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-MSE 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 (m1A, m6A, m2,2,7G, ac4C) 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.