A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe.
A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe.
复制标题
通过共价固定 DNA 肽探针进行 miRNA 定量的准直接 LC-MS/MS 靶向蛋白质组学方法
DOI：
10.1038/s41598-017-05495-7
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发表时间：
2017-07-18
影响因子：
4.6
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Liu L;Xu Q;Hao S;Chen Y
microRNAs（miRNAs）在调节基因表达方面起着至关重要的作用，并且与包括乳腺癌在内的多种癌症相关。其畸变的、独特的表达是一种潜在的临床诊断和治疗标志物。因此，准确测定miRNA的表达水平是其应用的先决条件。然而，目前可用于miRNA检测的测定通常需要预富集、扩增和标记步骤，并且大多数测定仅是半定量的。因此，我们开发了一种基于准直接液相色谱-串联质谱（LC-MS/MS）的靶向蛋白质组学方法，通过共价固定的DNA-肽探针将miRNA信号转化为报告肽的质量响应来定量靶向miRNA。具体地，含有靶向蛋白质组学选择的底物/报告肽GDRAVQLGVDPFR/AVQLGVDPFR和与靶miRNA互补的DNA序列（即，miR-21）首先使用PDITC固定在APMTS修饰的二氧化硅纳米颗粒上。在固定的探针被识别并与靶miRNA杂交后，使用MBN降解过量的探针，然后用胰蛋白酶消化杂交体。释放报告肽并使用LC-MS/MS定量。获得的LOQ为5 pM。最后，开发的检测方法用于乳腺细胞和组织样本中miR-21的定量分析。
MicroRNAs (miRNAs) play a vital role in regulating gene expression and are associated with a variety of cancers, including breast cancer. Their distorted and unique expression is a potential marker in clinical diagnoses and prognoses. Thus, accurate determination of miRNA expression levels is a prerequisite for their applications. However, the assays currently available for miRNA detection typically require pre-enrichment, amplification and labeling steps, and most of the assays are only semi-quantitative. Therefore, we developed a quasi-direct liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics approach to quantify target miRNA by innovatively converting the miRNA signal into the mass response of a reporter peptide via a covalently immobilized DNA-peptide probe. Specifically, the probe containing the targeted proteomics-selected substrate/reporter peptide, GDRAVQLGVDPFR/AVQLGVDPFR, and the DNA sequence complementary to the target miRNA (i.e., miR-21) was first immobilized on APMTS modified silica nanoparticles using PDITC. After the immobilized probe was recognized and hybridized with the target miRNA, the excess probe was degraded using MBN and followed by a trypsin digestion of the hybrids. The reporter peptide was released and quantified using LC-MS/MS. The obtained LOQ was 5 pM. Finally, the developed assay was used for the quantitative analysis of miR-21 in breast cells and tissue samples.
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