Wang Weijie, Kitova Elena N, Sun Jiangxiao, Klassen John S
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
J Am Soc Mass Spectrom. 2005 Oct;16(10):1583-94. doi: 10.1016/j.jasms.2005.05.008.
Gas-phase thermal dissociation experiments, implemented with blackbody infrared radiative dissociation (BIRD) and Fourier transform ion cyclotron resonance mass spectrometry, have been performed on a series of protonated and deprotonated 1:1 and protonated 1:2 protein-carbohydrate complexes formed by nonspecific interactions during the nanoflow electrospray (nanoES) ionization process. Nonspecific interactions between the proteins bovine carbonic anhydrase II (CA), bovine ubiquitin (Ubq), and bovine pancreatic trypsin inhibitor and several carbohydrates, ranging in size from mono- to tetrasaccharides, have been investigated. Over the range of temperatures studied (60-190 degrees C), BIRD of the protonated and deprotonated complexes proceeds exclusively by the loss of the carbohydrate in its neutral form. The rates of dissociation of the 1:1 complexes containing a mono- or disaccharide decrease with reaction time, suggesting the presence of two or more kinetically distinct structures produced during nanoES or by gas-phase processes. In contrast, the 1:1 complexes of the tri- and tetrasaccharides exhibit simple first-order dissociation kinetics, a result that, on its own, is suggestive of a single preferred carbohydrate binding site or multiple equivalent sites in the gas phase. A comparative analysis of the dissociation kinetics measured for protonated 1:1 and 1:2 complexes of Ubq with alphaTal[alphaAbe]alphaMan further supports the presence of a single preferred binding site. However, a similar analysis performed on the complexes of CA and alphaTal[alphaAbe]alphaMan suggests that equivalent but dependent carbohydrate binding sites exist in the gas phase. Analysis of the Arrhenius activation parameters (E(a) and A) determined for the dissociation of 1:1 complexes of CA with structurally related trisaccharides provides evidence that neutral intermolecular hydrogen bonds contribute, at least in part, to the stability of the gaseous complexes. Surprisingly, the E(a) values for the complexes of the same charge state are not sensitive to the structure (primary or higher order) of the protein, suggesting that the carbohydrates are able to form energetically equivalent interactions with the various functional groups presented by the protein. For a given protein-carbohydrate complex, the dissociation E(a) is sensitive to charge state, initially increasing and then decreasing with increasing charge. It is proposed that both ionic and neutral hydrogen bonds stabilize the nonspecific protein-carbohydrate complexes in the gas phase and that the relative contribution of the neutral and ionic interactions is strongly influenced by the charge state of the complex, with neutral interactions dominating at low charge states and ionic interactions dominating at high charge states.
利用黑体红外辐射解离（BIRD）和傅里叶变换离子回旋共振质谱进行了气相热解离实验，研究对象是在纳流电喷雾（nanoES）电离过程中通过非特异性相互作用形成的一系列质子化和去质子化的1:1以及质子化的1:2蛋白质 - 碳水化合物复合物。研究了蛋白质牛碳酸酐酶II（CA）、牛泛素（Ubq）和牛胰蛋白酶抑制剂与几种碳水化合物之间的非特异性相互作用，这些碳水化合物的大小从单糖到四糖不等。在所研究的温度范围（60 - 190摄氏度）内，质子化和去质子化复合物的BIRD过程仅通过中性形式的碳水化合物的损失来进行。含有单糖或二糖的1:1复合物的解离速率随反应时间降低，这表明在nanoES过程中或气相过程中产生了两种或更多种动力学上不同的结构。相比之下，三糖和四糖的1:1复合物表现出简单的一级解离动力学，这一结果本身表明在气相中存在单个优选的碳水化合物结合位点或多个等效位点。对Ubq与αTal[αAbe]αMan的质子化1:1和1:2复合物的解离动力学进行的比较分析进一步支持了单个优选结合位点的存在。然而，对CA与αTal[αAbe]αMan的复合物进行的类似分析表明，在气相中存在等效但相互依赖的碳水化合物结合位点。对CA与结构相关的三糖的1:1复合物解离所确定的阿累尼乌斯活化参数（E(a)和A）的分析提供了证据，表明中性分子间氢键至少部分地有助于气态复合物的稳定性。令人惊讶的是，相同电荷态复合物的E(a)值对蛋白质的结构（一级或更高阶）不敏感，这表明碳水化合物能够与蛋白质呈现的各种官能团形成能量上等效的相互作用。对于给定的蛋白质 - 碳水化合物复合物，解离E(a)对电荷态敏感，最初随电荷增加而增加，然后随电荷增加而降低。有人提出，离子键和中性氢键都能稳定气相中的非特异性蛋白质 - 碳水化合物复合物，并且中性和离子相互作用的相对贡献受复合物电荷态的强烈影响，在低电荷态时中性相互作用占主导，在高电荷态时离子相互作用占主导。
