Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, United States; Greenlight Biosciences, Medford, MA, 02155, United States.
Department of Chemistry & Chemical Biology, Northeastern University, Boston, MA 02115, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 1;1188:123044. doi: 10.1016/j.jchromb.2021.123044. Epub 2021 Nov 20.
Nucleotides exemplify some of the building blocks of life, comprising DNA & RNA, participating in processes such as cell signaling and metabolism, and serving as carriers of metabolic energy. The quantification of these compounds in biological samples is critical for researchers to understand complex systems. Herein, we demonstrate an anion exchange chromatography method utilizing a pH range of 8 to 10, which provides superior resolution and selectivity to previously reported methods and, more importantly, gives the flexibility to shift analyte selectivity if resolution between analytes is not optimal. We have applied the method to study the kinetics of the nucleotide pool in a bacterial cell-free lysate system that is producing RNA. Sample to sample runtimes are less than 18 min and recoveries greater than 96% were observed for all analytes through our methanol quench protocol with day-to-day variabilities less than 5%. This method reliably detects and quantifies all analytes that were expected to be observed in the process and helps lay the groundwork for future nucleotide research.
核苷酸是生命的组成部分之一，包括 DNA 和 RNA，参与细胞信号传递和代谢等过程，并作为代谢能量的载体。在生物样本中对这些化合物进行定量分析对于研究人员理解复杂系统至关重要。在此，我们展示了一种利用 pH 值 8 到 10 的阴离子交换色谱法，与之前报道的方法相比，该方法提供了更高的分辨率和选择性，更重要的是，如果分析物之间的分辨率不理想，则具有改变分析物选择性的灵活性。我们已经将该方法应用于研究在产生 RNA 的细菌无细胞裂解物系统中核苷酸库的动力学。通过我们的甲醇淬灭方案，所有分析物的样品到样品运行时间都小于 18 分钟，回收率均大于 96%，并且日间变异性小于 5%。该方法可靠地检测和定量了该过程中预计会观察到的所有分析物，为未来的核苷酸研究奠定了基础。
