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.

中文翻译：

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DNA肽探针和液相色谱-串联质谱（LC-MS / MS）的组合：一种用于多重MicroRNA定量分析的准靶蛋白质组学方法

microRNA（miRNA）在癌症中的畸变和独特表达使其成为有吸引力的生物标志物来源。有许多证据表明，一组称为“ miRNA指纹”的miRNA比作为生物标志物的单个miRNA更具特异性和信息量。因此，用于同时定量多种miRNA的多重测定在临床实践中可能更为有效。然而，当前可用的测定法通常需要预富集，扩增和标记步骤，并且它们中的大多数是半定量的或缺乏多重能力。在这项研究中，我们通过结合DNA肽探针和液相色谱-串联质谱（LC-MS / MS），开发了用于多重miRNA定量的准目标蛋白质组学测定方法。具体而言，靶标miRNA（即miR-21，miR-let7a，miR-200c，通过将miRNA与DNA肽探针杂交并随后进行胰蛋白酶消化以释放肽，将miR-125a和miR-15b）转化为报告肽的质量反应。在仔细优化与结合，结合，杂交和多反应监测（MRM）检测相关的条件后，对每种miRNA的检测方法进行了验证，所有miRNA的定量限（LOQ）均可达到1 pM。此外，对复杂试验中DNA肽探针之间的串扰进行了精确评估。使用这种准靶向蛋白质组学测定法，可以测定3种人类乳腺细胞系和36对匹配的乳腺组织样本中的靶miRNA水平。最后，还进行了单纯检测和qRT-PCR进行比较。