A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification.
A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification.
复制标题
DNA 肽探针与液相色谱-串联质谱 (LC-MS/MS) 的组合：多重 MicroRNA 定量的准靶向蛋白质组学方法
作者：
Xu F;Zhou W;Cao J;Xu Q;Jiang D;Chen Y
癌症中 microRNA (miRNA) 的扭曲和独特表达使其成为有吸引力的生物标志物来源。有大量证据表明，一组 miRNA（称为“miRNA 指纹”）比单个 miRNA 作为生物标志物更具特异性和信息量。因此，同时定量多个 miRNA 的多重检测在临床实践中可能更有效。然而，目前可用的检测通常需要预富集、扩增和标记步骤，并且大多数是半定量的或缺乏多重检测能力。在这项研究中，我们开发了一种准靶向蛋白质组学检测方法，通过结合 DNA-肽探针和液相色谱-串联质谱 (LC-MS/MS) 来进行多重 miRNA 定量。具体来说，通过 miRNA 与 DNA 肽探针杂交以及随后的胰蛋白酶消化以释放肽，将目标 miRNA（即 miR-21、miR-let7a、miR-200c、miR-125a 和 miR-15b）的信号转化为报告肽的质量响应。经过仔细优化与结合、缀合、杂交和多反应监测 (MRM) 检测相关的条件后，对每个 miRNA 的测定进行了验证，所有 miRNA 的定量限 (LOQ) 均可达到 1 pM。此外，还对多重测定中 DNA-肽探针之间的串扰进行了复杂的评估。使用这种准靶向蛋白质组学测定，在 3 个人类乳腺细胞系和 36 对匹配的乳腺组织样本中测定了目标 miRNA 的水平。最后，还进行了单纯法测定和qRT-PCR进行比较。该方法将靶向蛋白质组学策略移植到 miRNA 定量中，并可能为多重 miRNA 分析提供新的方法。
The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarker. There is much evidence indicating that a panel of miRNAs, termed “miRNA fingerprints”, is more specific and informative than an individual miRNA as biomarker. Thus, multiplex assays for simultaneous quantification of multiple miRNAs could be more potent in clinical practice. However, current available assays normally require pre-enrichment, amplification and labeling steps, and most of them are semi-quantitative or lack of multiplexing capability. In this study, we developed a quasi-targeted proteomics assay for multiplexed miRNA quantification by a combination of DNA-peptide probes and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, the signal of target miRNAs (i.e., miR-21, miR-let7a, miR-200c, miR-125a and miR-15b) was converted into the mass response of reporter peptides by hybridization of miRNAs with DNA-peptide probes and subsequent tryptic digestion to release the peptides. After a careful optimization of conditions related to binding, conjugation, hybridization and multiple reaction monitoring (MRM) detection, the assay was validated for each miRNA and the limit of quantification (LOQ) for all the miRNAs can achieve 1 pM. Moreover, crosstalk between DNA-peptide probes in multiplex assay was sophisticatedly evaluated. Using this quasi-targeted proteomics assay, the level of target miRNAs was determined in 3 human breast cell lines and 36 matched pairs of breast tissue samples. Finally, simplex assay and qRT-PCR were also performed for a comparison. This approach grafts the strategy of targeted proteomics into miRNA quantification and may offer a new way for multiplexed miRNA profiling.