Proteome Center Rostock, University Medicine Rostock, 18059 Rostock, Germany.
School of Pharmacy, University of Ghana, P. O. Box LG53 Legon, Ghana.
Molecules. 2020 Oct 17;25(20):4776. doi: 10.3390/molecules25204776.
Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10 was calculated for the troponin I epitope peptide-antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.
电喷雾质谱法用于测定气相中免疫复合物解离反应的表观结合能和准平衡离解常数。肌红蛋白是一种天然的蛋白质-配体复合物，已被用于开发该程序，该程序从确定平均电荷态和归一化平均离子强度开始。从质谱数据计算并随后外推至室温以模拟中性和静止肌红蛋白的碰撞条件，得出气相血红素解离过程的表观离解常数 KD m0g#=3.60×10。类似地，在室温下测定了 RNAse S 的 KD m0g#=4.03×10。该方案用两种由抗原肽和单克隆抗体组成的免疫复合物进行了测试。对于 FLAG 肽从抗 FLAG 抗体复合物中的表位肽解离反应，计算出表观气相解离常数 KD m0g#=4.04×10。同样，对于肌钙蛋白 I 表位肽-抗肌钙蛋白 I 抗体免疫复合物解离，计算出表观 KD m0g#=4.58×10。电喷雾质谱法是一种快速方法，对于鉴定蛋白质结合配体或测定表观气相蛋白质-配体复合物结合强度，都只需要少量样品。
