Mass Spectrometric Techniques for Label-free High-Throughput Screening in Drug Discovery

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• 作者：Thomas P. Roddy ; Christopher R. Horvath ; Steven J. Stout ; Kristin L. Kenney ; Pei-I Ho ; Ji-Hu Zhang ; Chad Vickers ; Virendar Kaushik ; Brian Hubbard ; Y. Karen Wang
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：November 1, 2007
• 年：2007
• 卷：79
• 期：21
• 页码：8207 - 8213
• 全文大小：270K
• 年卷期：v.79,no.21(November 1, 2007)
• ISSN：1520-6882

文摘

High-throughput screening (HTS) is an important tool forfinding active compounds to initiate medicinal chemistryprograms in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeledreagents and/or coupling assays to permit quantitationof enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is analternative that is not susceptible to the limitationsimposed by labeling and coupling enzymes. MS-HTS offersa selective and sensitive analytical method for unlabeledsubstrates and products. Furthermore, method development times are reduced without the need to incorporatelabels or coupling assays. MS-HTS also permits screeningof targets that are difficult or impossible to screen by othertechniques. For example, enzymes that are challengingto purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrixof membraneous enzymes. The high selectivity of tandemmass spectrometry (MS/MS) enables these screens toproceed with low levels of background noise to sensitivelydiscover interesting hits even with relatively weak activity.In this article, we describe three techniques that we haveadapted for large-scale (~175 000 sample) compoundlibrary screening, including four-way parallel multiplexedelectrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggeredgradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injectionMS/MS following 384-well plate solid-phase extraction(SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of lessthan 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scalescreens.