Origin and number of charges observed on multiply-protonated native proteins produced by ESI
Origin and number of charges observed on multiply-protonated native proteins produced by ESI
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DOI：
10.1016/s1387-3806(02)00588-2
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发表时间：
2002-08-01
影响因子：
1.8
通讯作者：
Kebarle, P
中科院分区：
文献类型：
--
作者：
Felitsyn, N;Peschke, M;Kebarle, P
天然蛋白质，特别是天然非共价键合的蛋白质-蛋白质和蛋白质-底物复合物是非常感兴趣的，并通过ESI-MS方法进行了深入研究。这些离子上的多重电荷不仅有助于降低m/z值，而且在这些络合物的化学行为中也起着重要的作用，从文献和目前的工作中提出的证据支持电荷残留模型（CRM）作为带电的球状蛋白质在气相中形成的模式。在ESI过程中，最终形成仅包含一个蛋白质分子的非常小的水滴。这些液滴的表面由于溶液中也存在的盐而被过量的小离子充电。因此，在正离子模式中，并且当缓冲液（乙酸铵）是所使用的主要电解质时，过量的小正离子是NH 4+离子。液滴中的水分蒸发后会产生球状蛋白质残留物。蛋白质通过过量的正离子如NH 4+而带电。可利用的NH 4+离子的数目Z（CRM）可以基于CRM来预测。蛋白质为了能够容纳所有提供的质子，必须具有位于蛋白质表面的足够数量的碱性侧链。发现大多数蛋白质具有足够的碱性位点来保持电荷Z（CRM）。这些酶的实例是碳酸酐酶和细胞色素c。对于这些蛋白质，用ESI-MS观察到的电荷被发现接近等于电荷Z（CRM）。一些不寻常的蛋白质，如胃蛋白酶，具有太少的碱性侧链，远小于电荷的数量，Z（CRM）提供。对于这些蛋白质，蛋白质上可用的碱性位点的数量决定了CRM提供的电荷中有多少将被保留。可以计算出碱性中心的数目，并发现与质谱中观察到的电荷相一致。也可以根据CRM进行其他预测。因此，水滴的蒸发将导致在蛋白质上形成中性（不带电）加合物，这是由于缓冲液的中性组分。加合物的近似数目可以预测。还可以预测在质谱仪的去溶剂化阶段，哪些缓冲液将导致难以除去的加合物。(C)2002 Elsevier Science B. V.保留所有权利。
Native proteins and particularly native non-covalently bonded protein-protein and protein-substrate complexes are of great interest and are intensely studied by ESI-MS methods. The multiple charges on these ions are not only useful in lowering the m/z values but play also an important role in the chemical behavior of these complexes.Evidence from the literature and the present work is presented which supports the charge residue model (CRM) as the mode of formation of the charged globular proteins in the gas phase. Very small water droplets which contain only one protein molecule are ultimately formed in the ESI process. The surface of these droplets is charged by an excess of small ions due to a salt which is also present in the solution. Thus, in the positive ion mode, and when the buffer (ammonium acetate) is the main electrolyte used, the excess small positive ions are NH4+ ions. Evaporation of the water in the droplet leads to a residue which is the globular protein. The protein is charged by the excess positive ions such as NH4+. The number of NH4+ ions available, Z(CRM), can be predicted on the basis of CRM. The proteins in order to be able to hold all of the protons provided must have a sufficient number of basic side chains located at the surface of the protein. It is found that most proteins have more than enough basic sites to hold the charge, Z(CRM). Examples for these are carbonic anhydrase and cytochrome c. For these proteins the charge observed with ESI-MS is found to be close to equal to the charge, Z(CRM) suppliedSome unusual proteins such as pepsin, have too few basic side chains, much less than the number of charges, Z(CRM) provided. For these proteins the number of basic sites available on the protein determine how many of the charges provided by CRM will be retained. The number of basic sites can be evaluated and is found in agreement with the observed charges in the mass spectrum.Other predictions can also be made on the basis of the CRM. Thus, evaporation of the water droplet will lead to formation of neutral (uncharged) adducts on the protein, which are due to neutral components of the buffer. The approximate number of adducts can be predicted. Predictions can also be made which buffers will lead to adducts difficult to get rid off, in the desolvation stage of the mass spectrometer. (C) 2002 Elsevier Science B.V. All rights reserved.