Photoinduced Online Enrichment-Deglycosylation of Glycolipids for Enhancing Lipid Coverage and Identification in...

Single-cell lipidomics provides important information for molecular mechanisms of living processes and diseases at the individual cell level. However, single-cell lipidomic mass spectrometry (MS) techniques suffer from low lipid coverage and incomplete structural elucidation, especially for poorly ionizable glycosphingolipids (GSLs). Herein, a photoinduced enrichment-deglycosylation method of GSLs was developed and introduced into an ambient liquid extraction MS system for enhancing detection coverage and identification accuracy of GSLs in single-cell MS. GSL standards were selectively adsorbed on TiO₂ in ammonia-added protic solvents. Under UV irradiation, the adsorbed GSLs would lose one hexosyl group (deglycosylation), and the products (>70% conversion efficiency) were desorbed from TiO₂. By coating porous TiO₂ into the capillary of the ambient liquid extraction MS system, online adsorption of GSLs and their separation from high-abundance phospholipids were achieved, largely reducing ion suppression. By UV irradiation, captured GSLs were rapidly deglycosylated and photodesorbed from TiO₂ coating without solvent switching, resulting in 6-fold enrichment. With the new method, the detection coverage of GSLs was enhanced 9-fold without losing other lipidomes, compared with the conventional method. Moreover, deglycosylated GSLs from photodesorption had more MS/MS fragments than intact GSLs, facilitating detailed fatty acyl and sphingosine chain elucidation. Seven deglycosylated GSL peaks were identified with the confirmed hydroxyl group location in the fatty acyl chain, while only 1 was identified for intact GSL. The new method was applied to the single-cell lipidomics study of two types of nerve cells. Totally, 31 lipids including 11 GSLs were identified in a single cell, and 5 hexosylceramides were found significantly altered after neuron injury.