Sah Samyukta, Yun Sylvia R, Gaul David A, Botros Andro, Park Eun Young, Kim Olga, Kim Jaeyeon, Fernández Facundo M
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Metabolites. 2022 Jun 9;12(6):532. doi: 10.3390/metabo12060532.
The lack of effective screening strategies for high-grade serous carcinoma (HGSC), a subtype of ovarian cancer (OC) responsible for 70-80% of OC related deaths, emphasizes the need for new diagnostic markers and a better understanding of disease pathogenesis. Capillary electrophoresis (CE) coupled with high-resolution mass spectrometry (HRMS) offers high selectivity and sensitivity for ionic compounds, thereby enhancing biomarker discovery. Recent advances in CE-MS include small, chip-based CE systems coupled with nanoelectrospray ionization (nanoESI) to provide rapid, high-resolution analysis of biological specimens. Here, we describe the development of a targeted microchip (µ) CE-HRMS method, with an acquisition time of only 3 min and sample injection volume of 4nL, to analyze 40 target metabolites in serum samples from a triple-mutant (TKO) mouse model of HGSC. Extracted ion electropherograms showed sharp, baseline resolved peak shapes, even for structural isomers such as leucine and isoleucine. All calibration curves of the analytes maintained good linearity with an average R of 0.994, while detection limits were in the nM range. Thirty metabolites were detected in mouse serum with recoveries ranging from 78 to 120%, indicating minimal ionization suppression and good accuracy. We applied the µCE-HRMS method to biweekly-collected serum samples from TKO and TKO control mice. A time-resolved analysis revealed characteristic temporal trends for amino acids, nucleosides, and amino acid derivatives. These metabolic alterations are indicative of altered nucleotide biosynthesis and amino acid metabolism in HGSC development and progression. A comparison of the µCE-HRMS dataset with non-targeted ultra-high performance liquid chromatography (UHPLC)-MS results showed identical temporal trends for the five metabolites detected with both platforms, indicating the µCE-HRMS method performed satisfactorily in terms of capturing metabolic reprogramming due to HGSC progression while reducing the total data collection time three-fold.
高级别浆液性癌（HGSC）是卵巢癌（OC）的一种亚型，占OC相关死亡的70 - 80%，缺乏有效的筛查策略凸显了对新诊断标志物的需求以及对疾病发病机制的更好理解。毛细管电泳（CE）与高分辨率质谱（HRMS）联用对离子化合物具有高选择性和灵敏度，从而有助于生物标志物的发现。CE - MS的最新进展包括基于芯片的小型CE系统与纳米电喷雾电离（nanoESI）联用，可对生物样本进行快速、高分辨率分析。在此，我们描述了一种靶向微芯片（µ）CE - HRMS方法的开发，该方法进样时间仅为3分钟，进样体积为4nL，用于分析HGSC三突变（TKO）小鼠模型血清样本中的40种目标代谢物。提取离子电泳图显示出尖锐的、基线分离的峰形，即使对于亮氨酸和异亮氨酸等结构异构体也是如此。所有分析物的校准曲线均保持良好的线性，平均R为0.994，而检测限在纳摩尔范围内。在小鼠血清中检测到30种代谢物，回收率在78%至120%之间，表明电离抑制最小且准确性良好。我们将µCE - HRMS方法应用于每两周收集一次的TKO和TKO对照小鼠的血清样本。时间分辨分析揭示了氨基酸、核苷和氨基酸衍生物的特征性时间趋势。这些代谢改变表明HGSC发生和发展过程中核苷酸生物合成和氨基酸代谢发生了改变。将µCE - HRMS数据集与非靶向超高效液相色谱（UHPLC）- MS结果进行比较，结果显示两个平台检测到的五种代谢物具有相同的时间趋势，这表明µCE - HRMS方法在捕捉HGSC进展导致的代谢重编程方面表现令人满意，同时将总数据收集时间缩短了三倍。
