Yin Sheng, Xie Yongming, Loo Joseph A
Department of Chemistry and Biochemistry, University of California-Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095, USA.
J Am Soc Mass Spectrom. 2008 Aug;19(8):1199-208. doi: 10.1016/j.jasms.2008.05.012. Epub 2008 May 28.
Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.
非共价蛋白质-配体复合物可通过电喷雾电离质谱（ESI-MS）轻松检测到。通过ESI-MS方法可以轻松确定配体结合化学计量。然而，检测非共价蛋白质-配体复合物的能力取决于复合物在气相环境中的稳定性。溶液结合亲和力可能是也可能不是其在真空中稳定性的准确预测指标。通过ESI-MS检测了由与核糖核酸酶A（RNase A）和核糖核酸酶S（RNase S）结合的胞苷核苷酸组成的复合物，并通过MS/MS进行了进一步分析。RNase A和RNase S具有相似的结构和生物活性。RNase A经枯草杆菌蛋白酶切割产生一个S肽和一个S蛋白；S肽和S蛋白通过疏水相互作用相互作用，溶液结合常数在纳摩尔范围内，从而产生有活性的RNase S。胞苷核苷酸通过静电相互作用与核糖核酸酶结合，溶液结合常数在微摩尔范围内。1:1的RNase A-CDP和CTP复合物的碰撞激活解离（CAD）导致核苷酸配体的共价磷酸键断裂，从复合物中释放出CMP。RNase S-CDP和CTP复合物的CAD使S肽与剩余的S蛋白/核苷酸复合物解离；S蛋白/核苷酸复合物的进一步解离使核苷酸的共价磷酸键断裂，随后释放出CMP。尽管溶液结合常数有利于S蛋白/S肽复合物，但在气相解离实验中，CDP/CTP仍通过静电作用与S蛋白结合。这项研究突出了气相中静电相互作用的内在稳定性以及非共价复合物在溶液和气相稳定性方面可能存在的显著差异。
