Effects of buffer loading for electrospray ionization mass spectrometry of a noncovalent protein complex that requires high concentrations of essential salts.
Effects of buffer loading for electrospray ionization mass spectrometry of a noncovalent protein complex that requires high concentrations of essential salts.
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DOI：
10.1016/j.jasms.2010.02.003
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发表时间：
2010-06
影响因子：
3.2
通讯作者：
Williams ER
中科院分区：
文献类型：
--
作者：
Sterling HJ;Batchelor JD;Wemmer DE;Williams ER
电喷雾离子化（ESI）质谱（MS）是分析生物大分子复合物活性形式的有效方法。然而，这些溶液通常含有高浓度的盐和/或去污剂，其通过使离子形成的可再现性降低而对ESI性能产生不利影响，从而导致严重的内收或离子抑制。许多用于从非挥发性添加剂中分离复合物的方法常规地与ESI-MS一起使用，但是这些方法可能不适合于需要这种稳定剂来进行活性的复合物。在这里，缓冲液加载使用浓度范围从0.22至1.41 M的乙酸铵的ESI质谱的溶液中含有的结构域截断突变体的σ54激活剂从Aquifex aeolicus的影响进行了研究。这种44.9 kDa的蛋白质需要存在毫摩尔浓度的Mg 2+、BeF 3 −和ADP（在160 °C下）才能组装成活性同源六聚体。乙酸铵的加入可以提高信号的稳定性和重现性，并可以显著降低加合和背景信号。然而，在较高浓度下，六聚体的相对离子丰度减少，而构成单体的相对离子丰度增加。这些结果与通过ATP水解测量的酶活性的损失一致，并且表明高浓度的乙酸铵干扰六聚体的组装。这表明，用乙酸铵加载的缓冲液对于获得需要高浓度必需盐的复合物的ESI信号是有效的，但是可以通过在高浓度下破坏关键的静电相互作用来干扰复合物的形成和/或使复合物不稳定。
Electrospray ionization (ESI) mass spectrometry (MS) is a powerful method for analyzing the active forms of macromolecular complexes of biomolecules. However, these solutions often contain high concentrations of salts and/or detergents that adversely effect ESI performance by making ion formation less reproducible, causing severe adduction or ion suppression. Many methods for separating complexes from nonvolatile additives are routinely used with ESI-MS, but these methods may not be appropriate for complexes that require such stabilizers for activity. Here, the effects of buffer loading using concentrations of ammonium acetate ranging from 0.22 to 1.41 M on the ESI mass spectra of a solution containing a domain truncation mutant of a σ54 activator from Aquifex aeolicus were studied. This 44.9 kDa protein requires the presence of millimolar concentrations of Mg2+, BeF3−, and ADP, (at ∼60 °C) to assemble into an active homo-hexamer. Addition of ammonium acetate can improve signal stability and reproducibility, and can significantly lower adduction and background signals. However, at higher concentrations, the relative ion abundance of the hexamer is diminished, while that of the constituent monomer is enhanced. These results are consistent with loss of enzymatic activity as measured by ATP hydrolysis and indicate that the high concentration of ammonium acetate interferes with assembly of the hexamer. This shows that buffer loading with ammonium acetate is effective for obtaining ESI signal for complexes that require high concentrations of essential salts, but can interfere with formation of, and/or destabilize complexes by disrupting crucial electrostatic interactions at high concentration.
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DOI：
10.1016/j.jasms.2009.06.012
发表时间：
2009-10
影响因子：
3.2
作者：
Sterling HJ;Williams ER
通讯作者：
Williams ER
影响因子：
2.9
作者：
Bishop, GR;Davidson, VL
通讯作者：
Davidson, VL
DOI：
10.1073/pnas.240326597
发表时间：
2000-12-19
影响因子：
11.1
作者：
Fändrich, M;Tito, MA;Robinson, CV
通讯作者：
Robinson, CV
影响因子：
2.9
作者：
Fedosova, NU;Esmann, M
通讯作者：
Esmann, M
影响因子：
14.8
作者：
Hernandez, Helena;Robinson, Carol V.
通讯作者：
Robinson, Carol V.