Elucidating Protein-Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion.
Elucidating Protein-Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion.
复制标题
DOI：
10.1021/acs.analchem.2c04266
复制
发表时间：
2023-01
影响因子：
7.4
通讯作者：
Mulin Fang;Oliver Wu;Kellye A. Cupp-Sutton;Kenneth Smith;Siyi Wu
中科院分区：
文献类型：
--
作者：
Mulin Fang;Oliver Wu;Kellye A. Cupp-Sutton;Kenneth Smith;Siyi Wu
氢-氘交换与质谱 (HDX-MS) 结合是表征蛋白质-配体相互作用的强大技术。目前，在 HDX-MS 分析应用到细胞裂解物等高度复杂的生物样品中的蛋白质-配体相互作用方面，越来越需要突破。然而，此类系统中的 HDX-MS 分析由于样品复杂性高且传统使用短 LC 梯度而导致 LC 分离能力有限，因而面临极端的光谱复杂性。在这里，我们引入了蛋白质热耗竭 (PTD) 来降低大肠杆菌细胞裂解液中的蛋白质复杂性，以便我们的零下温度长梯度 UPLC-HDX-MS 平台 (PTD-HDX-MS) 能够促进细胞裂解液中蛋白质-配体相互作用的高通量分析。我们将牛碳酸酐酶 II (CaII) 及其抑制剂乙酰唑酰胺 (AZM) 掺入大肠杆菌细胞裂解物中作为我们研究的模型系统。我们证明，60°C 下的 PTD 大大降低了细胞裂解物中的蛋白质复杂性，而 AZM 靶向的 CaII 复合物由于结合后热稳定性的提高而保留在溶液中。利用 PTD 降低样品复杂性，并使用零下温度长梯度 UPLC 提高 LC 分离能力，我们通过对数百种蛋白质中的数千种氘代肽进行高通量 HDX-MS 分析，成功阐明了大肠杆菌细胞裂解液中 AZM 和 CaII 之间的相互作用位点。我们的结果凸显了 PTD-HDX-MS 平台在高度复杂的生物样品（例如细胞裂解物）中配体靶标的鉴定和蛋白质-配体相互作用的表征方面的巨大前景。
Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein-ligand interactions. Currently, there is a growing need for breakthroughs in the application of HDX-MS analysis to protein-ligand interactions in highly complex biological samples such as cell lysates. However, HDX-MS analysis in such systems suffers from extreme spectral complexity as a result of high sample complexity and limited LC separation power due to the traditional use of short LC gradients. Here, we introduced protein thermal depletion (PTD) to reduce protein complexity in E. coli cell lysate for our subzero-temperature long gradient UPLC-HDX-MS platform (PTD-HDX-MS) to facilitate high-throughput analysis of protein-ligand interactions in cell lysates. We spiked bovine carbonic anhydrase II (CaII) and its inhibitor acetazolamide (AZM) into E. coli cell lysate as a model system in our study. We demonstrated that PTD at 60 °C greatly reduces protein complexity in cell lysates, while the AZM-targeted CaII complex remains in solution due to improved thermal stability upon binding. Using both PTD to reduce sample complexity and subzero-temperature long gradient UPLC to boost LC separation power, we successfully elucidated the interaction sites between AZM and CaII in E. coli cell lysate from the high-throughput HDX-MS analysis of thousands of deuterated peptides from hundreds of proteins. Our results highlight the great promise of the PTD-HDX-MS platform for the identification of ligand targets and characterization of protein-ligand interactions in highly complex biological samples such as cell lysates.