis of Three Peptide Components in Centipede by UFLC-MS/MS and Its Application in Identification of Centipede
-
摘要:
目的 建立超高效液相色谱-质谱联用(UFLC-MS/MS)法分析蜈蚣中3种酶解多肽成分TD1(LEEDLERSEERL)、TD2(EEKDKALQNAEGEVAAL)、TD3(MILPTGASSF), 比较不同品种蜈蚣中肽类成分的差异, 寻找蜈蚣特异性多肽, 用于蜈蚣的鉴别。 方法 3种肽类成分的UFLC-MS/MS法, 色谱柱为XSelect HSS T3(4.6 mm×150 mm, 3.5 μm); 流动相为乙腈-0.1%甲酸溶液，梯度洗脱。采用三重四极杆质谱检测器, 电喷雾离子化(ESI), 正离子模式下多反应监测(MRM)进行信号采集。样品采用正交试验方法, 对蛋白提取方法进行优化, 最终确定提取方法为蛋白裂解液超声处理60 min。得到的蜈蚣蛋白提取溶液经胃蛋白酶酶解并脱盐后, 注入液质联用仪分析。测定了20批蜈蚣及8批其他动物药中3种肽类成分, 并对结果进行比较。 结果 在地龙、僵蚕、水蛭、土鳖虫和全蝎中未检出TD2和TD3, TD1与TD2在少棘巨蜈蚣与哈氏蜈蚣、墨江蜈蚣中存在较大差异, TD3在少棘巨蜈蚣与黑头蜈蚣、多棘蜈蚣中有较大差异。 结论 该方法可用于区分少棘巨蜈蚣与地龙、僵蚕、水蛭、土鳖虫和全蝎, 同时通过比较3种肽段的差异, 区分药典品种少棘巨蜈蚣和4种常见非药典品种蜈蚣, 为蜈蚣的质量评价和基础研究提供参考。 -
关键词:
- 蜈蚣 /
- 特异性多肽 /
- UFLC-MS/MS /
- 鉴别
Abstract:OBJECTIVE To analyze three enzymatic peptide components TD1 (LEEDLERSEERL), TD2 (EEKDKALQNAEGEVAAL) and TD3 (MILPTGASSF) in centipedes by UFLC-MS/MS method for comparing the differences of peptide components in different species of centipedes and identification of centipedes. METHODS UFLC-MS/MS of the three peptide components was performed on an XSelect HSS T3 (4.6 mm×150 mm, 3.5 μm) column and the mobile phase was acetonitrile -0.1% formic acid solution with gradient elution. A triple quadrupole mass spectrometer detector with electrospray ionization (ESI) and multiple reaction monitoring (MRM) in positive ion mode were used for signal acquisition. The samples were subjected to orthogonal experimental methods to optimize the protein extraction method, and the final extraction method was determined by sonicating the protein lysate for 60 min to obtain the centipede protein extraction solution, which was enzymatic hydrolyzed and desalted before injecting. Three peptide components were determined in 20 batches of centipedes and 8 batches of other animal medicines and the results were compared. RESULTS TD2 and TD3 were not detected in earthworm, stiff silkworm, leech, ground beetle and scorpion. TD1 and TD2 were more different in S. subspinipes mutilans L.Koch and S. subspinipes dehaani Brandt and S. mojiangica Zhang et chi, and TD3 was more different in S. subspinipes mutilans L.Koch and S. negrocapitis Zhang et Wang and S. subspinipes multidens Newport. CONCLUSION This method can be used to distinguish the S. subspinipes mutilans L. Koch from those of earthworm, stiff silkworm, leech, ground beetle and scorpion. By comparing the differences of three peptides, Scolopendra subspinipes mutilans L.Koch can be distinguished with other four common non-pharmacopoeial species, providing a reference for quality evaluation and basic research of centipedes. -
Key words:
- centipede /
- characterized peptides /
- UFLC-MS/MS /
- identification
-
图 3 少棘巨蜈蚣与其他动物药3种肽段提取离子色谱图
注: A.TD1;B.TD2;C.TD3
Figure 3. Extracted ion chromatograms of three peptides in Scolopendra subspinipes mutilans L. Koch and other animal medicines
图 4 少棘巨蜈蚣与其他4种非药典蜈蚣品种3种肽段提取离子色谱图
注: A.TD1;B.TD2;C.TD3
Figure 4. Extracted ion chromatograms of three peptides in the Scolopendra subspinipes mutilans L. Koch and other four non-pharmacopoeia species
表 1 20批蜈蚣及8批其他动物药来源信息表
Table 1. Source information for 20 batches of centipedes and 8 batches of other animal drugs
编号 品种 拉丁名 批号 产地 1 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ1 陕西 2 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ2 湖北 3 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ3 安徽 4 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ4 四川 5 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ5 湖北 6 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ6 湖南 7 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ7 湖北 8 少棘巨蜈蚣 Scolopendra subspinipes mutilans L.Koch SJ8 湖北 9 多棘蜈蚣 S. subspinipes multidens Newport DJ1 广西 10 多棘蜈蚣 S. subspinipes multidens Newport DJ2 湖北 11 多棘蜈蚣 S. subspinipes multidens Newport DJ3 广西 12 多棘蜈蚣 S. subspinipes multidens Newport DJ4 广西 13 多棘蜈蚣 S. subspinipes multidens Newport DJ5 广西 14 黑头蜈蚣 S. negrocapitis Zhang et Wang HT1 安徽 15 黑头蜈蚣 S. negrocapitis Zhang et Wang HT2 广西 16 黑头蜈蚣 S. negrocapitis Zhang et Wang HT3 湖北 17 墨江蜈蚣 S. mojiangica Zhang et chi MJ1 云南 18 墨江蜈蚣 S. mojiangica Zhang et chi MJ2 云南 19 哈氏蜈蚣 S. subspinipes dehaani Brandt HS1 广西 20 哈氏蜈蚣 S. subspinipes dehaani Brandt HS2 印度尼西亚 21 广地龙 Pheretima aspergillum (E.Perrier) GDL1 广东 22 广地龙 Pheretima aspergillum (E.Perrier) GDL2 广西 23 沪地龙 Pheretima pectinifera Michaelsen HDL1 上海 24 僵蚕 Bombyx Batryticatus JC1 浙江 25 僵蚕 Bombyx Batryticatus JC2 浙江 26 水蛭 Whitmania pigra Whitman SZ 江苏 27 土鳖虫 Eupolyphaga/Steleophaga TBC 江苏 28 全蝎 Buthus martensii Karsch QX 山东 
下载: 导出CSV
表 2 3种多肽的信息表
Table 2. Information for 3 peptides
编号 肽段 分子量 纯度/% TD1 LEEDLERSEERL 1 517.60 98.84 TD2 EEKDKALQNAEGEVAAL 1 814.95 98.95 TD3 MILPTGASSF 1 023.21 99.32
下载: 导出CSV
表 3 梯度洗脱表
Table 3. Gradient elution table
时间/min 流动相A/% 流动相B/% 0~5 20→50 80→50 5~7 50 50 7~8 50→20 50→80 8~9 20 80
下载: 导出CSV
表 4 3种肽段优化的质谱参数
Table 4. Optimized mass spectrometry parameters for 3 peptides
编号 分子量 Q1 m/z(z) Q3 m/z(z) 去簇电压/eV 碰撞电压/eV 碰撞室出口电压/eV TD1 1 517.60 506.59(3+) 638.4(2+) 104.6 24.0 34.2 TD2 1 814.95 605.64(3+) 562.0(3+) 80.2 17.0 35.1 TD3 1 023.21 1 023.5 666.1 160.0 49.9 42.0
下载: 导出CSV
表 5 正交试验因素水平表
Table 5. Orthogonal test factor levels
序号 A(提取方式) B(提取溶剂) C(沉淀方式) 1 4 ℃冷浸 水 80%乙醇 2 60 ℃热浸 1%碳酸氢铵 80%丙酮 3 超声处理 蛋白裂解液 80%硫酸铵
下载: 导出CSV
表 6 蜈蚣蛋白提取条件的正交试验结果
Table 6. Results of orthogonal experiments for protein extraction conditions
序号 A(提取方式) B(提取溶剂) C(沉淀方式) 综合评分 1 1 1 1 22.38 2 1 2 2 32.83 3 1 3 3 165.52 4 2 1 3 46.74 5 2 2 1 44.29 6 2 3 2 130.56 7 3 1 2 9.87 8 3 2 3 11.11 9 3 3 1 194.13 K1 220.74 79.00 260.81 K2 221.60 88.23 173.27 K3 215.11 490.22 223.37 R 6.48 411.22 87.54 注: 综合评分=TD1相对含量×0.3+TD2相对含量×0.3+TD3相对含量×0.4。
下载: 导出CSV
表 7 蜈蚣中3个肽段的线性关系考察结果
Table 7. Investigation results of linear relationship of three peptides in centipede
编号 标准曲线 线性范围/(μg·mL-1) R2 定量限/(μg·mL-1) 检测限/(μg·mL-1) TD1 Y=330.13X-1 805 0.006 0~0.2 0.998 6 0.006 0 0.003 0 TD2 Y=441.71X-2 670.1 0.006 0~0.2 0.998 7 0.004 5 0.001 5 TD3 Y=779.56X-2 094.1 0.001 5~0.2 0.999 2 0.001 5 0.000 5
下载: 导出CSV
表 8 28批样品中3种肽段的含量
Table 8. Contents of 3 peptides in 28 batches of samples
编号 品种 批号 TD1/(μg·g-1) TD2/(μg·g-1) TD3/(μg·g-1) 1 少棘巨蜈蚣 SJ1 1.97 1.10 0.23 2 少棘巨蜈蚣 SJ2 1.86 1.52 0.13 3 少棘巨蜈蚣 SJ3 2.24 1.14 0.13 4 少棘巨蜈蚣 SJ4 0.57 1.65 0.08 5 少棘巨蜈蚣 SJ5 3.29 2.48 0.08 6 少棘巨蜈蚣 SJ6 4.23 1.36 0.09 7 少棘巨蜈蚣 SJ7 4.68 1.84 0.69 8 少棘巨蜈蚣 SJ8 4.94 3.13 0.29 9 多棘蜈蚣 DJ1 2.81 0.73 - 10 多棘蜈蚣 DJ2 10.09 0.68 - 11 多棘蜈蚣 DJ3 7.10 0.78 - 12 多棘蜈蚣 DJ4 9.71 2.96 - 13 多棘蜈蚣 DJ5 4.94 0.30 - 14 黑头蜈蚣 HT1 23.79 3.50 - 15 黑头蜈蚣 HT2 9.85 1.15 - 16 黑头蜈蚣 HT3 10.13 0.75 - 17 墨江蜈蚣 MJ1 - - 0.09 18 墨江蜈蚣 MJ2 - - - 19 哈氏蜈蚣 HS1 0.26 0.13 - 20 哈氏蜈蚣 HS2 0.37 - 0.18 21 广地龙 GDL1 - - - 22 广地龙 GDL2 - - - 23 沪地龙 HDL1 - - - 24 僵蚕 JC1 - - - 25 僵蚕 JC2 - - - 26 水蛭 SZ - - - 27 土鳖虫 TBC 11.58 - - 28 全蝎 QX 5.06 - - 注: “-”表示未检出。
下载: 导出CSV
-
[1] 国家药典委员会. 中华人民共和国药典: 一部[S]. 北京: 中国医药科技出版社, 2020: 372, 188-189, 197-198.National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: Ⅰ[S]. Beijing: China medical science and technology press, 2020: 372, 188-189, 197-198. [2] 李晓东, 李欣, 屈桂群, 等. 药用蜈蚣的生物学研究进展[J]. 大众科技, 2013, 15(9): 85-88. https://www.cnki.com.cn/Article/CJFDTOTAL-DZJI201309031.htmLI XD, LI X, QU GQ, et al. Advances in biological studies of medical centipede in China[J]. Pop Sci Technol, 2013, 15(9): 85-88. https://www.cnki.com.cn/Article/CJFDTOTAL-DZJI201309031.htm [3] 康四和. 中国蜈蚣属动物分类鉴定及药用蜈蚣药材质量评价研究[D]. 武汉: 湖北中医药大学, 2018.KANG SH. Taxonomy and identification of the genus Scolopendra and quality evaluation of centipede medicinal materials in China[D]. Wuhan: Hubei University of Chinese Medicine, 2018. [4] 康四和, 邓海英, 刘义梅, 等. 我国药用蜈蚣资源与品质研究现状及对策[J]. 中南民族大学学报(自然科学版), 2016, 35(2): 61-66. https://www.cnki.com.cn/Article/CJFDTOTAL-ZNZK201602014.htmKANG SH, DENG HY, LIU YM, et al. Status of resources and quality of medicinal centipede in China[J]. J South Central Univ Natl Nat Sci Ed, 2016, 35(2): 61-66. https://www.cnki.com.cn/Article/CJFDTOTAL-ZNZK201602014.htm [5] 刘兵, 谭竹钧, 孔祥平, 等. 少棘蜈蚣活性蛋白对舌癌细胞Tea-8113的抑制作用研究[J]. 时珍国医国药, 2013, 24(6): 1543-1544. https://www.cnki.com.cn/Article/CJFDTOTAL-SZGY201306121.htmLIU B, TAN ZJ, KONG XP, et al. Study on the inhibitory effect of active protein from Scolopendra subspinipes mutilans to human tongue cancer cell line Tea-8113[J]. Lishizhen Med Mater Med Res, 2013, 24(6): 1543-1544. https://www.cnki.com.cn/Article/CJFDTOTAL-SZGY201306121.htm [6] LEE JH, KIM IW, KIM SH, et al. Anticancer activity of the antimicrobial peptide scolopendrasin Ⅶ derived from the centipede, Scolopendra subspinipes mutilans[J]. J Microbiol Biotechnol, 2015, 25(8): 1275-1280. doi: 10.4014/jmb.1503.03091 [7] 姜丽, 王玉蓉, 赵韶华, 等. 蜈蚣胃蛋白酶解物体外抗凝活性肽类的分离与分析[J]. 中国中药杂志, 2012, 37(3): 298-301. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZY201203011.htmJIANG L, WANG YR, ZHAO SH, et al. Study on separation and analysis of anticoagulant compounds for anticoagulant activity in vitro on mixture of peptide from pepsin enzymolysis of centipede[J]. China J Chin Mater Med, 2012, 37(3): 298-301. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZY201203011.htm [8] 陈少鹏, 韩雅莉, 郭桅, 等. 少棘蜈蚣纤溶活性蛋白的抗血栓作用[J]. 中国药理学通报, 2007, 23(8): 1088-1092. https://www.cnki.com.cn/Article/CJFDTOTAL-YAOL200708026.htmCHEN SP, HAN YL, GUO W, et al. Study on the antithrombotic effect of fibrinolytic proteins from Scolopendra subspinipes mutilans[J]. Chin Pharmacol Bull, 2007, 23(8): 1088-1092. https://www.cnki.com.cn/Article/CJFDTOTAL-YAOL200708026.htm [9] LEE W, LEE J, KULKARNI R, et al. Antithrombotic and antiplatelet activities of small-molecule alkaloids from Scolopendra subspinipes mutilans[J]. Sci Rep, 2016, 6: 21956. [10] LEE W, HWANG JS, LEE DG. A novel antimicrobial peptide, scolopendin, from Scolopendra subspinipes mutilans and its microbicidal mechanism[J]. Biochimie, 2015, 118: 176-184. [11] LEE B, HWANG JS, LEE DG. Antibacterial action of lactoferricin B like peptide against Escherichia coli: Reactive oxygen species-induced apoptosis-like death[J]. J Appl Microbiol, 2020, 129(2): 287-295. [12] LEE JH, KIM IW, KIM MA, et al. Antimicrobial activity of the scolopendrasin V peptide identified from the centipede Scolopendra subspinipes mutilans[J]. J Microbiol Biotechnol, 2017, 27(1): 43-48. [13] CHAPARRO-AGUIRRE E, SEGURA-RAMIREZ PJ, ALVES FL, et al. Antimicrobial activity and mechanism of action of a novel peptide present in the ecdysis process of centipede Scolopendra subspinipes subspinipes[J]. Sci Rep, 2019, 9(1): 13631. [14] 梁瑞强, 孙姗姗, 曹进, 等. 基于蛋白质组学技术测定全蝎中β-肌动蛋白的含量[J]. 世界中医药, 2020, 15(13): 1849-1856. https://www.cnki.com.cn/Article/CJFDTOTAL-SJZA202013003.htmLIANG RQ, SUN SS, CAO J, et al. Determination of β-actin in scorpion based on proteomics[J]. World Chin Med, 2020, 15(13): 1849-1856. https://www.cnki.com.cn/Article/CJFDTOTAL-SJZA202013003.htm [15] LIU R, HUANG Y, XU HK, et al. A strategy for identifying species-specific peptide biomarkers in deer-hide gelatin using untargeted and targeted mass spectrometry approaches[J]. Anal Chim Acta, 2019, 1092: 32-41. [16] 陈霞, 文红梅, 刘睿, 等. 纳升级反相液相色谱-串联质谱法分析蜈蚣提取蛋白质[J]. 分析化学, 2014, 42(2): 239-243. https://www.cnki.com.cn/Article/CJFDTOTAL-FXHX201402016.htmCHEN X, WEN HM, LIU R, et al. Analysis of extracted proteins of Scolopendra by Nanoflow reversed phase liquid chromatography-tandem mass spectrometry[J]. Chin J Anal Chem, 2014, 42(2): 239-243. https://www.cnki.com.cn/Article/CJFDTOTAL-FXHX201402016.htm -
点击查看大图
图(4) / 表(8)
计量
- 文章访问数: 47
- HTML全文浏览量: 4
- PDF下载量: 7
- 被引次数: 0
