Liquid extraction surface analysis (LESA) coupled to native mass spectrometry (MS) presents unique analytical opportunities due to its sensitivity, speed, and automation. Here, we examine whether this tool can be used to quantitatively probe protein–ligand interactions through calculation of equilibrium dissociation constants (K_d values). We performed native LESA MS analyses for a well-characterized system comprising bovine carbonic anhydrase II and the ligands chlorothiazide, dansylamide, and sulfanilamide, and compared the results with those obtained from direct infusion mass spectrometry and surface plasmon resonance measurements. Two LESA approaches were considered: In one approach, the protein and ligand were premixed in solution before being deposited and dried onto a solid substrate for LESA sampling, and in the second, the protein alone was dried onto the substrate and the ligand was included in the LESA sampling solvent. Good agreement was found between the K_d values derived from direct infusion MS and LESA MS when the protein and ligand were premixed; however, K_d values determined from LESA MS measurements where the ligand was in the sampling solvent were inconsistent. Our results suggest that LESA MS is a suitable tool for quantitative analysis of protein–ligand interactions when the dried sample comprises both protein and ligand.

中文翻译：

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LESA质谱法对三种碳酸酐酶抑制剂的定量表征

液体萃取表面分析 (LESA) 与原生质谱 (MS) 相结合，由于其灵敏度、速度和自动化，提供了独特的分析机会。在这里，我们通过计算平衡解离常数（K _d值）。我们对包含牛碳酸酐酶 II 和配体氯噻嗪、丹磺酰胺和磺胺的系统进行了天然 LESA MS 分析，并将结果与​​直接输注质谱法和表面等离子体共振测量获得的结果进行了比较。考虑了两种 LESA 方法：在一种方法中，将蛋白质和配体在溶液中预混合，然后沉积并干燥到用于 LESA 采样的固体基质上，在第二种方法中，仅将蛋白质干燥到基质上并将配体包含在LESA 取样溶剂。当蛋白质和配体预混合时，直接输注 MS 和 LESA MS 得到的K _d值有很好的一致性；然而_，Kd在配体位于采样溶剂中的情况下，从 LESA MS 测量确定的值不一致。我们的结果表明，当干燥样品同时包含蛋白质和配体时，LESA MS 是定量分析蛋白质-配体相互作用的合适工具。