跳至主要内容

A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification

文献作者：Feifei Xu, Weixian Zhou, Jianxiang Cao, Qingqing Xu, Dechen Jiang and Yun Chen   《THERANOSTICS》

ABSTRACTS
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.
KEY WORDS
Quasi-Targeted Proteomics, MicroRNAs, Multiplex Assay, Reporter Peptides, DNA-peptide Probes, Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).
SCREENSHOT

RELATED PRODUCTS
Peptides including maleimide-modified substrate peptide, reporter peptide and internal standard containing stable-isotope labeled amino acids were developed by ChinaPeptides Co., Ltd. (Shanghai, China).
CHAINING
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562220/
标签：

评论

发表评论

此博客中的热门博文

The Catalytically Inactive Mutation of the Ubiquitin-Conjugating Enzyme CDC34 Affects its Stability and Cell Proliferation

Author：Xun Liu, Yang Zhang, Zhanhong Hu, Qian Li, Lu Yang,  Guoqiang Xu     《The Protein Journal》 ABSTRACTS The ubiquitin proteasome system (UPS) plays important roles in the regulation of protein stability, localization, and activity. A myriad of studies have focused on the functions of ubiquitin ligases E3s and deubiquitinating enzymes DUBs due to their specificity in the recognition of downstream substrates. However, the roles of the most ubiquitin-conjugating enzymes E2s are not completely understood except that they transport the activated ubiquitin and form E2–E3 protein complexes. Ubiquitin-conjugating enzyme CDC34 can promote the degradation of downstream targets through the UPS whereas its non-catalytic functions are still elusive. Here, we find that mutation of the catalytically active cysteine to serine (C93S) results in the reduced ubiquitination, increased stability, and attenuated degradation rate of CDC34. Through semi-quantitative proteom...
发表评论
阅读全文

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

Author：Liang Liu, Qingqing Xu, Shuai Hao & Yun Chen     《Scientific Reports》 ABSTRACTS 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-...
发表评论
阅读全文

Preparation of an antimicrobial surface by direct assembly of antimicrobial peptide with its surface binding activity

Author：Junjian Chen, Yuchen Zhu, Yancheng Song, Lin Wang,  Jiezhao Zhan, Jingcai He, Jian Zheng, Chunting Zhong, Xuetao Shi, Sa Liu, Li Ren and Yingjun Wang     《Journal of Materials Chemistry B》 ABSTRACTS Antimicrobial peptides (AMPs) are a broad prospect for clinical application against bacterial infections of biomaterials. However, a bottleneck exists as there is a lack of simple technology to prepare AMPs on biomaterials with sufficient activity, as the activity of AMP is dependent on the correct orientation on the biomaterial. In the present study, based on the conventional AMP (Tet213: KRWWKWWRRC) and surface binding peptide (SKHKGGKHKGGKHKG), we designed an Anchor-AMP that could be directly assembled onto the surface of the biomaterial and also showed excellent antimicrobial activity. By characterizing the surface using a quartz crystal microbalance with dissipation (QCM-D), contact angle, atom force microscopy (AFM) and X-ray photoelectron spectr...
发表评论
阅读全文