Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting
Infrared absorbance spectroscopy of aqueous proteins: Comparison of transmission and ATR data collection and analysis for secondary structure fitting
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DOI：
10.1002/chir.23002
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发表时间：
2018-07-20
期刊：
影响因子：
2
通讯作者：
Rodger A
中科院分区：
文献类型：
--
作者：
Corujo MP;Sklepari M;Ang DL;Millichip M;Reason A;Goodchild SC;Wormell P;Amarasinghe DP;Lindo V;Chmel NP;Rodger A
蛋白质在水溶液中的衰减全反射（ATR）红外吸收光谱比透射光谱更容易进行，其中短路径长度的细胞需要可重复地组装。然而，所得ATR红外光谱的形状随样品的折射率和仪器配置而变化。折射率又取决于样品的吸光度。在这项工作中，它表明，室温三甘氨酸硫酸盐检测器和ZnSe ATR单元可以用来收集可重复的蛋白质光谱。还给出了一种将蛋白质ATR光谱转换为透射光谱形状的简单方法，该方法通过将Kramers-Krönig确定的水的折射率近似为跨酰胺I和II区域的四个线性分量的总和来进行。晶体表面的光强（45°入射）及其远离表面的衰减速率被确定为波数相关折射率以及来自表面的渐逝波的衰减的函数。结果是每个波数处的单个校正因子。光谱在1600 cm−1和1700 cm−1之间归一化为最大值1，并使用BioTools参考集应用自组织图谱二级结构拟合算法SOMSpec。由此产生的二级结构估计是令人鼓舞的ATR光谱的生物制药表征和质量控制应用的未来。
Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.
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影响因子：
2
作者：
Hall, Vincent;Sklepari, Meropi;Rodger, Alison
通讯作者：
Rodger, Alison
影响因子：
5.6
作者：
FERMI, G;PERUTZ, MF;FOURME, R
通讯作者：
FOURME, R
影响因子：
2.9
作者：
SREERAMA, N;WOODY, RW
通讯作者：
WOODY, RW
影响因子：
--
作者：
HARRICK, NJ
通讯作者：
HARRICK, NJ
影响因子：
--
作者：
HARRICK, NJ
通讯作者：
HARRICK, NJ