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

doi:10.7538/zpxb.youxian.2016.0037

CORC > 北京大学 > 化学与分子工程学院

	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
	聂洪港 ; 刘冉冉 ; 杨悠悠 ; 刘虎威 ; 白玉
刊名	质谱学报
	2016
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 two-dimensional liquid chromatography mass spectrometry Madin-Darby canine kidney (MDCK) cells aristolochic acid lipidomics
DOI	10.7538/zpxb.youxian.2016.0037
英文摘要	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 classes are 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/z value 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(d17:1/12:0), PE(14:0/14:0), LPE(17:1), PS(14:0/14:0), LPC(17:0), PC(14:1/14:1), SM(d18:1/17:0), were selected to be separated in five fractions for the evaluation of this method. The linear regression coefficients (R2=0.991 7-0.998 3), the limit of detection (2-5 ��g/L) and the relative standard deviation of peak area (0.9%-7.7%) and retention time (0.01%-0.11%) were all satisfactory. To investigate the lipid changes in MDCK cells dosed with aristolochic acid (I), 16 MDCK cell samples (each containing 5��105cells) were randomly separated into a dosed group (n=8) and a control group (n=8). All 16 samples were detected by the 2D LC/MS method. The dosed group and control group were alternately injected to reduce systemic error. During the sequence, one blank sample was injected after every three injections, and no significant carryover of lipids was observed. MS data of all 16 samples were extracted by Mass-Hunter Qualitative Analysis software and analyzed by Mass Profiler Professional software. Through setting threshold parameters, the software presented a list of potential biomarkers whose absolute fold-change of peak area was larger than 2 and p value less than 0.05. By the above-mentioned approach, 15 changed lipid species were confirmed, as their concentrations in the dosed group were 2-4 times of those in the control group. The results would contribute to the study on therapeutic and toxicological mechanisms of aristolochic acids and revealed that this two-dimensional liquid chromatography quadrupole time-of-flight mass spectrometry method was a promising tool for lipidomics research. ? 2016, Editorial Board of Journal of Chinese Mass Spectrometry Society. All right reserved.; 国家自然科学基金项目; EI; 中文核心期刊要目总览(PKU); 中国科技核心期刊(ISTIC); 中国科学引文数据库(CSCD); 4; 289-300; 37
语种	中文
内容类型	期刊论文
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/494070]
专题	化学与分子工程学院
推荐引用方式 GB/T 7714	聂洪港,刘冉冉,杨悠悠,等. 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[J]. 质谱学报,2016.
APA	聂洪港,刘冉冉,杨悠悠,刘虎威,&白玉.(2016).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.质谱学报.
MLA	聂洪港,et al."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".质谱学报 (2016).