Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia.
Department of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Croatia.
Anal Bioanal Chem. 2024 Oct;416(24):5377-5386. doi: 10.1007/s00216-024-05473-9. Epub 2024 Aug 22.
Manganese ion homeostasis is vital for bacteria and is achieved via manganese-dependent transcription factors. Manganese mediation of transcription factor attachment to the corresponding oligonucleotide sequences can be investigated, e.g. via electrophoretic mobility shift assays (EMSA). Formation of specific biocomplexes leads to differences in the migration pattern upon gel electrophoresis. Focusing on electrophoresis in the gas-phase, applying a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) also known as nES differential mobility analyzer (nES DMA), and on transcription factors (MntR proteins) from Bacillus subtilis and Mycobacterium tuberculosis, we took interest in the gas-phase electrophoresis of the corresponding biospecific complexes. We compared nES GEMMA, separating analytes in the nanometer regime (a few to several hundred nm in diameter) in the gas-phase in their native state according to particle size, to EMSA data. Indeed we were able to demonstrate manganese-mediated attachment of MntR to target genomic sequences with both analytical techniques. Despite some inherent pitfalls of the nES GEMMA method like analyte/instrument surface interactions, we were able to detect the target complexes. Moreover, we were able to calculate the molecular weight (MW) of the obtained species by application of a correlation function based on nES GEMMA obtained data. As gas-phase electrophoresis also offers the possibility of offline hyphenation to orthogonal analysis techniques, we are confident that nES GEMMA measurements are not just complementary to EMSA, but will offer the possibility of further in-depth characterization of biocomplexes in the future.
锰离子内稳态对细菌至关重要，是通过锰依赖的转录因子实现的。可以通过电泳迁移率变动分析（EMSA）等方法来研究锰对转录因子与相应寡核苷酸序列结合的介导作用。特定生物复合物的形成会导致凝胶电泳迁移模式的差异。本研究关注气相电泳，应用纳升电喷雾气相电泳迁移率分子分析仪（nES GEMMA），又称纳升差分迁移率分析仪（nES DMA），以及枯草芽孢杆菌和结核分枝杆菌的 MntR 蛋白，研究相应生物特异性复合物的气相电泳。我们将 nES GEMMA 与 EMSA 数据进行了比较，nES GEMMA 可根据粒径在纳米级（直径为数纳米至数百纳米）将气相中的分析物在其天然状态下进行分离。实际上，我们能够利用这两种分析技术证明锰介导的 MntR 与靶基因组序列的结合。尽管 nES GEMMA 方法存在一些固有缺陷，如分析物/仪器表面相互作用，但我们仍能够检测到目标复合物。此外，我们还可以通过应用基于 nES GEMMA 获得的数据的相关函数来计算获得的物种的分子量（MW）。由于气相电泳还提供了与正交分析技术离线连接的可能性，我们相信 nES GEMMA 测量不仅是 EMSA 的补充，而且还将为未来进一步深入研究生物复合物提供可能。
