Nguyen Giang T H, Tran Thinh N, Podgorski Matthew N, Bell Stephen G, Supuran Claudiu T, Donald William A
School of Chemistry, University of New South Wales, Dalton Building, Sydney, New South Wales 2052, Australia.
School of Electrical Engineering and Telecommunications, University of New South Wales, Sydney, New South Wales 2052, Australia.
ACS Cent Sci. 2019 Feb 27;5(2):308-318. doi: 10.1021/acscentsci.8b00787. Epub 2019 Jan 14.
Electrospray ionization (ESI) mass spectrometry (MS) is a crucial method for rapidly determining the interactions between small molecules and proteins with ultrahigh sensitivity. However, nonvolatile molecules and salts that are often necessary to stabilize the native structures of protein-ligand complexes can readily adduct to protein ions, broaden spectral peaks, and lower signal-to-noise ratios in native MS. ESI emitters with narrow tip diameters (∼250 nm) were used to significantly reduce the extent of adduction of salt and nonvolatile molecules to protein complexes to more accurately measure ligand-protein binding constants than by use of conventional larger-bore emitters under these conditions. As a result of decreased salt adduction, peaks corresponding to protein-ligand complexes that differ in relative molecular weight by as low as 0.06% can be readily resolved. For low-molecular-weight anion ligands formed from sodium salts, anion-bound and unbound protein ions that differ in relative mass by 0.2% were completely baseline resolved using nanoscale emitters, which was not possible under these conditions using conventional emitters. Owing to the improved spectral resolution obtained using narrow-bore emitters and an analytically derived equation, values were simultaneously obtained for at least six ligands to a single druggable protein target from one spectrum for the first time. This research suggests that ligand-protein binding constants can be directly and accurately measured from solutions with high concentrations of nonvolatile buffers and salts by native MS.
电喷雾电离（ESI）质谱（MS）是一种至关重要的方法，可用于以超高灵敏度快速测定小分子与蛋白质之间的相互作用。然而，通常用于稳定蛋白质 - 配体复合物天然结构所必需的非挥发性分子和盐，很容易与蛋白质离子加合，使光谱峰变宽，并降低天然质谱中的信噪比。使用尖端直径较窄（约250纳米）的电喷雾电离发射器，可显著降低盐和非挥发性分子与蛋白质复合物的加合程度，从而在这些条件下比使用传统的大口径发射器更准确地测量配体 - 蛋白质结合常数。由于盐加合减少，相对分子量相差低至0.06%的蛋白质 - 配体复合物对应的峰可以很容易地分辨出来。对于由钠盐形成的低分子量阴离子配体，使用纳米级发射器可将相对质量相差0.2%的阴离子结合和未结合的蛋白质离子完全基线分离，而在这些条件下使用传统发射器则无法做到。由于使用窄口径发射器获得了更高的光谱分辨率以及一个通过分析推导的方程，首次从一个光谱中同时获得了针对单个可成药蛋白质靶点的至少六种配体的 值。这项研究表明，通过天然质谱可以直接且准确地从含有高浓度非挥发性缓冲剂和盐的溶液中测量配体 - 蛋白质结合常数。
