HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.
HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.
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DOI：
10.1007/s13361-016-1472-3
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发表时间：
2016-11
影响因子：
3.2
通讯作者：
Desaire H
中科院分区：
文献类型：
--
作者：
Imaduwage KP;Go EP;Zhu Z;Desaire H
药物发现的一个主要挑战是鉴定与特定靶蛋白结合的高亲和力先导化合物。这些线索通常通过高通量筛选来识别。质谱法已成为药物筛选分析中的首选检测方法，因为不需要修改靶标和配体。无标记测定是有利的，因为它们可以比需要标记的测定更快地开发，并且它们消除了标记干扰结合事件的风险。然而，在常用的基于 MS 的筛查方法中，假阳性的检测是一个重大挑战。在这里，我们描述了一种旨在消除误报的检测策略。在这种方法中，蛋白质和配体一起孵育，非结合剂被分离以进行检测。与蛋白质一起孵育后，MS 无法检测到命中物（蛋白质结合物），但当孵育介质中不存在目标蛋白质时，MS 可以轻松识别命中物（蛋白质结合物）。该测定使用三种不同的蛋白质和数百种非抑制剂进行了验证；在任何实验中均未发现误报。通过改变所用蛋白质的量和固定方法，可以调整测定法以选择具有特定结合亲和力的配体。例如，该方法选择性检测 Ki 值为 0.2 μM、50 pM 和 700 pM 的抑制剂。这些发现表明，本文描述的方法优于传统的基于 MS 的筛选方法。
A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM and 700 pM. These findings demonstrate that the approach described here compares favorably to traditional MS based screening methods.
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影响因子：
2
作者：
Lim, Mark J.;Liu, Ziying;Rothschild, Kenneth J.
通讯作者：
Rothschild, Kenneth J.
影响因子：
3
作者：
Makley LN;Gestwicki JE
通讯作者：
Gestwicki JE
DOI：
10.1073/pnas.220403997
发表时间：
2000-10-24
影响因子：
11.1
作者：
Cancilla, MT;Leavell, MD;Leary, JA
通讯作者：
Leary, JA
影响因子：
7.4
作者：
O'Connell, Thomas N.;Ramsay, Jason;Stroh, Justin G.
通讯作者：
Stroh, Justin G.
影响因子：
--
作者：
Brown, N;Zehender, H;Jacoby, E
通讯作者：
Jacoby, E