Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers
Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers
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DOI：
10.1074/mcp.m116.064758
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发表时间：
2017-05-01
影响因子：
7
通讯作者：
Yang, Wei-Ting Ou
中科院分区：
文献类型：
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作者：
Chen, Yi-Ting;Chen, Hsiao-Wei;Yang, Wei-Ting Ou
多肽的多重（选择）反应监测（MRM/SRM）是一种不断发展的靶蛋白定量技术，因为它比基于抗体的技术更稳健、精确、准确、高通量和多重功能。该技术已在临床上应用于不同类型液体中多种靶蛋白的大规模定量。然而，以前基于磁共振成像的研究很少关注样品制备工作流程和唾液中蛋白质的精确定量分析性能，唾液是一种非侵入性的采样体液。在这项研究中，我们评估了一种简单而稳健的基于多反应监测（MRM）的靶向蛋白质组学方法，该方法结合了液相色谱和质谱检测（LC-MRM/MS）。该平台用于定量评估一组56种唾液蛋白的生物标志物潜力，这些蛋白先前与人类癌症相关。为了进一步加强该技术在唾液样品分析中的发展，我们优化了唾液蛋白消化的工作流程，并评估了分析临床样品的定量性能、稳健性和技术局限性。使用临床特征良好的两个独立临床样本组（总n = 119），我们定量表征了对照组和口腔鳞状细胞癌（OSCC）患者唾液标本中的这些蛋白质生物标志物候选物。结果清楚地显示，与对照组相比，OSCC患者唾液样本中大多数靶向蛋白显著升高。总体而言，该平台能够分析最高度复用的唾液蛋白生物标志物，突出了MRM在口腔癌生物标志物研究中的临床应用。
Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research.