Department of Chemistry , Purdue University , West Lafayette , Indiana 47907 , United States.
Physical Sciences Division , Pacific Northwest National Laboratory , Richland Washington 99352 , United States.
Anal Chem. 2018 Jul 3;90(13):7937-7945. doi: 10.1021/acs.analchem.8b00551. Epub 2018 Jun 18.
Quantitative live cell mass spectrometry analysis at a subcellular level requires the precisely controlled extraction of subpicoliter volumes of material from the cell, sensitive analysis of the extracted analytes, and their accurate quantification without prior separation. In this study, we demonstrate that localized electroosmotic extraction provides a direct path to addressing this challenge. Specifically, we demonstrate quantitative mass spectrometry analysis of biomolecules in picoliter volumes extracted from live cells. Electroosmotic extraction was performed using two electrodes and a finely pulled nanopipette with tip diameter of <1 μm containing a hydrophobic electrolyte compatible with mass spectrometry analysis. The electroosmotic drag was used to drive analytes out of the cell into the nanopipette. Analyte molecules extracted both from solutions and cell samples were analyzed using nanoelectrospray ionization (nanoESI) directly from the nanopipette into a mass spectrometer. More than 50 metabolites including sugars and flavonoids were detected in positive mode in 2-5 pL volumes of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential extraction of a known volume of the aqueous solution containing glucose- d standard of known concentration. We found that the ratio of the signal of glucose to glucose- d increased linearly with glucose concentration. This observation indicates that the approach developed in this study enables quantitative analysis of small volumes of metabolites extracted from cells. Furthermore, we observed efficient separation of hydrophilic and hydrophobic analytes through partitioning into the aqueous and hydrophobic electrolyte phase, respectively, which provides additional important information on the molecular properties of extracted metabolites.
在亚细胞水平进行定量活细胞质谱分析需要精确控制从小体积（亚皮升）的细胞中提取物质，对提取的分析物进行灵敏分析，并在无需预先分离的情况下对其进行准确量化。在本研究中，我们证明局部电渗流提取为解决这一挑战提供了直接途径。具体而言，我们证明了从小鼠肝脏细胞中提取的皮升体积的生物分子的定量质谱分析。电渗流提取使用两个电极和一个具有 <1 μm 尖端直径的精细拉制的纳米吸管进行，该纳米吸管中含有一种与质谱分析兼容的疏水电解质。电渗流拖曳用于将分析物从细胞中拖出到纳米吸管中。从溶液和细胞样品中提取的分析物分子直接从纳米吸管通过纳喷雾电离（nanoESI）进入质谱仪进行分析。从洋葱细胞的细胞质中提取的 2-5 pL 体积的细胞内物质中检测到 50 多种代谢物，包括糖和类黄酮。通过顺序提取含有已知浓度葡萄糖- d 标准品的已知体积的水溶液来进行提取葡萄糖的定量分析。我们发现葡萄糖信号与葡萄糖- d 的比值随葡萄糖浓度呈线性增加。这一观察结果表明，本研究中开发的方法可用于对从小鼠肝脏细胞中提取的代谢物的小体积进行定量分析。此外，我们观察到亲水和疏水分析物通过分别分配到水相和疏水电解质相而实现有效的分离，这为提取代谢物的分子特性提供了重要的附加信息。
