Background

Tissue metabolomics analysis, alongside genomics and proteomics, offers crucial insights into the regulatory mechanisms of tumorigenesis. To enhance metabolite detection sensitivity, chemical isotope labeling (CIL) techniques, such as dansylation, have been developed to improve metabolite separation and ionization in mass spectrometry (MS). However, the dissolution of hydrophobic derivatized metabolites in solvents with high acetonitrile content limits the use of liquid chromatography (LC) systems with small-volume reversed-phase (RP) columns. In this study, we established a nano-LC-MS system with an online dilution design to address this issue, enabling sensitive analysis of oral cancer tissue metabolomes.

Results

Our nano-LC system features a flow path design with online sample dilution before an RP trap column and backflushing of the trap column before entering the analytical column. Compared to other nano-LC systems, both with and without online dilution designs, our system demonstrates the superiority of the T-connector-based dilution method. Using only 1/20th of the sample required for popular micro-LC systems, our nano-LC detects a larger number of peak pairs with similar recovery rates for both hydrophilic and hydrophobic metabolites, ensuring unbiased results. Thirty-two matched pairs of oral squamous cell carcinoma (OSCC) tissue samples and adjacent noncancerous tissues (ANTs) underwent high-throughput CIL-metabolome analysis using our nano-LC system. Compared to our previous micro-LC methods, the nano-LC-MS system exhibits enhanced detection sensitivity, significantly reducing sample requirements.

Significance

Our findings highlight the efficacy of our platform for metabolomic analysis with limited sample amounts. The nano-LC system's ability to analyze samples dissolved in strong eluents suggests potential applications for handling other hydrophobic compounds using RPLC or other separation methods facing similar solvent incompatibility issues. This approach holds promise for identifying novel metabolite biomarkers for oral cancers, advancing our understanding of tumorigenesis, and enhancing clinical applications.

中文翻译：

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增强型 nano-LC-MS 用于分析溶于高洗脱强度溶剂中的丹酰化口腔癌组织代谢组

 背景

组织代谢组学分析与基因组学和蛋白质组学一起，为肿瘤发生的调控机制提供了重要的见解。为了提高代谢物检测的灵敏度，已经开发了化学同位素标记 （CIL） 技术，例如丹酰化，以改善质谱 （MS） 中的代谢物分离和电离。然而，疏水性衍生代谢物在乙腈含量高的溶剂中的溶解限制了使用小容量反相 （RP） 色谱柱的液相色谱 （LC） 系统。在这项研究中，我们建立了一个具有在线稀释设计的 nano-LC-MS 系统来解决这个问题，从而能够对口腔癌组织代谢组进行灵敏分析。

 结果

我们的纳微液相色谱系统采用流路设计，在 RP 捕集柱之前在线稀释样品，并在进入分析柱之前对捕集柱进行反吹。与其他纳离子色谱系统相比，无论是否采用在线稀释设计，我们的系统都证明了基于 T 型连接器的稀释方法的优越性。我们的纳微液相色谱系统仅使用常用微量液相色谱系统所需样品的 1/20，即可检测到大量峰对，亲水性和疏水性代谢物的回收率相似，确保结果无偏差。使用我们的纳米液相色谱系统，对 32 对匹配的口腔鳞状细胞癌 （OSCC） 组织样品和相邻的非癌组织 （ANT） 进行了高通量 CIL 代谢组分析。与我们之前的微量液相色谱方法相比，纳粹 LC-MS 系统表现出更高的检测灵敏度，显著降低了样品要求。

 意义

我们的研究结果强调了我们的平台在有限样本量下进行代谢组学分析的有效性。纳流色谱系统能够分析溶解在强洗脱液中的样品，这表明使用 RPLC 或其他面临类似溶剂不相容性问题的分离方法处理其他疏水性化合物的潜在应用。这种方法有望识别口腔癌的新型代谢物生物标志物，促进我们对肿瘤发生的理解，并增强临床应用。