BH+/MH+-matching method for discovery of cis-diol-containing modified nucleosides in urine by ribose-targeted solid phase extraction followed by dual-mass spectrometry platform identification

Profiling of new modified nucleosides from urine plays an important role in exploring biomarkers for cancer. However, limitations from the nature of the compound, bio-sample, instrumentation, and analytical method pose great challenges to achieving a comprehensive analysis of urinary nucleosides. Herein, a method of BH + /MH + -matching (BH + , protonated nucleobase ion; MH + , protonated precursor ion) was developed to discover novel modified nucleosides from human urine samples based on solid phase extraction targeted toward specific modified nucleosides combined with ultra-performance liquid chromatography coupled with dual-mass spectrometry platforms. Firstly, nucleosides containing 2,3-diol structure on ribose were effectively enriched by PBA (Phenylboronic Acid) cartridges. Secondly, a novel method, “BH + /MH + -matching” was established to achieve rapid screening of modified nucleosides. Based on the in-source fragmentation pattern of nucleosides, a series of putative modified nucleosides were rationally designed and characterized by matching the daughter ion BH + and its parent ion MH + in UPLC-MS E spectra. Finally, as a complement to UPLC Q-TOF/MS, UPLC Q-Trap/MS was employed to validate the structure of putative compounds by MRM-IDA-EPI mode. Using the strategy, 12 new cis -diol-containing nucleoside analogs were successfully characterized, which were formed by modified base (m 1 A, m 6 A, m 2,2,7 G, ac 4 C) and modified ribose containing C5′- O -formylation or C5′- O -methylation. Taken together, the results demonstrated our strategy could efficiently support the rapid discovery of cis -diol-containing nucleosides with modifications on either ribose or base moiety (or both), which exhibited a promising perspective in the future application of biochemical analysis and clinical diagnosis.