College of Chemistry, Sichuan University, Chengdu, 610065, China.
Anal Bioanal Chem. 2013 Oct;405(25):8131-6. doi: 10.1007/s00216-013-7260-z. Epub 2013 Aug 9.
A fast microchip electrophoresis-nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 μM. Limits of detection were found to be 0.37 μM for Asp and 0.33 μM for Glu (S/N = 3). Assay repeatability (relative standard deviation, n = 6) was 4.2 and 4.5%, for Asp and Glu at 5.0 μM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.
建立了一种快速微芯片电泳-纳喷雾电离-质谱联用（MCE-nanoESI-MS）方法，用于分析生物样品中的氨基酸。该平台采用了带有整体纳喷雾发射器的玻璃/聚二甲基硅氧烷混合微芯片。所提出的 MCE-nanoESI-MS 分析方法对氨基酸具有高分离效率。包含 Lys、Arg、Val、Tyr 和 Glu 的氨基酸混合物在 120 s 内完成基线分离，理论塔板数>7500。该方法用于研究化学刺激下细胞释放兴奋性氨基酸（即天冬氨酸（Asp）和谷氨酸（Glu））。在 1.00 至 150.0 μM 的浓度范围内，均获得了 Asp 和 Glu 的线性校准曲线。Asp 和 Glu 的检测限分别为 0.37 μM 和 0.33 μM（S/N = 3）。在 5.0 μM 时，Asp 和 Glu 的测定重复性（相对标准偏差，n = 6）分别为 4.2%和 4.5%。在细胞释放研究中，将 PC-12 神经细胞在不同浓度的酒精中孵育 1 小时。通过所提出的方法测量细胞内外的 Asp 和 Glu 水平。结果清楚地表明，乙醇促进了细胞中外源和内源 Asp 和 Glu 的释放。
