Department of Pharmaceutical and Biomedical Sciences, The University of Georgia College of Pharmacy, 250 West Green Street, Athens, Georgia 30602-2352, United States.
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Ain Shams University, Organization of African Unity Street, Abassia, Cairo 11566, Egypt.
J Am Soc Mass Spectrom. 2021 Feb 3;32(2):497-508. doi: 10.1021/jasms.0c00380. Epub 2021 Jan 21.
Improving the mobile phase of electrospray oligonucleotides has been a major focus in the field of oligonucleotides. These improved mobile phases should reduce the charge state envelope of oligonucleotides coupled with electrospray ionization, which is key to reducing spectral complexity and increasing sensitivity. Traditional mobile phase compositions with fluorinated alcohol and alkylamine, like hexafluoroisopropanol (HFIP) and triethylamine (TEA), have a large amount of cationic adduction and many charge states. Utilizing different fluorinated alcohol and alkylamine combinations, like nonafluoro--butyl alcohol (NFTB) and octylamine (OA), can selectively reduce the charge states analyzed. Other classes of biomolecules have been analyzed with anionic salts to stabilize complexes, increase the molecular peak detection, and even provide unique structural information about these molecules; however, there have been no studies using anionic salts with oligonucleotides. Our experiments systematically study the stability and binding of ammonium anionic salt. We show that anions selectively bind low charge states of these oligonucleotides. Ion-mobility measurements are made to determine the collision cross section (CCS) of these oligonucleotides with anion adduction. We utilize both a nucleic acid exact hard sphere simulation (EHSS) calibration and a protein calibration. We are able to show that NFTB/OA is a good choice for the study of oligonucleotides with reduced charge states for the binding of anionic salts and the determination of CCS using ion mobility.
改进电喷雾寡核苷酸的流动相一直是寡核苷酸领域的主要关注点。这些改进的流动相应能减少与电喷雾电离偶联的寡核苷酸的电荷状态包络，这是降低光谱复杂性和提高灵敏度的关键。具有氟化醇和烷基胺的传统流动相组成，如六氟异丙醇 (HFIP) 和三乙胺 (TEA)，具有大量的阳离子加合物和许多电荷状态。利用不同的氟化醇和烷基胺组合，如全氟丁醇 (NFTB) 和辛胺 (OA)，可以选择性地减少分析的电荷状态。已经有其他类别的生物分子被阴离子盐分析，以稳定复合物，增加分子峰检测，甚至提供这些分子的独特结构信息；然而，还没有关于寡核苷酸与阴离子盐的研究。我们的实验系统地研究了铵阴离子盐的稳定性和结合。我们表明，阴离子选择性地结合这些寡核苷酸的低电荷状态。进行离子迁移率测量以确定这些带有阴离子加合物的寡核苷酸的碰撞截面 (CCS)。我们同时使用核酸精确硬球模拟 (EHSS) 校准和蛋白质校准。我们能够表明，NFTB/OA 是研究带阴离子盐的寡核苷酸的一个很好的选择，用于减少电荷状态的结合和使用离子迁移率确定 CCS。
