蛋白质十二烷基硫酸钠毛细管凝胶电泳中的电迁移分散,Analytical Chemistry

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蛋白质十二烷基硫酸钠毛细管凝胶电泳中的电迁移分散
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-12 , DOI: 10.1021/acs.analchem.2c02348
Csenge Filep ₁ , András Guttman _{1,

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Affiliation

使用硼酸盐交联葡聚糖筛分基质在十二烷基硫酸钠毛细管凝胶电泳 (SDS-CGE) 中研究治疗性单克隆抗体奥马珠单抗的轻链和重链亚基峰的电迁移分散。增加硼酸含量 (340–640 mM) 会导致所有凝胶-缓冲液组合物中的低 (2%) 和高 (10%) 葡聚糖浓度凝胶的电迁移色散变化。在重链片段的情况下，硼酸盐浓度升高导致拖尾减少和前沿增加，分别使用较高和较低的葡聚糖浓度凝胶。另一方面，对于本研究中检测的两种葡聚糖浓度，轻链片段的前沿随着硼酸盐浓度的增加而增加。凝胶-缓冲系统中甘油成分水平的增加与两种葡聚糖浓度中硼酸盐浓度的增加所产生的效果相同。检测到的电迁移分散体被认为是形成单体和二聚甘油-硼酸盐复合物作为共离子成分的结果，迁移速度比非共轭四羟基硼酸盐慢。此外，四羟基硼酸根阴离子与葡聚糖聚合物的葡萄糖结构单元的络合将其迁移率降低到几乎为零，从而进一步降低了共离子物质的最终有效迁移率。我们认为，随着硼酸浓度的增加，在 SDS-CGE 中观察到的单克隆抗体片段的前沿和/或拖尾峰形状可以被认为是多种影响的结果，包括 pH、筛分基质孔径、粘度和迁移率的变化共离子硼酸盐加合物与凝胶缓冲剂成分的变化。虽然电迁移分散介导的谱带展宽通常可以通过使共离子物质的有效迁移率与感兴趣的分析物分子相匹配来最小化，但在硼酸盐交联葡聚糖凝胶的情况下，硼酸浓度的优化需要特别考虑其凝胶交联功能。对于 IgG 分析物的轻链和重链片段，分别使用 10% 葡聚糖/340 mM 硼酸和 10% 葡聚糖/640 mM 硼酸凝胶缓冲系统获得最佳峰形。基于这一观察，我们在这里介绍了蛋白质 SDS-CGE 中硼酸盐梯度介导的瞬态迁移率匹配的概念。这种新方法在一次运行中为远距离迁移的 IgG 亚基带来了接近最佳的峰形，并解开了长期寻找的可能的解决方案，以进行毛细管孔径梯度凝胶电泳。

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Electromigration Dispersion in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Proteins

The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340–640 mM) caused electromigration dispersion shifts for both low (2%)- and high (10%)-dextran-concentration gels in all gel–buffer compositions. In case of the heavy-chain fragment, elevated borate concentrations resulted in decreasing tailing and increasing fronting with the use of higher- and lower-dextran-concentration gels, respectively. The light-chain fragment, on the other hand, exhibited increased fronting with increasing borate concentration for both dextran concentrations examined in this study. Increase of the glycerol ingredient level in the gel–buffer system caused the same effect as the increasing borate concentration in both dextran concentrations. The detected electromigration dispersion was considered as the result of the formation of monomeric and dimeric glycerol-borate complexes as co-ionic constituents, migrating slower than that of the unconjugated tetrahydroxyborate. In addition, complexation of the tetrahydroxyborate anion with the glucose building blocks of the dextran polymer decreased its mobility to practically zero, contributing to further decrease in the resultant effective mobility of the co-ionic species. We suggest that the observed fronting and/or tailing peak shapes of the monoclonal antibody fragments in SDS–CGE at increasing boric acid concentrations can be considered as the result of multiple effects including changes in pH, sieving matrix pore size, viscosity, and the mobility variation of the co-ionic borate adducts with the gel–buffer ingredients. While electromigration dispersion-mediated band broadening, in general, can be minimized via matching the effective mobility of the co-ionic species to the analyte molecules of interest, in case of borate cross-linked dextran gels, optimization of the boric acid concentration required special consideration of its gel cross-linking function. For the light- and heavy-chain fragments of the IgG analyte, best peak shapes were attained with the use of 10% dextran/340 mM boric acid and 10% dextran/640 mM boric acid-containing gel–buffer systems, respectively. Based on this observation, here we introduce the concept of borate-gradient-mediated transient mobility matching in SDS–CGE of proteins. This novel approach resulted in close to optimal peak shapes for the distantly migrating IgG subunits within a single run, as well as unraveled the long-sought possible solution to perform capillary pore-size-gradient gel electrophoresis.

更新日期：2022-09-12

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