Insight into Heterogeneous Distribution of Protein Aggregates at the Surface Layer Using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Imaging.
Insight into Heterogeneous Distribution of Protein Aggregates at the Surface Layer Using Attenuated Total Reflection-Fourier Transform Infrared Spectroscopic Imaging.
复制标题
使用衰减全反射傅立叶变换红外光谱成像深入了解表面层蛋白质聚集体的不均匀分布。
DOI：
10.1021/acs.analchem.0c00250
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发表时间：
2020
影响因子：
7.4
通讯作者：
Tiernan H
中科院分区：
文献类型：
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作者：
Tiernan H
单克隆抗体（mAb）在过去几十年中已被用作治疗剂。有必要研究这些单克隆抗体在应激条件下的稳定性，并阐明聚集机制，作为开发方法的一种手段，最大限度地减少了问题。衰减全反射（ATR）-FTIR光谱成像允许在约0.5-5 μm的穿透深度处探测样品，这使得它适合于研究聚集的蛋白质，当聚集为靠近ZnSe内反射元件（IRE）表面的层时。在这里，宏观ATR-FTIR光谱成像，沿着与一个可变的入射角配件，已被用来区分蛋白质的二级结构在本体溶液和那些沉淀到或附近的ZnSe IRE表面。从单个威尔斯孔中的蛋白质样品获得的IgG光谱已被平均、提取和预处理，并且蛋白质样品的酰胺I带被比较并进一步分析以揭示在ZnSe IRE表面的蛋白质分布。这些发现显示了在ZnSe IRE表面（0.5-5 μm）处的IgG聚集体的深度分布，并且不遵循随着入射角增加或穿透深度增加而减少蛋白质存在的趋势，这表明聚集体在z方向上的不规则分布。
Monoclonal antibodies (mAbs) have been used as therapeutics for the last few decades. It is necessary to investigate the stability of these mAbs under stress conditions and to elucidate aggregation mechanisms as a means of developing approaches which minimize the problem. Attenuated total reflection (ATR)-FTIR spectroscopic imaging allows probing of a sample at a depth of penetration of around 0.5–5 μm, which makes it suitable for the study of aggregated proteins when accumulated as a layer close to the surface of the ZnSe internal reflection element (IRE). Here, macro ATR-FTIR spectroscopic imaging, along with a variable angle of incidence accessory, have been used to differentiate between the secondary structure of proteins in bulk solution and those that have precipitated onto or near the ZnSe IRE surface. IgG spectra obtained from protein samples in individual wells have been averaged, extracted, and preprocessed, and the Amide I bands of the protein samples were compared and further analyzed to reveal protein distribution at the ZnSe IRE surface. These findings show depth profiling of IgG aggregates at the ZnSe IRE surface (0.5–5 μm) and do not follow a trend of decreasing protein presence with an increasing angle of incidence or increasing depth of penetration, suggesting an irregular distribution of aggregates in thez-direction.