背景:秀丽隐杆线虫中的极性代谢物(C.秀丽隐杆线虫)主要使用亲水相互作用液相色谱与质谱联用(HILIC-MS)进行分析。与质谱联用的毛细管电泳(CE-MS)代表另一种适用于极性和带电分析物的互补分析平台。
目的:我们比较了CE-MS和HILIC-MS,以分析与秀丽隐杆线虫相关的60种参考标准,并特别研究了CE分离的优势。此外,我们采用CE-MS作为补充分析方法来研究秀丽隐杆线虫样品中的极性代谢物,特别是在长寿的背景下,以解决其代谢组的不同部分。
方法:我们使用HILIC-MS和CE-MS使用Q-ToF-MS仪器分析了来自秀丽隐杆线虫daf-2功能丧失突变体和野生型(WT)样品的60个参考标准以及代谢物提取物。
结果:CE分离显示较窄的峰宽和在不同浓度下估计的响应函数的更好的线性,这与MS信号的较低饱和度有关。此外,与HILIC-MS相比,CE在化合物的分离中表现出明显的选择性,为目标化合物的分析提供补充信息。对daf-2突变体和WT样品的秀丽隐杆线虫代谢产物的分析揭示了通过HILIC-MS鉴定的共有代谢产物的显著改变。以及不同代谢物的存在。
结论:CE-MS已成功应用于秀丽隐杆线虫代谢组学,能够恢复已知的以及识别新的推定的长寿生物标志物。
BACKGROUND: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes.
OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome.
METHODS: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument.
RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites.
CONCLUSIONS: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.