Automated MALDI Target Preparation Concept: Providing Ultra-High-Throughput Mass Spectrometry-Based Screening for Drug Discovery
Automated MALDI Target Preparation Concept: Providing Ultra-High-Throughput Mass Spectrometry-Based Screening for Drug Discovery
复制标题
DOI：
10.1177/2472630318791981
复制
发表时间：
2019-04-01
期刊：
影响因子：
2.7
通讯作者：
Buettner, Frank H.
中科院分区：
文献类型：
--
作者：
Winter, Martin;Ries, Robert;Buettner, Frank H.
通过直接分析底物到产物的转化来破译酶反应的无标记质谱学(MS)为药物发现活动中更具生理学相关性的分析提供了下一步。减少遭受复合干扰的风险，以及减少对定制信号调解器的必要性，强调了无标签读数的宝贵作用。然而，由于缺乏与高通量筛查(HTS)兼容的样品引入，基于MS的检测迄今尚未满足高通量筛查(HTS)的要求。在这项研究中，我们报告了一种内部构建的全自动液体处理概念，将生化分析与基质辅助激光解吸/电离飞行时间法串联起来，以缩小这一技术差距。集成的重新格式化从384孔格式到1536孔格式，使得自动斑点的周期为0.6S/样品，MS分析的周期为0.4S/样品，符合高温超导兼容性的要求。对蛋白质酪氨酸磷酸酶1B(PTP1B)抑制剂筛选(4896种化合物)的深入检测，以及对斑点质量、定量准确性和仪器稳健性的深入研究，证明了大量询问的HTS整合无标记MS读数的潜力。总体而言，提供的数据表明，引入的自动化概念使HTS在药物发现活动中以及在其他需要超高速MS检测的研究领域都可以访问无标签的MS读数。
Label-free, mass spectrometric (MS) deciphering of enzymatic reactions by direct analysis of substrate-to-product conversion provides the next step toward more physiological relevant assays within drug discovery campaigns. Reduced risk of suffering from compound interference combined with diminished necessity for tailored signal mediators emphasizes the valuable role of label-free readouts. However, MS-based detection has not hitherto met high-throughput screening (HTS) requirements because of the lack of HTS-compatible sample introduction. In the present study, we report on a fully automated liquid-handling concept built in-house to concatenate biochemical assays with matrix-assisted laser desorption/ionization time-of-flight closing this technological gap. The integrated reformatting from 384- to 1536-well format enables cycle times of 0.6 s/sample for automated spotting and 0.4 s/sample for MS analysis, matching the requirements of HTS compatibility. In-depth examination of spotting quality, quantification accuracy, and instrument robustness together with the implementation of a protein tyrosine phosphatase 1B (PTP1B) inhibitor screening (4896 compounds) demonstrate the potential of the heavily inquired HTS integration of the label-free MS readout. Overall, the presented data demonstrate that the introduced automation concept makes label-free MS-based readouts accessible for HTS within drug discovery campaigns but also in other research areas requiring ultrafast MS-based detection.