Smith Brandye M, Franzen Stefan
Department of Chemistry, North Carolina State University, Raleigh 27695-8204, USA.
Anal Chem. 2002 Aug 15;74(16):4076-80. doi: 10.1021/ac020103v.
The application of single-pass attenuated total reflection Fourier transform infrared (ATR-FT-IR) microscopy was investigated for secondary structure analysis of 15 representative proteins in H2O solution. This is the first reported application of single-pass ATR-FT-IR for protein analysis; thus, the method was validated using transmission FT-IR and multipass ATR-FT-IR as referee methods. The single-pass ATR-FT-IR technique was advantageous since the single-pass geometry permits rapid secondary structure analysis on small volumes of protein in H2O solution without the use of demountable thin path length sample cells. Moreover, the fact that H2O backgrounds were small allowed the simultaneous observation of the amide I-III, A, and B regions without having to perform H2O subtraction. A comparison of replicate protein spectra indicated that the single-pass ATR-FT-IR method yields more reproducible data than those acquired by transmission FT-IR. The observed trends for the amide I-III and A bands obtained by single-pass ATR-FT-IR agreed with those in the literature for conventional transmission FT-IR.
研究了单通道衰减全反射傅里叶变换红外（ATR-FT-IR）显微镜在H2O溶液中对15种代表性蛋白质二级结构分析中的应用。这是首次报道将单通道ATR-FT-IR用于蛋白质分析；因此，该方法使用透射FT-IR和多通道ATR-FT-IR作为参考方法进行了验证。单通道ATR-FT-IR技术具有优势，因为单通道几何结构允许在不使用可拆卸的薄光程样品池的情况下，对H2O溶液中的少量蛋白质进行快速二级结构分析。此外，H2O背景较小这一事实使得能够同时观察酰胺I-III、A和B区域，而无需进行H2O扣除。对重复蛋白质光谱的比较表明，单通道ATR-FT-IR方法产生的数据比透射FT-IR获得的数据更具可重复性。单通道ATR-FT-IR获得的酰胺I-III和A带的观察趋势与传统透射FT-IR文献中的趋势一致。
