Preserving the structure of adsorbed protein films for time-of-flight secondary ion mass spectrometry analysis.
Preserving the structure of adsorbed protein films for time-of-flight secondary ion mass spectrometry analysis.
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保留吸附蛋白质膜的结构，用于飞行时间二次离子质谱分析。
DOI：
10.1002/jbm.a.10063
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Castner,DavidG
中科院分区：
文献类型：
--
作者：
Xia,Nan;Castner,DavidG
吸附的蛋白质膜的表征与超真空（UHV）表面分析技术需要脱水的样品，这可能会导致显着的蛋白质结构的改变。在干燥过程中需要保持吸附的蛋白质膜的结构，因此可以在更能代表蛋白质在水性环境中的实际结构的状态下进行UHV分析。在这项研究中，两种方法，海藻糖保护和戊二醛固定，探讨其在保留吸附蛋白质结构的可行性，一个强大的UHV表面分析技术，飞行时间二次离子质谱（ToF‐西姆斯）。在我们之前的研究中，海藻糖保护显示出西姆斯分析的一些前景，并在本研究中用模型蛋白纤维蛋白原进一步检查。使用主成分分析（PCA）和静态西姆斯分析的组合，我们发现海藻糖保护可以减少干燥时纤维蛋白原的构象变化，并防止其解折叠和暴露疏水结构域。此外，当吸附的蛋白质膜变得更致密时，干燥引起的蛋白质结构变化减少。因此，海藻糖涂层提供的保护在较低的蛋白质表面浓度下更显着。另一种方法，戊二醛固定，首次用于ToF‐西姆斯分析。赖氨酸的α-氨基被确定为蛋白质结构中对戊二醛固定的主要反应基团。在干燥前和干燥后戊二醛固定的纤维蛋白原薄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薄膜之间观察到的结构差异相似。发现戊二醛固定是西姆斯分析中海藻糖保护的一种可行的替代稳定方法。© 2003威利期刊公司J Biomed Mater Res 67A：179-190，2003
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 ϵ‐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. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 179–190, 2003
电子显微镜固定装置
影响因子：
5
作者：
M. Hayat
通讯作者：
M. Hayat
美国与 IIASA 的联系
影响因子：
56.9
作者：
W. Carey
通讯作者：
W. Carey
影响因子：
7.4
作者：
MANTUS, DS;RATNER, BD;MOULDER, JF
通讯作者：
MOULDER, JF