Analysis of nucleotide pools in bacteria using HPLC-MS in HILIC mode

TALANTA

卷:205

Article

Zbornikova, Eva^1,2  Knejzlik, Zdenek¹  Hauryliuk, Vasili^3,4,5  Krasny, Libor⁶  Rejman, Dominik¹ 

[1] Acad Sci Czech Republ, Inst Organ Chem & Biochem, Vvi, Flemingovonam 2, CZ-16610 Prague 6, Czech Republic

[2] Charles Univ Prague, Dept Analyt Chem, Fac Sci, Hlavova 8, Prague 2, Czech Republic

[3] Umea Univ, Dept Mol Biol, Bldg 6K,6L Univ Hosp Area, SE-90187 Umea, Sweden

[4] Umea Univ, Lab Mol Infect Med Sweden MIMS, Univ Hosp Area, Bldg 6K & 6L, SE-90187 Umea, Sweden

[5] Univ Tartu, Inst Technol, EE-50411 Tartu, Estonia

[6] Czech Acad Sci, Inst Microbiol, Vvi, Vldenska 1083, Prague 14220 4, Czech Republic

关键词: Nucleotide;    HPLC-MS;    HILIC;    ppGpp;    Stringent response;    Escherichia coli;   

DOI  :  10.1016/j.talanta.2019.120161

来源: Elsevier

【 摘 要 】

Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of > 25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.

【 授权许可】

Free