Free energies of protein-protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods
Free energies of protein-protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods
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DOI：
10.1110/ps.062083406
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发表时间：
2006-06-01
期刊：
影响因子：
8
通讯作者：
Kirsch, Jack F.
中科院分区：
文献类型：
--
作者：
Krishnaswamy, Sanjay R.;Williams, Evan R.;Kirsch, Jack F.
通过选择解淀粉芽孢杆菌蛋白barstar （b*）和RNAase barnase （bn）的变体，证明了电喷雾电离质谱（ESIMS）在测量紧密结合的蛋白-蛋白复合物的相对溶液相亲和力方面的优势，它们形成具有皮摩尔到纳摩尔解离常数范围的蛋白-蛋白复合物。一种新的化学退火程序快速建立平衡的溶液中含有竞争的b*变体与限制bn。络合物的相对离子丰度和竞争的未结合单体的相对离子丰度反映了这些各自物种的相对溶液相浓度。在ESI或质量检测过程中，复合物均未发生可测量的解离，也没有证据表明在蛋白质浓度< 25 μ m时存在非特异性结合。得到的δ δ G值与液相酶测定值平均偏差0.26 kcal/mol。结果表明，可以从质量和电荷态分布的差异中获得蛋白质构象和共价修饰的信息。这种方法可以作为一种快速和精确的方法来询问在皮摩尔到纳摩尔范围内具有亲和力的复合物的蛋白质-蛋白质结合表面。
The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solution-phase affinities of tightly bound protein-protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form protein protein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution-phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations < 25 mu M. Differences in Delta Delta G of dissociation between variants were determined with precisions < 0.1 kcal/mol. The Delta Delta G values obtained deviate on average by 0.26 kcal/mol from those measured with a solution-phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein-protein-binding surfaces for complexes that have affinities in the picomolar to nanomolar range.