High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics.
High-Performance Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Exosome Metabolomics.
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DOI：
10.1021/acs.analchem.8b01726
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发表时间：
2018-07-17
影响因子：
7.4
通讯作者：
Li L
中科院分区：
文献类型：
--
作者：
Luo X;An M;Cuneo KC;Lubman DM;Li L
体液（如血液）中的循环外泌体作为癌症诊断和监测的化学生物标志物的丰富来源正在被积极研究。尽管核酸分析是发现外泌体中循环生物标志物的主要工具，但代谢组学具有扩大生物标志物化学多样性的潜力，这些生物标志物可能易于快速检测。然而，只有微量的外泌体可以从少量患者血液中分离出来，因此需要一种非常敏感的技术来分析外泌体的代谢组。在本报告中，我们提出了一个工作流程，包括使用2ml人血清作为起始材料进行超离心分离外泌体的多个循环，在50%甲醇/水中进行冻融循环进行外泌体裂解和代谢物提取，代谢物的差异化学同位素标记（CIL）以加强液相色谱（LC）分离和改进质谱（MS）检测，以及使用captivspray进行纳米流LC-MS （nLC-MS）分析。作为一项原理证明，我们使用丹酰化标记分析了从5名胰腺癌患者化疗前后的血液样本中分离的两组外泌体样本中的胺和酚亚代谢组。第一组样品平均检测到的峰对或代谢物数为1964±60个，共检测到2446对峰对（n = 10）；第二组样品平均检测到1948±117个，共检测到2511对峰对（n = 10）。在第一组和第二组中分别鉴定出101和94个代谢物，其中1580和1590个峰对的质量与MyCompoundID代谢组数据库中代谢物的质量精确匹配。分析12c标记的单个外泌体样本与13c标记的混合样本（作为内标）的混合物，可以相对量化治疗前后采集的血液样本外泌体的代谢组学变化。
Circulating exosomes in bodily fluids such as blood are being actively studied as a rich source of chemical biomarkers for cancer diagnosis and monitoring. Although nucleic acid analysis is a primary tool for the discovery of circulating biomarkers in exosomes, metabolomics holds the potential of expanding the chemical diversity of biomarkers that may be easy and rapid to detect. However, only trace amounts of exosomes can be isolated from a small volume of patient blood, and thus a very sensitive technique is required to analyze the metabolome of exosomes. In this report, we present a workflow that involves multiple cycles of ultracentrifugation for exosome isolation using a starting material of 2 mL of human serum, freeze – thaw-cycles in 50% methanol/water for exosome lysis and metabolite extraction, differential chemical isotope labeling (CIL) of metabolites for enhancing liquid chromatography (LC) separation and improving mass spectrometry (MS) detection, and nanoflow LC-MS (nLC-MS) with captivespray for analysis. As a proof-of-principle, we used dansylation labeling to analyze the amine- and phenol-submetabolomes in two sets of exosome samples isolated from the blood samples of five pancreatic cancer patients before and after chemotherapy treatment. The average number of peak pairs or metabolites detected was 1964 ± 60 per sample for a total of 2446 peak pairs (n = 10) in the first set and 1948 ± 117 per sample for a total of 2511 peak pairs (n = 10) in the second set. There were 101 and 94 metabolites positively identified in the first and second set, respectively, and 1580 and 1590 peak pairs with accurate masses matching those of metabolites in the MyCompoundID metabolome database. Analyzing the mixtures of 12C-labeled individual exosome samples spiked with a 13C-labeled pooled sample which served as an internal standard allowed relative quantification of metabolomic changes of exosomes of blood samples collected before and after treatment.
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影响因子：
9.7
作者：
Lai X;Wang M;McElyea SD;Sherman S;House M;Korc M
通讯作者：
Korc M
影响因子：
2.9
作者：
Kim J;Tan Z;Lubman DM
通讯作者：
Lubman DM
影响因子：
3.7
作者：
Altadill, Tatiana;Campoy, Irene;Cheema, Amrita K.
通讯作者：
Cheema, Amrita K.
影响因子：
3.7
作者：
Buratta S;Urbanelli L;Sagini K;Giovagnoli S;Caponi S;Fioretto D;Mitro N;Caruso D;Emiliani C
通讯作者：
Emiliani C
影响因子：
4.4
作者：
An M;Zhu J;Wu J;Cuneo KC;Lubman DM
通讯作者：
Lubman DM