二维液相色谱-质谱法研究犬肾小管上皮细胞脂质组成及马兜铃酸(Ⅰ)对其影响  

Lipid Profiling of Madin-Darby Canine Kidney Cells and Its Lipid Changes Induced by Treatment of Aristolochic Acid(Ⅰ)Using Two Dimensional Liquid Chromatography-Mass Spectrometry

作　　者：聂洪港[1] 刘冉冉[2] 杨悠悠 刘虎威[2] 白玉[2] 

机构地区：[1]北京大学分析测试中心,北京100871 [2]北京分子科学国家实验室,生物有机与分子工程教育部重点实验室,北京大学化学与分子工程学院分析化学研究所,北京100871 [3]中粮营养健康研究院,营养健康与食品安全北京市重点实验室,北京102209

出　　处：《质谱学报》2016年第4期289-300,共12页Journal of Chinese Mass Spectrometry Society

基　　金：国家自然科学基金项目(21175005;21405006;21527809)资助

摘　　要：采用在线正反相二维液相色谱-质谱联用技术研究了犬肾小管上皮细胞脂质组成及马兜铃酸（Ⅰ）对其影响。二维色谱的第一维用于分离不同种类脂质,第二维用于分离同类脂质的不同分子,进而利用高分辨质谱对脂质分子进行检测。该方法减少了共流出,降低了电离抑制,提高了灵敏度与准确性。借助精确质荷比检索数据库、高分辨二级质谱和当量碳数与保留时间规律等方法检测了犬肾小管上皮细胞中13类脂质的1 416个脂质分子。选取11种外源性脂质标准品进行方法验证,方法的线性关系、检测限、重复性均满足检测要求。在此基础上,考察了犬肾小管上皮细胞暴露于马兜铃酸（Ⅰ）后的脂质变化情况,对改变含量2~4倍的15个脂质分子进行了鉴定。该实验结果可为马兜铃酸的毒理、病理研究和相关疾病的临床诊断提供丰富的信息,并展现了二维液相色谱-质谱法在脂质组学研究中广阔的应用前景。The mechanisms of lipids separation are as following：different lipid classesare separated by adsorption mechanisms and eluted out of the column in normal-phase liquid chromatography（NPLC）,and individual molecular species are separated based on hydrophobicity in reversed-phase liquid chromatography（RPLC）.In RPLC,the elution sequence of lipid molecules is determined by both the chain length and the degree of unsaturation in the fatty-acyl chains.To avoid co-elution of molecular species,an online,normal-phase and reversed-phase two-dimensional（2D）liquid chromatography（LC）quadrupole time-of-flight mass spectrometry（Q TOF-MS）system was developed for the lipid profiling of Madin-Darby canine kidney（MDCK）cells and the investigation of the lipid changes in MDCK cells treated with aristolochic acid（Ⅰ）.Different lipid classes in MDCK cells were separated in the first dimension of the two-dimensional liquid chromatograph system and lipid molecular species were further separated in the second dimension followed by mass spectrometry detection,so that the ion suppression effects were reduced while the detection sensitivity was improved.All lipids in MDCK cells were identified with high accuracy mass values measured by Agilent 6530 accurate mass Q TOF-MS.The abundant molecular species were confirmed by targeted MS/MS,meanwhile the retention time and low abundance lipid molecules were identified with m/zvalue and the retention time based on the correlation between the equivalent carbon number（ECN）and the retention time.The measured accurate masses were applied for preliminary identification using the online database with a mass tolerance of less than ±0.005 on the basis of the predicted elemental composition.1 416 endogenous lipid species from 13 lipid classes were identified by accurate masses,tandem mass spectra and the retention time.11 exogenous lipid standards from different classes,including FA 17∶0,Hemi BMP（17∶0）,LPG（17∶1）,PG（14∶0/14∶0）,Sphingosyl PE（d17∶1/12�

关 键 词：二维液相色谱-质谱 犬肾小管上皮细胞 马兜铃酸 脂质组学 

分 类 号：O657.63[理学—分析化学]