Poly(ethylene glycol)-Based Stable Isotope Labeling Reagents for the Quantitative Analysis of Low Molecular Weight Metabolites by LC-MS
Poly(ethylene glycol)-Based Stable Isotope Labeling Reagents for the Quantitative Analysis of Low Molecular Weight Metabolites by LC-MS
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DOI：
10.1021/ac801215c
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发表时间：
2008-12-01
影响因子：
7.4
通讯作者：
Bischoff, Rainer
中科院分区：
文献类型：
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作者：
Abello, Nicolas;Geurink, Paul P.;Bischoff, Rainer
稳定同位素标记（SIL）与液相色谱-质谱联用是目前应用最广泛的定量分析方法之一，因为其灵敏度高，能够处理极其复杂的生物样品。然而，用于代谢物分析的SIL方法仍然经常在多路复用、衍生分析物的色谱特性或其电离效率方面受到限制。在这里，我们描述了一个新的家庭的试剂的SIL的含伯胺的化合物的基础上五氟苯基活化酯的C-13-含聚（乙二醇）链（PEG），解决了这些缺点。PEG链的连续积累允许引入各种数目的C-13原子，从而打开了扩展的多路复用可能性。PEG衍生物，而亲水性分子，如氨基酸和谷胱甘肽被成功地保留在一个标准的C-18反相柱，其鉴定是促进基于m/z值和保留时间，使用提取的离子色谱图。由于PEG衍生化导致的质量增加将低分子量代谢物信号移出质谱的通常有噪声的低m/z区域，这导致总体灵敏度和选择性增强。此外，由于移动的相中乙腈含量较高，在增加的保留时间下洗脱导致有效的电喷雾电离。该方法已成功地应用于定量细胞内的氨基酸和谷胱甘肽在暴露于香烟烟雾诱导的氧化应激的人肺上皮细胞模型。结果表明，相对于空气对照和香烟烟雾提取物，当A549细胞暴露于气相香烟烟雾时，大多数氨基酸的浓度增加，并且游离的含巯基物质（例如，谷胱甘肽）减少，但二硫键形成没有增加。这些标记试剂也应该证明可用于标记肽和其他含有伯胺官能团的化合物。
Stable isotope labeling (SIL) in combination with liquid chromatography-mass spectrometry is one of the most widely used quantitative analytical methods due to its sensitivity and ability to deal with extremely complex biological samples. However, SIL methods for metabolite analysis are still often limited in terms of multiplexing, the chromatographic properties of the derivatized analytes, or their ionization efficiency. Here we describe a new family of reagents for the SIL of primary amine-containing compounds based on pentafluorophenyl-activated esters of C-13-containing poly(ethylene glycol) chains (PEG) that addresses these shortcomings. A sequential buildup of the PEG chain allowed the introduction of various numbers of C-13 atoms opening extended multiplexing possibilities. The PEG derivatives of rather hydrophilic molecules such as amino acids and glutathione were successfully retained on a standard C-18 reversed-phase column, and their identification was facilitated based on m/z values and retention times using extracted ion chromatograms. The mass increase due to PEG derivatization moved low molecular weight metabolite signals out of the often noisy, low m/z region of the mass spectra, which resulted in enhanced overall sensitivity and selectivity. Furthermore, elution at increased retention times resulted in efficient electrospray ionization due to the higher acetonitrile content in the mobile phase. The method was successfully applied to the quantification of intracellular amino acids and glutathione in a cellular model of human lung epithelium exposed to cigarette smoke-induced oxidative stress. It was shown that the concentration of most amino acids increased upon exposure of A549 cells to gas-phase cigarette smoke with respect to air control and cigarette smoke extract and that free thiol-containing species (e.g., glutathione) decreased although disulfide bond formation was not increased. These labeling reagents should also prove useful for the labeling of peptides and other compounds containing primary amine functionalities.