A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe

Abstract 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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Liang Liu, Qingqing Xu, Shuai Hao, Yun Chen
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/60fe382e5c5c426cae8c6a6f5e41945a
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:60fe382e5c5c426cae8c6a6f5e41945a
record_format	dspace
spelling	oai:doaj.org-article:60fe382e5c5c426cae8c6a6f5e41945a2021-12-02T16:08:24ZA Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe10.1038/s41598-017-05495-72045-2322https://doaj.org/article/60fe382e5c5c426cae8c6a6f5e41945a2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-05495-7https://doaj.org/toc/2045-2322Abstract 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.Liang LiuQingqing XuShuai HaoYun ChenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-12 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Liang Liu Qingqing Xu Shuai Hao Yun Chen A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe
description	Abstract 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.
format	article
author	Liang Liu Qingqing Xu Shuai Hao Yun Chen
author_facet	Liang Liu Qingqing Xu Shuai Hao Yun Chen
author_sort	Liang Liu
title	A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe
title_short	A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe
title_full	A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe
title_fullStr	A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe
title_full_unstemmed	A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe
title_sort	quasi-direct lc-ms/ms-based targeted proteomics approach for mirna quantification via a covalently immobilized dna-peptide probe
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/60fe382e5c5c426cae8c6a6f5e41945a
work_keys_str_mv	AT liangliu aquasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT qingqingxu aquasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT shuaihao aquasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT yunchen aquasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT liangliu quasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT qingqingxu quasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT shuaihao quasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe AT yunchen quasidirectlcmsmsbasedtargetedproteomicsapproachformirnaquantificationviaacovalentlyimmobilizeddnapeptideprobe
_version_	1718384471963598848

A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe

Ejemplares similares