High-Throughput Amenable MALDI-MS Detection of RNA and DNA with On-Surface Analyte Enrichment Using Fluorous Partitioning.
High-Throughput Amenable MALDI-MS Detection of RNA and DNA with On-Surface Analyte Enrichment Using Fluorous Partitioning.
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DOI：
10.1177/2472555220958391
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发表时间：
2021-01
期刊：
影响因子：
3.1
通讯作者：
Yu, Marvin S.
中科院分区：
文献类型：
--
作者：
Emanuelson, Cole;Ankenbruck, Nicholas;Deiters, Alexander;Yu, Marvin S.
高通量基质辅助激光解吸/电离质谱（HT-MALDI-MS）作为使用基于光的检测方法的分析的信息丰富的替代方法，在药物发现行业内获得了相当大的关注。迄今为止，这些努力主要集中在使用蛋白质或肽底物的测定。通过HT-MALDI-MS进行RNA或DNA分析的方法尚未被广泛报道，这是由于与寡核苷酸的MALDI-MS相关的挑战，包括形成多个盐加合物的倾向、低电离电位和易于片段化。这项工作的目的是开发一个适合于RNA和DNA底物的HT-MS分析的平台，通过将表面样品制备与软电离相结合来克服这些障碍。这是通过将氟标记的寡核苷酸选择性固定在氟改性的MS靶板上，然后进行表面富集、基质添加和直接激光解吸/电离（称为氟HT-MS（F-HT-MS））来实现的。这项工作已经产生了一些方法，通过这些方法，可以使用适合于完全自动化的程序从典型的测定缓冲液系统中以纳摩尔浓度检测RNA和DNA底物。这些方案被应用于miRNA生物合成测定，证明了其在RNA过程中的潜力，从而填补了RNA药物发现中的一个突出空白：缺乏体外功能测定。
High-throughput matrix-assisted laser desorption/ionization mass spectrometry (HT-MALDI-MS) has garnered considerable attention within the drug discovery industry as an information-rich alternative to assays using light-based detection methods. To date, these efforts have been primarily focused on assays using protein or peptide substrates. Methods for RNA or DNA analysis by HT-MALDI-MS have not been extensively reported due to the challenges associated with MALDI-MS of oligonucleotides, including the propensity to form multiple salt adducts, low ionization potential, and ease of fragmentation. The objective of this work was to develop a platform suitable for HT-MS analysis of RNA and DNA substrates that overcomes these hurdles by combining on-surface sample preparation with soft ionization. This has been accomplished through the selective immobilization of fluorous-tagged oligonucleotides on a fluorous-modified MS target plate, followed by on-surface enrichment, matrix addition, and direct laser desorption/ionization, a process dubbed fluorous HT-MS (F-HT-MS). The work has resulted in methods by which RNA and DNA substrates can be detected at nanomolar concentrations from a typical assay buffer system using procedures that are amenable to full automation. The protocols were applied to an miRNA biogenesis assay, demonstrating its potential for RNA processes and thereby filling a prominent gap in RNA drug discovery: the paucity of in vitro functional assays.
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DOI：
10.1039/c7cc00288b
发表时间：
2017-03-09
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Flynn GE;Withers JM;Macias G;Sperling JR;Henry SL;Cooper JM;Burley GA;Clark AW
通讯作者：
Clark AW
影响因子：
3.1
作者：
Chandler, Jessica;Haslam, Carl;Marshall, Peter
通讯作者：
Marshall, Peter
DOI：
10.1002/wrna.1477
发表时间：
2018-07
期刊：
Wiley interdisciplinary reviews. RNA
影响因子：
--
作者：
Donlic A;Hargrove AE
通讯作者：
Hargrove AE
影响因子：
1.9
作者：
Collet, Beatrice Y. M.;Nagashima, Tadamichi;Pohl, Nicola L. B.
通讯作者：
Pohl, Nicola L. B.
影响因子：
4.4
作者：
Go, Eden P.;Uritboonthai, Wilasinee;Siuzdak, Gary
通讯作者：
Siuzdak, Gary