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Novel Stable Isotope-Resolved Metabolomics Method for a Small Number of Cells Using Chip-Based Nanoelectrospray Mass Spectrometry
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-10-04 , DOI: 10.1021/acs.analchem.1c01507 Di Yu 1, 2 , Fujian Zheng 1, 2 , Lichao Wang 1 , Chao Li 3 , Xin Lu 1 , Xiaohui Lin 3 , Lina Zhou 1 , Guowang Xu 1, 2
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-10-04 , DOI: 10.1021/acs.analchem.1c01507 Di Yu 1, 2 , Fujian Zheng 1, 2 , Lichao Wang 1 , Chao Li 3 , Xin Lu 1 , Xiaohui Lin 3 , Lina Zhou 1 , Guowang Xu 1, 2
Affiliation
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Stable isotope-resolved metabolomics (SIRM) can provide metabolic conversion information of specific targets; it is a powerful tool for cell metabolism studies. The common analytical platform for SIRM is chromatography-mass spectrometry, which requires a large number of cells and is not suitable for precious rare cell analysis. To study a small number of cells, we established a novel SIRM method using chip-based nanoelectrospray mass spectrometry (MS). 13C-glutamine was taken as an example; the unlabeled and 13C-labeled cells were cultured and extracted in a 96-well plate and then directly injected into MS and analyzed in full scan mode and parallel reaction monitoring (PRM) mode targeting 44 glutamine-derived metabolites and their isotopologues. To define focused metabolite-related MS2 fragments produced in the PRM, a new strategy was proposed including MS2 exact m/z matching, MS2 false positive filtering, and MS2 fragment grouping to remove the interfering MS2 ions. In total, 292 and 349 pairs of paired MS2 ions were obtained in positive and negative ionization modes, respectively. By searching spectra databases, 31 targeted metabolites with their isotopologues were identified and their characteristic product ions were confirmed for MS2 quantification. The relative quantification was achieved by MS2 quantification, which showed better sensitivity and accuracy than common MS1-based quantification. Finally, this method was applied to isocitrate dehydrogenase I-mutated glioma cells for revealing the effects of triptolide on glioma cell metabolism using U-13C-glutamine as a labeling substrate.
中文翻译：
使用基于芯片的纳米电喷雾质谱法对少量细胞进行新型稳定同位素解析代谢组学方法
稳定同位素分辨代谢组学（SIRM）可以提供特定靶点的代谢转化信息；它是细胞代谢研究的有力工具。SIRM常用的分析平台是层析-质谱，需要大量细胞，不适合珍贵稀有细胞分析。为了研究少量细胞，我们使用基于芯片的纳米电喷雾质谱 (MS) 建立了一种新的 SIRM 方法。以13 C-谷氨酰胺为例；未标记的和13在 96 孔板中培养和提取 C 标记的细胞，然后直接注射到 MS 中，并以全扫描模式和平行反应监测 (PRM) 模式针对 44 种谷氨酰胺衍生代谢物及其同位素体进行分析。为了定义 PRM 中产生的与代谢物相关的重点 MS2 片段，提出了一种新策略，包括 MS2 精确m / z匹配、MS2 误报过滤和 MS2 碎片分组以去除干扰 MS2 离子。在正离子和负离子模式下，总共分别获得了 292 和 349 对配对 MS2 离子。通过搜索光谱数据库，确定了 31 种目标代谢物及其同位素体，并确认了它们的特征产物离子用于 MS2 定量。相对定量是通过 MS2 定量实现的，其显示出比常见的基于 MS1 的定量更好的灵敏度和准确度。最后，将该方法应用于异柠檬酸脱氢酶 I 突变的神经胶质瘤细胞，以使用 U- 13 C-谷氨酰胺作为标记底物揭示雷公藤内酯对神经胶质瘤细胞代谢的影响。
更新日期：2021-10-19
中文翻译：
使用基于芯片的纳米电喷雾质谱法对少量细胞进行新型稳定同位素解析代谢组学方法
稳定同位素分辨代谢组学（SIRM）可以提供特定靶点的代谢转化信息；它是细胞代谢研究的有力工具。SIRM常用的分析平台是层析-质谱，需要大量细胞，不适合珍贵稀有细胞分析。为了研究少量细胞，我们使用基于芯片的纳米电喷雾质谱 (MS) 建立了一种新的 SIRM 方法。以13 C-谷氨酰胺为例；未标记的和13在 96 孔板中培养和提取 C 标记的细胞，然后直接注射到 MS 中，并以全扫描模式和平行反应监测 (PRM) 模式针对 44 种谷氨酰胺衍生代谢物及其同位素体进行分析。为了定义 PRM 中产生的与代谢物相关的重点 MS2 片段，提出了一种新策略，包括 MS2 精确m / z匹配、MS2 误报过滤和 MS2 碎片分组以去除干扰 MS2 离子。在正离子和负离子模式下，总共分别获得了 292 和 349 对配对 MS2 离子。通过搜索光谱数据库，确定了 31 种目标代谢物及其同位素体，并确认了它们的特征产物离子用于 MS2 定量。相对定量是通过 MS2 定量实现的，其显示出比常见的基于 MS1 的定量更好的灵敏度和准确度。最后，将该方法应用于异柠檬酸脱氢酶 I 突变的神经胶质瘤细胞，以使用 U- 13 C-谷氨酰胺作为标记底物揭示雷公藤内酯对神经胶质瘤细胞代谢的影响。
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