Citation: QIN Mei-Ling, GAO Huan-Huan, CHAI Shuang-Shuang, HE Qiao, ZHANG Han-Tong, MA You-Ning. Metabolic Profiling Analysis of Rice Leaf Based on Hydrophilic Interaction Chromatography Combined with Reversed Phase Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry. Chinese Journal of Analytical Chemistry, 2018, 46(4): 479-485. doi: 10.11895/j.issn.0253-3820.171370 [复制]
Metabolic Profiling Analysis of Rice Leaf Based on Hydrophilic Interaction Chromatography Combined with Reversed Phase Liquid Chromatography Quadrupole-Time-of-Flight Mass Spectrometry
采用四极杆-飞行时间液相色谱-质谱联用（Q-TOF LC/MS）方法，比较不同提取方法对水稻叶片中代谢物提取结果的影响，建立了HSS T3和XBridge Amide相结合的水稻叶片代谢轮廓分析方法。以检出峰数目、初步鉴定的代谢物数量、种类以及代谢途径为指标，探讨了甲醇-氯仿-水，甲醇-氯仿-氨水，甲醇-甲基叔丁基醚-水3种提取方法和不同色谱系统对水稻叶片中代谢物提取及分离的效果。结果表明，甲醇-氯仿-水对水稻叶片中代谢物的检出覆盖率最高，提取到的独有代谢物最多，分别为苯甲酸、木樨草素、α-亚麻酸、乌头酸、赤霉素A12醛、异牧荆素、L-谷氨酸；HSS T3和XBridge Amide色谱柱同时应用实现了对极性不同的代谢物较全面的检测，初步鉴定到16种有机酸、17种核苷酸、21种氨基酸、66种脂肪酸、11种磷脂、7种鞘脂，XBridge Amide在分离检测磷脂和鞘脂类化合物方面具有明显优势；鉴定到的代谢物所涉及的代谢途径为嘌呤代谢、嘧啶代谢、三羧酸循环、精氨酸代谢、脂肪酸代谢、磷脂代谢、鞘脂代谢、苯丙氨酸代谢以及维生素B2的合成。两种色谱柱初步鉴定到的代谢物和涉及的代谢途径均存在一定的互补性，本方法可望用于水稻代谢表型的差异性研究。
A metabolic profiling analysis method for metabolomic studies of rice leaf was established based on HSS T3 combined with XBridge Amide Q-TOF LC/MS by comparing the influences of different extraction methods in rice leaves of metabolites. The extraction and separation of rice leaf metabolites using three different methods including methanol-chloroform-water, methanol-chloroform-ammonia, methanol-methyl tert-butyl ether -water and different chromatographic systems were compared by the numbers of peaks, identified metabolites and the metabolic pathways. The results showed that the method of methanol-chloroform-water reached the highest coverage rate of metabolites in rice leaves, and the maximum number of unique metabolites including prephenic acid, luteolin, α-linolenic acid, aconitic acid, gibberellin A12 aldehyde, isovitexin, L-Glutamate were detected. Metabolites with different polarity in rice leaf could be detected by HSS T3 and XBridge Amide. A total of 16 kinds of organic acids, 17 kinds of nucleotides, 21 kinds of amino acids, 66 kinds of fatty acids, 11 kinds of phospholipids and 7 kinds of sphingolipids were identified. XBridge Amide had an absolute advantage in detecting phospholipids and sphingolipids. The metabolic pathways involved purine metabolism, pyrimidine metabolism, tricarboxylic acid cycle, arginine metabolism, fatty acid metabolism, phospholipid metabolism, sphingolipid metabolism, phenylalanine metabolism and vitamin B2 synthesis. It showed certain complementarity between the two columns in identifying metabolites and involved the metabolic pathways. The established method is expected to be useful for the metabolomic studies of rice.
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