蛋白質(zhì)組學(xué)方法探究唐草片對微粒體CYP450酶影響

1、蛋白質(zhì)組學(xué)方法探究唐草片對微粒體cyp450酶影響摘要艾滋?。╝ids）患者常用唐草片進(jìn)行輔助甚 至替代治療，然而中藥成分復(fù)雜，其對抗病毒藥物的影響及 其作用機(jī)制尚不清楚ocyp450酶是藥物的主要代謝酶，因此, 研究中藥唐草片與依非韋倫聯(lián)合用藥前后對cyp450酶的調(diào) 控具有重要意義。蛋白質(zhì)組學(xué)以其高通量高靈敏度的特點(diǎn)被 廣泛用于代謝酶的研究。該文采用差速離心法分離肝微粒 體，sds-page分離其蛋白質(zhì)，切取cyp450所在的3條電泳 帶，用液相色譜串聯(lián)質(zhì)譜進(jìn)行鑒定，一共鑒定了 16個cyp450 同工酶。為了定量分析唐草片對cyp450酶的影響，采用基 于質(zhì)譜的多反應(yīng)監(jiān)測技術(shù)（mrm）o

2、根據(jù)蛋白質(zhì)的質(zhì)譜鑒定結(jié) 果，選擇cyp2cllo其特征多肽通過expasy blast搜索獲得。 片斷離子的m/z 1. 5 mrm定量cyp450酶cyp2c11被選 做mrm定量，采用hplc （shimadzuhplc,日本）串聯(lián)質(zhì)譜 （applied biosystems api3200 ,美國）進(jìn)行定量分析 8-10 o 合成內(nèi)標(biāo)肽（is） elnnalqnlarti （esat-6）（該 多肽來自結(jié)核分泌抗原6 kda的6476位氨基酸序列）。用 100 u l 2% acn和0. 1%甲酸從第2條帶提取cyp2c11多肽, 且加入10 ul的1 mg - l-1內(nèi)標(biāo) （is）,吸

3、取45 ul樣 品用于質(zhì)譜分析。acclaim pepmap c18色譜柱（150 mmx 1. 0 mm, 5 u m）,流速為0. 06 ml min-1 o色譜梯度為0 12 min, 4%-45% acn （含 0.1%甲酸）；12-20 min, 45% acn（含 0.1%甲酸）；20-25 min, 80% acn （含 0.1%甲酸）和 2550 min, 4% acn （含0.1%甲酸）。質(zhì)譜參數(shù)為mrm模式， 離子源電壓 5 500 v； cad 60； dp 50； ep 8 和 ce35o cyp2c11 的特征肽通過expasy blast搜索獲得。mrm定量的離子對

4、選 自hct離子腓質(zhì)譜鑒定的結(jié)果，且片斷離子的m/z 6 lane c s, nisar s, griffiths w j, et al. identification of cytochrome p450 enzymes in human colorectal metastases and the surrounding liver: a proteomic approach j. eur j cancer, 2004,40： 2127.7 zhang l, peng x, zhang z, et al. subcellularproteome analysis unraveled anne

5、xin a2 related to immune liver fibrosis j. j cell biochem, 2010,110：219.8 zhang l, jia x, zhang x, et al. alpha-1antitrypsin variants in plasma from hiv-infected patients revealed by proteomic and glycoproteomic analysis j electrophoresis, 2010,31： 34379 xu m y, qu y, jia x f, et al. serum proteomic

6、 mrm identify peptide ions of transferrin as new fibrosis markers in chronic hepatitis bj. biomedpharmacother, 2013,67： 561.10 劉永福，賈小芳，騰珍林，等液質(zhì)聯(lián)用多反 應(yīng)監(jiān)測法定量目標(biāo)多肽或蛋白質(zhì)j.中國生物化學(xué)與分子 生物學(xué)報(bào)，2012, 28 (1)： 86.proteomic study on effect of tangcao pill on microsome cyp450zhang li-jun , jia xiao-fang , yin lin, liu

7、xiao-qian , shen yin-zhong , lu hong-zhou , cheng neng-neng(1. college of pharmacy , fudan university , shanghai 200032, china；2. shanghai public hea1th clinical center, fudan university, shanghai 201508, china)abstract tangcao pill is commonly applied in adjuvant and even alternative therapy for pa

8、tients with aids. however, the herb contains complex ingredients, but with unknown effect against anti-hiv drug and unknown function. because cyp450 emzyme is the main metabolic enzymes of the drug, it is of important significance to study the regulation of cyp450 enzymes before and after the combin

9、ed administration of tangcao pill and efv. proteomics, due to its high throughoutand high sensitivity, has been widely applied in cyp450 enzyme study. in this paper, liver microsomes were separated through differential centrifugation. their proteins were separated through sds-page. the three protein

10、 bands that cyp450 enzymes were located were cut and identified by liquid chromatography tandem mass spectrometry. totally 16 cyp450 isoenzymes were identified furtherinore , in order to make a quantitative analysis on the effect of tang herb on cyp450 emzyme, the multi pie reaction monito ring (mrm

11、 ) technology based on ms was adopted. the cyp2c11 was selected based on the resuits of the mass spectrum identification of proteins the characteristic polypeptides were obtained through searching expasy blast database the m/z of the fragment ions was less than 800. in the paper, the m/z of ion pair

12、s of cyp2c11 were 711. 5/232. 1 ,711.5/319.2 ,711.5/466.2 and711. 5/595. 3, and the m/z of esat-6 (in ter nal sta ndard, is) were 735. 5/215. 3,735. 5/389. 3,735. 5/460. 3 and735 5/524. 3. the relative peak (analyte/is) area was adopted for the relative quantitative analysis compared with the efv single administration group, the efv and tangcao pill combined administration group showed a 1.6-fold increase in cyp2c11. the resuits of the paper indicated that tangcao pill