2D-Infrared Spectroscopy of Proteins in Water: Using the Solvent Thermal Response as an Internal Standard
2D-Infrared Spectroscopy of Proteins in Water: Using the Solvent Thermal Response as an Internal Standard
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DOI：
10.1021/acs.analchem.9b05601
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发表时间：
2020-02-18
影响因子：
7.4
通讯作者：
Hunt, Neil T.
中科院分区：
文献类型：
--
作者：
Hume, Samantha;Greetham, Gregory M.;Hunt, Neil T.
超快二维红外（2D-IR）光谱现在可以在几秒钟内获得，开辟了生物医学和制药领域相关的高通量筛选应用的可能性。然而，通过不同样品和不同仪器上记录的2D-IR光谱确定定量信息是困难的，因为光束对准、激光强度和样品条件的变化。最近，我们证明，2D-IR光谱的蛋白质酰胺I带可以在水溶液（H2O），而不是氘代（D2 O）溶剂中进行，我们现在报告的方法，使用的幅度相关的热响应H2O作为2D-IR光谱的内部归一化标准。使用与蛋白质信号在时间上分离的水响应来归一化光谱，可以显著降低测量到测量波动对数据的影响。我们证明，这种归一化方法可以创建用于测量绝对蛋白质浓度的校准曲线，并有利于可重复的差谱方法。这些进展使强大的数据处理策略，将是必不可少的实现自动化光谱分析工具的大规模2D-IR筛选含蛋白质的溶液和生物流体的研究取得了重大进展。
Ultrafast two-dimensional infrared (2D-IR) spectra can now be obtained in a matter of seconds, opening up the possibility of high-throughput screening applications of relevance to the biomedical and pharmaceutical sectors. Determining quantitative information from 2D-IR spectra recorded on different samples and different instruments is however made difficult by variations in beam alignment, laser intensity, and sample conditions. Recently, we demonstrated that 2D-IR spectroscopy of the protein amide I band can be performed in aqueous (H2O) rather than deuterated (D2O) solvents, and we now report a method that uses the magnitude of the associated thermal response of H2O as an internal normalization standard for 2D-IR spectra. Using the water response, which is temporally separated from the protein signal, to normalize the spectra allows significant reduction of the impact of measurement-to-measurement fluctuations on the data. We demonstrate that this normalization method enables creation of calibration curves for measurement of absolute protein concentrations and facilitates reproducible difference spectroscopy methodologies. These advances make significant progress toward the robust data handling strategies that will be essential for the realization of automated spectral analysis tools for large scale 2D-IR screening studies of protein-containing solutions and biofluids.