EMD Millipore Corporation, Bedford, Massachusetts 01730, United States.
Anal Chem. 2012 Sep 4;84(17):7367-73. doi: 10.1021/ac300994d. Epub 2012 Aug 15.
Confocal Raman microscopy is a nondestructive analytical technique that combines the chemical information from vibrational spectroscopy with the spatial resolution of confocal microscopy. It was applied, for the first time, to measure conformation and distribution of protein adsorbed in wetted chromatographic particles. Monoclonal antibody was loaded into the Fractogel EMD SO(3) (M) cation exchanger at 2 mS/cm or 10 mS/cm. Amide I and III frequencies in the Raman spectrum of the adsorbed protein suggest that there are no detectable changes of the original β-sheet conformation in the chromatographic particles. Protein depth profile measurements indicate that, when the conductivity is increased from 2 mS/cm to 10 mS/cm, there is a change in mass transport mechanism for protein adsorption, from the shrinking-core model to the homogeneous-diffusion model. In this study, the use of confocal Raman microscopy to measure protein distribution in chromatographic particles fundamentally agrees with previous confocal laser scanning microscopic investigations, but confocal Raman spectroscopy enjoys additional advantages: use of unlabeled protein to eliminate fluorescent labeling, ability for characterization of protein secondary structure, and ability for spectral normalization to provide a nondestructive experimental approach to correct light attenuation effects caused by refractive index (RI) mismatching in semiopaque chromatographic particles.
共焦拉曼显微镜是一种非破坏性分析技术，它将振动光谱的化学信息与共焦显微镜的空间分辨率相结合。首次将其应用于测量在润湿的色谱颗粒中吸附的蛋白质的构象和分布。单克隆抗体以 2 mS/cm 或 10 mS/cm 的浓度加载到 Fractogel EMD SO(3) (M)阳离子交换剂中。吸附蛋白质的拉曼光谱中的酰胺 I 和 III 频率表明，在色谱颗粒中没有检测到原始β-折叠构象的变化。蛋白质深度剖面测量表明，当电导率从 2 mS/cm 增加到 10 mS/cm 时，蛋白质吸附的传质机制发生变化，从收缩核模型变为均相扩散模型。在这项研究中，使用共焦拉曼显微镜测量色谱颗粒中的蛋白质分布与以前的共焦激光扫描显微镜研究基本一致，但共焦拉曼光谱具有额外的优势：使用未标记的蛋白质消除荧光标记、能够对蛋白质二级结构进行表征，以及能够进行光谱归一化，为校正半不透明色谱颗粒中折射率（RI）不匹配引起的光衰减效应提供了一种非破坏性的实验方法。
