Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses
Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses
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DOI：
10.1002/jms.208
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发表时间：
2001-09-01
影响因子：
2.3
通讯作者：
Allmaier, G
中科院分区：
文献类型：
--
作者：
Bacher, G;Szymanski, WW;Allmaier, G
本研究探讨了一种新的电喷雾为基础的方法，称为气相电泳迁移率分子分析（GEMMA），允许分子量测定肽，蛋白质和非共价生物复合物高达2 MDa（免疫球蛋白M的二聚体）的潜力。大分子离子是通过纳米电喷雾离子化（ESI）在“锥形射流模式”下形成的。所产生的单分散液滴/离子的多电荷状态通过双极电离空气（由α粒子源产生）来减少，以仅产生单电荷的正离子和负离子以及中性离子。这些离子随后在大气压下使用纳米微分迁移率分析仪根据它们在空气中的电泳迁移率进行分离。最后，使用标准凝聚粒子计数器检测离子。通过应用密立根方程将数据表示为电泳迁移率直径。测量的电泳迁移率直径，或密立根直径，32个明确定义的蛋白质，对它们的分子量在3.5至1920 kDa的范围内绘制，并表现出良好的平方相关系数（r（2）= 0.999）。这一发现允许通过这种新技术精确测定大（糖）蛋白和非共价生物复合物的分子量，在飞摩尔水平上的质量准确度为+/-5.6%至2 MDa。从由此获得的弱结合的大蛋白质复合物的分子量，例如，可以导出完整复合物的结合化学计量和作为pH的函数的复合物稳定性。特定的蛋白质复合物，如抗生物素蛋白或过氧化氢酶同源四聚体的例子，用于说明高质量的生物特异性复合物的表征技术的潜力。电荷减少的纳米ESI GEMMA的当前和未来的应用，如化学反应监测（免疫球蛋白G的还原过程）或完整的病毒，超分子复合物的大小测定的讨论，并监测部分解离的人鼻病毒，提供。版权所有（C）2001约翰威利父子有限公司
This study explores the potential of a novel electrospray based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet mode'. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha -particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided. Copyright (C) 2001 John Wiley & Sons, Ltd.