NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
Methods Mol Biol. 2024;2823:291-310. doi: 10.1007/978-1-0716-3922-1_18.
We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms.
我们提出了一种新的方法来确定体外靶向 KRAS4B 的化合物的结合和特异性。通过采用自上而下的质谱（MS）分析，该方法可以直接分析和准确鉴定化合物处理样本中每个 KRAS4B 物种。此外，通过靶向 MS2 碎片分析，我们可以将每个化合物分子精确地定位到给定 KRAS4B 蛋白水解产物的特定残基上。该方法可以全面评估化合物的特异性，清楚地检测非特异性结合事件，并确定其发生的顺序和频率。我们提供了两个使用公开可用的化合物的方法的概念验证示例，以及详细的样品制备、自上而下的 MS 数据采集、靶向蛋白水解产物 MS2 片段分析和结果数据分析协议。我们的结果表明了 KRAS4B-化合物结合的浓度依赖性，并强调了自上而下的 MS 可以直接映射化合物结合位置，而不会破坏 KRAS4B 的一级结构。我们希望这种新方法可以帮助加速鉴定新的针对 KRAS4B 和其他 RAS 同工型的成功靶向抑制剂。
