Selective Preconcentration and Mass Spectrometry Profiling of Charged Extracellular Modified Ribonucleosides by...

Extracellular modified ribonucleosides have recently emerged as potent cell-to-cell signaling molecules, but their low abundance in complex biofluids has impeded progress toward understanding the full complement of their functions. A major bottleneck is the lack of sample preparation techniques that can extract, purify, and preconcentrate extracellular modified nucleosides from biological fluids. Here, we address this limitation by developing a borate complexation, temperature-assisted ionic liquid microextraction (BTAIL-ME) technique. BTAIL-ME capitalizes on the formation of boronate esters with the cis-diols of ribonucleosides and the in situ production of an IL solvent to permit rapid and selective extraction of modified ribonucleosides. We synthesized and investigated the extraction performance of 8 IL solvents and optimized key parameters of BTAIL-ME, including temperature, extraction time, and IL concentration. The optimized BTAIL-ME method leveraged a dicationic IL 1,10-di-(3-benzylimidazolium)-decane bis-[(trifluoromethyl)-sulfonyl]-imide ([C₁₀(BzIM)₂]²⁺ 2-[NTf₂]^-) that displayed preconcentration factors of approximately 27-fold for N7-methylguanosine (m⁷G) and 30-fold for N1-methyladenosine (m¹A), compared to no enrichment of these nucleosides using conventional solid-phase microextraction approaches for nanomolar concentration samples. Importantly, for broad applicability of BTAIL-ME, we developed a mass spectrometry-compatible method to recover nucleosides from the IL phase into an ammonium acetate buffer for direct injection into liquid chromatography-mass spectrometry (LC-MS) instrumentation. We then applied BTAIL-ME to investigate extracellular modified nucleosides released from primary cultures of Aplysia californica neural tissues, which enabled downstream LC-MS detection of three methylated, positively charged ribonucleosides. BTAIL-ME introduces an extraction approach for profiling select extracellular modified ribonucleosides by LC-MS, paving the way toward understanding their functions in biological systems.