Discovery Technologies, Thousand Oaks, CA, USA.
Research Automation Technologies, Thousand Oaks, CA, USA.
SLAS Discov. 2021 Feb;26(2):230-241. doi: 10.1177/2472555220979596. Epub 2020 Dec 17.
Affinity selection mass spectrometry (MS) or, simply, affinity mass spectrometry (AMS) is a label-free technology that has been used to identify high-affinity ligands of target proteins of interest by screening against small-molecule compound libraries and identifying molecules that are enriched in the presence of the target protein. We have previously applied Agilent Technology's (Santa Clara, CA) RapidFire solid-phase extraction (SPE)-based high-throughput MS technology to screen small-molecule libraries using AMS. However, SPE-based technologies rely on fluidics for desalting and separation prior to mass analysis with attendant high solvent consumption, relatively high sample volume requirements, risk of sample carryover, and frequent maintenance. To address these challenges, we have established an AMS platform using a laser diode thermal desorption-atmospheric pressure chemical ionization (LDTD-APCI) ionization source (Phytronix, Quebec, Canada) coupled with a SCIEX 5600+ TripleTOF MS (Framingham, MA). We also validated a data-independent acquisition (DIA) Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) method for the robust detection and analysis of small-molecule affinity hits. An informatics platform developed in-house has resulted in a streamlined data analysis workflow for high-throughput AMS screening campaigns and reduced data processing time without compromising data quality. Finally, 68,000 compounds were screened in a single plate and affinity selected hits were confirmed in an orthogonal enzyme activity assay.
亲和选择质谱（MS），或简称亲和质谱（AMS），是一种无标记技术，已被用于通过筛选小分子化合物文库并鉴定在靶蛋白存在下富集的分子，来鉴定靶蛋白的高亲和力配体。我们之前曾应用安捷伦科技（加利福尼亚州圣克拉拉）的 RapidFire 基于固相萃取（SPE）的高通量 MS 技术，使用 AMS 筛选小分子文库。然而，基于 SPE 的技术依赖于流体力学进行脱盐和分离，然后进行质谱分析，因此溶剂消耗量大、对样品体积要求相对较高、存在样品交叉污染风险，并且需要频繁维护。为了解决这些挑战，我们建立了一个使用激光二极管热解吸-大气压化学电离（LDTD-APCI）电离源（Phytronix，魁北克省，加拿大）与 SCIEX 5600+ TripleTOF MS（弗雷明汉，马萨诸塞州）联用的 AMS 平台。我们还验证了一种用于稳健检测和分析小分子亲和命中物的无数据依赖采集（DIA）序贯窗口采集所有理论质量谱（SWATH-MS）方法。内部开发的信息学平台为高通量 AMS 筛选活动提供了简化的数据分析工作流程，并减少了数据处理时间，而不会影响数据质量。最后，在一个平板上筛选了 68000 种化合物，并在正交酶活性测定中确认了亲和选择的命中物。
