Capillary LC-MS for high sensitivity metabolomic analysis of single islets of Langerhans (Retracted Article. See vol 81, pg 9201, 2009)
Capillary LC-MS for high sensitivity metabolomic analysis of single islets of Langerhans (Retracted Article. See vol 81, pg 9201, 2009)
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DOI：
10.1021/ac800406f
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发表时间：
2008-05-15
影响因子：
7.4
通讯作者：
Kennedy, Robert T.
中科院分区：
文献类型：
--
作者：
Ni, Qihui;Reid, Kendra R.;Kennedy, Robert T.
以朗格汉斯胰岛为模型系统，探讨了电喷雾电离-质谱联用的反相填充毛细管液相色谱分析方法在微量组织样品中代谢物的测定。在全扫描模式下，采用75 μ m内径柱与四极离子阱质谱联用，糖酵解和三羧酸循环代谢物的检出限为0.1 ~ 33 fmol。通过快速冷冻和甲醇-水萃取对胰岛进行可重复处理，代谢产物损失小。该方法产生20 μ L的提取物，其中仅注射了15 μ L，这表明在同一胰岛上进行多次检测的潜力。可检测到约200种推定代谢物，其中22种通过保留时间和MS/MS光谱与标准物匹配鉴定。峰检测的相对标准偏差在7 - 18%之间，保存11天不受影响。该方法用于检测与细胞外胰岛葡萄糖浓度从3到20 mM增加相关的代谢变化，所得结果与已知的胰岛代谢基本一致。由于之前大多数关于胰岛代谢的研究一次只观察到少数化合物，并且需要更多的组织，因此这种测量方法代表了研究胰岛代谢和其他微尺度样本的重大进步。
Reversed-phase, packed capillary liquid chromatography interfaced by electrospray ionization to mass spectrometry was explored as an analytical method for determination of metabolites in microscale tissue samples using single islets of Langerhans as a model system. With the use of a 75 mu m inner diameter column coupled to a quadrupole ion trap mass spectrometer in full scan mode, detection limits of 0.1-33 fmol were achieved for glycoloytic and tricarboxylic acid cycle metabolites. Reproducible processing of islets for analysis with little loss of metabolites was performed by rapid freezing followed by methanol-water extraction. The method yielded 20 mu L of extract of which just 15 nL was injected suggesting the potential for performing multiple assays on the same islet. Approximately 200 presumed metabolites could be detected, of which 22 were identified by matching retention times and MS/MS spectra to standards. Relative standard deviations for peak detection was from 7 to 18% and was unaffected by storage for up to 11 days. The method was used to detect changes in metabolism associated with increasing extracellular islet glucose concentration from 3 to 20 mM yielding results largely consistent with known metabolism of islets. Because most previous studies of islet metabolism have only observed a few compounds at once and require far more tissue, this measurement method represents a significant advance for studies of metabolism of islets and other microscale samples.