Köhler Martin, Marchand Adrien, Hentzen Nina B, Egli Jasmine, Begley Alina I, Wennemers Helma, Zenobi Renato
Department of Chemistry and Applied Biosciences , ETH Zurich , 8093 , Zurich , Switzerland . Email:
Chem Sci. 2019 Sep 3;10(42):9829-9835. doi: 10.1039/c9sc03248g. eCollection 2019 Nov 14.
Collagen model peptides are useful for understanding the assembly and structure of collagen triple helices. The design of self-assembling heterotrimeric helices is particularly challenging and often affords mixtures of non-covalent assemblies that are difficult to characterize by conventional NMR and CD spectroscopic techniques. This can render a detailed understanding of the factors that control heterotrimer formation difficult and restrict rational design. Here, we present a novel method based on electrospray ionization mass spectrometry to investigate homo- and heterotrimeric collagen model peptides. Under native conditions, the high resolving power of mass spectrometry was used to access the stoichiometric composition of different triple helices in complex mixtures. A temperature-controlled electrospray ionization source was built to perform thermal denaturation experiments and provided melting temperatures of triple helices. These were found to be in good agreement with values obtained from CD spectroscopic measurements. Importantly, for mixtures of coexisting homo- and heterotrimers, which are difficult to analyze by conventional methods, our technique allowed for the identification and monitoring of the unfolding of each individual species. Their respective melting temperatures could easily be accessed in a single experiment, using small amounts of sample.
胶原蛋白模型肽对于理解胶原蛋白三螺旋的组装和结构很有用。自组装异源三聚体螺旋的设计尤其具有挑战性，并且常常会产生非共价组装的混合物，这些混合物难以通过传统的核磁共振（NMR）和圆二色（CD）光谱技术进行表征。这可能导致难以详细了解控制异源三聚体形成的因素，并限制合理设计。在此，我们提出了一种基于电喷雾电离质谱的新方法，用于研究同源和异源三聚体胶原蛋白模型肽。在天然条件下，利用质谱的高分辨能力来确定复杂混合物中不同三螺旋的化学计量组成。构建了一个温度可控的电喷雾电离源来进行热变性实验，并提供三螺旋的解链温度。发现这些温度与通过CD光谱测量获得的值高度一致。重要的是，对于传统方法难以分析的共存同源和异源三聚体混合物，我们的技术能够识别和监测每个单独物种的解折叠过程。使用少量样品，在单个实验中就可以轻松获得它们各自的解链温度。
