Single-Cell Mass Spectrometry with Enhanced Coverage of cis -Diol Metabolites via a Reactive Pulled Flowprobe in Static-Zone Enrichment Sampling Mode

Single-cell mass spectrometry (MS) faces significant challenges in covering low-abundance and poorly ionizable metabolites, hindering comprehensive single-cell metabolomics studies of disease and life processes. Herein, we proposed a new single-cell MS method based on a pulled flowprobe that enables online static-zone metabolite enrichment and derivatization of cis-diol metabolites, thereby greatly enhancing metabolite coverage. A stationary sample band was observed within the continuously flowing liquid microjunction of the pulled flowprobe when the inner capillary was substantially retracted inside the outer capillary. A larger retraction distance and lower solvent flowrate were found to benefit band formation, and the band served as a static zone to enrich analytes, achieving up to 50-fold signal enhancement. Then, a benzylboronic acid derivatization reagent was introduced into this band, where both the reagent and cis-diol metabolites were enriched to achieve static derivatization, yielding faster reaction kinetics (< 1/4 the time to reach maximum yield) and greatly reduced band diffusion/dilution (peak width: 30 s vs > 100 s) compared with the traditional dynamic online derivatization. Then, the new single-cell MS method with online enrichment and static-zone derivatization was established and enhanced overall metabolite coverage more than two-fold (53 vs 24), and specifically three-fold for cis-diol metabolites from a single cell, compared to the traditional method using dynamic derivatization. Finally, the new single-cell MS method was applied to the neuronal injury study, and six metabolites involved in oxidative stress, myelin damage, and DNA damage pathways were found to be significantly altered between injured and normal cells. These results highlight the advances of our method in expanding the molecular understanding at the single-cell level to uncover disease mechanisms.