Thermal dissociation of multimeric protein complexes by using nanoelectrospray mass spectrometry
Thermal dissociation of multimeric protein complexes by using nanoelectrospray mass spectrometry
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DOI：
10.1021/ac034132x
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发表时间：
2003-05-15
影响因子：
7.4
通讯作者：
Robinson, CV
中科院分区：
文献类型：
--
作者：
Benesch, JLP;Sobott, F;Robinson, CV
大分子系统在不同温度下的行为通常对其生物活性和功能至关重要。虽然个别蛋白质的热诱导的变化很容易通过许多光谱方法监测，但生物分子的非共价复合物的变化更难以解释。纳米电喷雾质谱在研究大的非共价复合物中正变得越来越强大，在这里，我们描述了一种新型探针的设计，表征和应用，该探针允许在电喷雾毛细管中对溶液进行热控制。的蛋白质溶菌酶的解折叠的转变温度是很容易获得的，并与荧光光谱法测量的密切相关，从而证明了这种方法的有效性。我们应用这种技术的研究200 kDa的复合物的小热休克蛋白TaHSP16.9，揭示了它的解离成亚寡聚物种和增加其大小和多分散性在高温下。与此相反，气相活化的这种复合物也进行，并产生一个解离途径从根本上不同于所观察到的热活化溶液中。因此，该探针允许以生物学相关的方式研究非共价复合物的可逆热诱导变化。
The behavior of macromolecular systems at different temperatures is often crucial to their biological activity and function. While heat-induced changes of individual proteins are readily monitored by a number of spectroscopic methods, changes in noncovalent complexes of biomolecules are more challenging to interpret. Nanoelectrospray mass spectrometry is becoming increasingly powerful in the study of large noncovalent complexes, and here we describe the design, characterization, and application of a novel probe that allows the thermocontrol of the solution in the electrospray capillary. The transition temperature for the unfolding of the protein lysozyme is readily obtained and correlates closely with that measured by fluorescence spectroscopy, thereby demonstrating the validity of this approach. We apply this technique to the study of the 200-kDa complex of the small heat shock protein TaHSP16.9, revealing both its dissociation into suboligomeric species and an increase in its size and polydispersity at elevated temperatures. In contrast, gas-phase activation of this complex is also carried out and yields a dissociation pathway fundamentally different from that observed for thermal activation in solution. As such, this probe allows the study of the reversible heat-induced changes of noncovalent complexes in a biologically relevant manner.