Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Psychology, University of Michigan, Ann Arbor, Michigan 48109, United States.
Anal Chem. 2024 Oct 15;96(41):16387-16396. doi: 10.1021/acs.analchem.4c03806. Epub 2024 Oct 3.
Metabolomic analysis of samples acquired from the brain extracellular space by microdialysis sampling can provide insights into chemical underpinnings of a given brain state and how it changes over time. Small sample volumes and low physiological concentrations have limited the identification of compounds from this compartment, so at present, we have scant knowledge of its composition. As a result, most measurements have limited depth of analysis. Here, we describe an approach to (1) identify hundreds of compounds in brain dialysate and (2) routinely detect many of these compounds in 5 μL microdialysis samples to enable deep monitoring of brain chemistry in time-resolved studies. Dialysate samples collected over 12 h were concentrated 10-fold and then analyzed using liquid chromatography with iterative tandem mass spectrometry (LC-MS/MS). Using this approach on dialysate from the rat striatum with both reversed-phase and hydrophilic interaction liquid chromatography yielded 479 unique compound identifications. 60% of the identified compounds could be detected in 5 μL of dialysate without further concentration using a single 20 min LC-MS analysis, showing that once identified, most compounds can be detected using small sample volumes and shorter analysis times compatible with routine monitoring. To detect more neurochemicals, LC-MS analysis of dialysate derivatized with light and isotopically labeled benzoyl chloride was employed. 872 nondegenerate benzoylated features were detected with this approach, including most small-molecule neurotransmitters and several metabolites involved in dopamine metabolism. This strategy allows deeper annotation of the brain extracellular space than previously possible and provides a launching point for defining the chemistry underlying brain states.
通过微透析取样从大脑细胞外空间获取的样本的代谢组学分析，可以深入了解特定大脑状态的化学基础以及其随时间的变化。由于样本体积小且生理浓度低，限制了从该隔室中鉴定化合物，因此目前我们对其组成知之甚少。因此，大多数测量的分析深度有限。在这里，我们描述了一种方法，（1）鉴定脑透析液中的数百种化合物，（2）常规检测 5μL 微透析样本中的许多这些化合物，以实现对时间分辨研究中大脑化学的深度监测。在 12 小时内收集的透析液浓缩 10 倍，然后使用具有反相和亲水相互作用液相色谱的液相色谱与迭代串联质谱法（LC-MS/MS）进行分析。使用这种方法对大鼠纹状体的透析液进行分析，使用反相和亲水相互作用液相色谱法可鉴定出 479 种独特的化合物。在不进一步浓缩的情况下，5μL 透析液中可检测到 60%的鉴定化合物，使用单次 20 分钟 LC-MS 分析即可检测到，这表明一旦鉴定出，大多数化合物可以使用小体积的样本和更短的分析时间进行检测，与常规监测兼容。为了检测更多的神经化学物质，使用用轻和同位素标记的苯甲酰氯衍生的透析液进行 LC-MS 分析。使用这种方法检测到 872 个非简并的苯甲酰化特征，包括大多数小分子神经递质和几种参与多巴胺代谢的代谢物。这种策略允许对大脑细胞外空间进行比以前更深入的注释，并为定义大脑状态的化学基础提供了起点。
