Catalina M Isabel, van den Heuvel Robert H H, van Duijn Esther, Heck Albert J R
Department of Biomolecular Mass Spectrometry, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Chemistry. 2005 Jan 21;11(3):960-8. doi: 10.1002/chem.200400395.
Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.
电喷雾电离质谱（ESI-MS）是结构生物学中用于研究球状蛋白质及其生物分子相互作用的重要工具。在电喷雾电离过程中，蛋白质会去溶剂化并带上多个电荷，这可能会影响其结构。减少电喷雾过程中获得的净电荷可能与球状蛋白质的研究相关。在本报告中，我们展示了一系列无机和有机气相碱对蛋白质及蛋白质复合物所带电荷数的影响。在所谓的“质子海绵”中鉴定出了具有非常强气相碱度（GB）的溶液添加剂。添加到蛋白质水溶液中的化合物的气相质子亲和力（PA）范围为700至1050 kJ mol⁻¹。圆二色性研究表明，在这些溶液中蛋白质保留了其球状结构。所研究的蛋白质大小范围从14.3 kDa的溶菌酶到800 kDa的十四聚体伴侣复合物GroEL。通过添加碱性最强的化合物而非更常用的醋酸铵添加剂，可在电喷雾过程中使蛋白质的电荷减少多达60%。这种去电荷过程可能是由于在最终去溶剂化之前，多质子化蛋白质离子与碱性添加剂之间发生了质子竞争事件。我们推测，在电离过程中获得相对较少电荷的此类球状蛋白质物种，其气相结构更类似于其溶液相结构。因此，这些碱性添加剂可用于通过质谱研究球状蛋白质的生物学相关特性。
