Xia Nan, Castner David G
National ESCA and Surface Analysis Center for Biomedical Problems, Department of Chemical Engineering, University of Washington, Box 351750, Seattle, Washington 98195-1750, USA.
J Biomed Mater Res A. 2003 Oct 1;67(1):179-90. doi: 10.1002/jbm.a.10063.
The characterization of adsorbed protein films with ultrahigh vacuum (UHV) surface analysis techniques requires dehydration of the samples, which can cause significant alterations in protein structure. It is desirable to preserve the structure of adsorbed protein films during drying, so UHV analysis could be done in a state that is more representative of proteins' actual structure in the aqueous environment. In this study, two methods, trehalose protection and glutaraldehyde fixation, were explored for their feasibility in preserving adsorbed protein structure for a powerful UHV surface analysis technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS). Trehalose protection had shown some promise for ToF-SIMS analysis in our previous study and was further examined with the model protein fibrinogen in this study. Using the combination of principal component analysis (PCA) and static ToF-SIMS analysis, we found that trehalose protection could reduce the conformation change of fibrinogen upon drying, and prevent it from unfolding and exposing hydrophobic domains. Moreover, when the adsorbed protein film became more densely packed, the drying-induced changes in protein structure were reduced. Thus, the protection afforded by trehalose coating was more significant at lower protein surface concentrations. The other method, glutaraldehyde fixation, was used in ToF-SIMS analysis for the first time. The epsilon-amino group of lysine was identified as the major reactive group in the protein structure toward glutaraldehyde fixation. Structural differences observed between fibrinogen films that were glutaraldehyde fixed before drying and after drying were similar to those observed between trehalose-protected and-unprotected dried fibrinogen films. Glutaraldehyde fixation was found to be a viable, alternative stabilizing method to trehalose protection for ToF-SIMS analysis.
用超高真空（UHV）表面分析技术表征吸附的蛋白质膜需要对样品进行脱水处理，这可能会导致蛋白质结构发生显著变化。在干燥过程中保持吸附蛋白质膜的结构是很有必要的，这样超高真空分析就可以在更能代表蛋白质在水环境中实际结构的状态下进行。在本研究中，探索了海藻糖保护和戊二醛固定这两种方法在为一种强大的超高真空表面分析技术——飞行时间二次离子质谱（ToF-SIMS）保留吸附蛋白质结构方面的可行性。在我们之前的研究中，海藻糖保护在ToF-SIMS分析中已显示出一定的前景，本研究中用模型蛋白纤维蛋白原对其进行了进一步研究。通过主成分分析（PCA）和静态ToF-SIMS分析相结合，我们发现海藻糖保护可以减少纤维蛋白原在干燥时的构象变化，并防止其展开和暴露疏水结构域。此外，当吸附的蛋白质膜变得更加紧密堆积时，干燥引起的蛋白质结构变化会减少。因此，在较低的蛋白质表面浓度下，海藻糖涂层提供的保护更为显著。另一种方法，戊二醛固定，首次用于ToF-SIMS分析。赖氨酸的ε-氨基被确定为蛋白质结构中对戊二醛固定起主要反应的基团。在干燥前和干燥后进行戊二醛固定的纤维蛋白原膜之间观察到的结构差异，与在海藻糖保护和未保护的干燥纤维蛋白原膜之间观察到的差异相似。戊二醛固定被发现是一种可行的、替代海藻糖保护的稳定方法，可用于ToF-SIMS分析。
