Monitoring Guanidinium-Induced Structural Changes in Ribonuclease Proteins Using Raman Spectroscopy and 2D Correlation Analysis
Monitoring Guanidinium-Induced Structural Changes in Ribonuclease Proteins Using Raman Spectroscopy and 2D Correlation Analysis
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DOI：
10.1021/ac303265q
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发表时间：
2013-04-02
影响因子：
7.4
通讯作者：
Goodacre, Royston
中科院分区：
文献类型：
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作者：
Brewster, Victoria L.;Ashton, Lorna;Goodacre, Royston
通过比较蛋白质的展开曲线来评估蛋白质的稳定性对于任何生物药物来说都是一个非常重要的表征和质量控制步骤，这通常是通过荧光光谱来衡量的。在这篇文章中，我们提出了拉曼光谱作为一种快速、非侵入性的替代分析方法，我们将证明这种方法具有更高的灵敏度，因此可以揭示用荧光测量无法观察到的非常微妙的蛋白质构象变化。拉曼光谱是一种强大的非破坏性方法，在蛋白质表征方面有着悠久的应用历史。在这项工作中，我们描述了如何使用拉曼显微镜作为一种快速和可靠的方法来跟踪蛋白质在化学变性剂存在下展开。我们将拉曼光谱数据与用荧光光谱分析的等效样品进行了比较，以验证拉曼方法的有效性。从拉曼和荧光展开曲线计算的[D](50)值和吉布斯自由能彼此关联很好，更重要的是与文献中发现的这些蛋白质的值相一致。此外，还对拉曼和荧光数据集进行了二维相关分析，以便进一步比较每种方法所指示的展开行为。由于许多生物药物是糖基化的，为了具有功能，我们比较了蛋白质(RNaseA)和糖蛋白(RNaseB)的拉曼光谱，并讨论了糖基化对蛋白质稳定性的影响。
Assessing the stability of proteins by comparing their unfolding profiles is a very important characterization and quality control step for any biopharmaceutical, and this is usually measured by fluorescence spectroscopy. In this paper we propose Raman spectroscopy as a rapid, noninvasive alternative analytical method and we shall show this has enhanced sensitivity and can therefore reveal very subtle protein conformational changes that are not observed with fluorescence measurements. Raman spectroscopy is a powerful nondestructive method that has a strong history of applications in protein characterization. In this work we describe how Raman microscopy can be used as a fast and reliable method of tracking protein unfolding in the presence of a chemical denaturant. We have compared Raman spectroscopic data to the equivalent samples analyzed using fluorescence spectroscopy in order to validate the Raman approach. Calculations from both Raman and fluorescence unfolding curves of [D](50) values and Gibbs free energy correlate well with each other and more importantly agree with the values found in the literature for these proteins. In addition, 2D correlation analysis has been performed on both Raman and fluorescence data sets in order to allow further comparisons of the unfolding behavior indicated by each method. As many biopharmaceuticals are glycosylated in order to be functional, we compare the unfolding profiles of a protein (RNase A) and a glycoprotein (RNase B) as measured by Raman spectroscopy and discuss the implications that glycosylation has on the stability of the protein.