Manchester Institute of Biotechnology, School of Chemistry, Faculty of Science and Engineering , University of Manchester , Manchester M1 7DN , U.K.
School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre , University of Manchester , Manchester M13 9PL , U.K.
Mol Pharm. 2018 Jul 2;15(7):2785-2796. doi: 10.1021/acs.molpharmaceut.8b00282. Epub 2018 Jun 12.
The ability to monitor the behavior of individual proteins in complex mixtures has many potential uses, ranging from analysis of protein interactions in highly concentrated solutions, modeling biological fluids or the intracellular environment, to optimizing biopharmaceutical co-formulations. Differential labeling NMR approaches, which traditionally use N or C isotope incorporation during recombinant expression, are not always practical in cases when endogenous proteins are obtained from an organism, or where the expression system does not allow for efficient labeling, especially for larger proteins. This study proposes differential labeling of proteins by covalent attachment of F groups with distinct chemical shifts, giving each protein a unique spectral signature which can be monitored by F NMR without signal overlap, even in complex mixtures, and without any interfering signals from the buffer or other unlabeled components. Parameters, such as signal intensities, translational diffusion coefficients, and transverse relaxation rates, which report on the behavior of individual proteins in the mixture, can be recorded even for proteins as large as antibodies at a wide range of concentrations.
在复杂混合物中监测单个蛋白质行为的能力具有许多潜在用途，从分析高浓度溶液中的蛋白质相互作用、模拟生物流体或细胞内环境，到优化生物制药共制剂。传统上，在从生物体获得内源性蛋白质或表达系统不允许进行有效标记的情况下，特别是对于较大的蛋白质，使用 N 或 C 同位素掺入的差异标记 NMR 方法并不总是实用。本研究提出了通过共价连接具有不同化学位移的 F 基团对蛋白质进行差异标记，使每种蛋白质具有独特的光谱特征，可以通过 F NMR 进行监测，即使在复杂混合物中也没有信号重叠，并且没有来自缓冲液或其他未标记成分的任何干扰信号。可以记录报告混合物中单个蛋白质行为的参数，如信号强度、平动扩散系数和横向弛豫率，即使对于抗体等大蛋白，也可以在很宽的浓度范围内记录。
