Mass spectrometric techniques for label-free high-throughput screening in drug discovery
Mass spectrometric techniques for label-free high-throughput screening in drug discovery
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DOI：
10.1021/ac062421q
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发表时间：
2007-11-01
影响因子：
7.4
通讯作者：
Wang, Y. Karen
中科院分区：
文献类型：
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作者：
Roddy, Thomas P.;Horvath, Christopher R.;Wang, Y. Karen
高通量筛选 (HTS) 是寻找活性化合物以启动药物发现研究中药物化学项目的重要工具。传统的 HTS 方法依靠荧光或放射性标记试剂和/或偶联测定来定量酶靶抑制或激活。基于质谱的高通量筛选 (MS-HTS) 是一种不易受到标记和偶联酶限制的替代方法。 MS-HTS 为未标记的底物和产物提供了一种选择性且灵敏的分析方法。此外，方法开发时间缩短，无需合并标记或偶联测定。 MS-HTS 还可以筛选其他技术难以或不可能筛选的靶标。例如，难以纯化的酶可能导致对不纯酶或膜酶基质的结构相似成分进行非特异性检测。串联质谱 (MS/MS) 的高选择性使这些筛选能够在低水平的背景噪声下进行，即使活性相对较弱，也能灵敏地发现感兴趣的命中。在本文中，我们描述了三种用于大规模（类似于 175 000 个样品）化合物库筛选的技术，包括四路平行多重电喷雾液相色谱串联质谱 (MUX-LC/MS/MS)、四路平行交错梯度液相色谱串联质谱 (LC/MS/MS) 和 384 孔板固相萃取后的八路交错流动注射 MS/MS （S：PE）。这些方法能够在 37 分钟内分析 384 孔板，典型分析时间不到 2 小时。本文通过两次大规模筛选的筛选数据证明了 MS-HTS 方法的质量。
High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (similar to 175 000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (S:PE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.