Glutathione-functionalized magnetic thioether-COFs for the simultaneous capture of urinary exosomes and enrichment of exosomal glycosylated and phosphorylated peptides

Abstract

Exosomes play an irreplaceable role in physiological and pathological processes, and the study of proteomics (especially protein post-translational modifications, PTMs) in exosomes can reveal the pathogenesis of diseases and screen therapeutic disease targets. The separation and enrichment process is an essential step in mass spectroscopy-based exosomal PTMs studies to reduce sample complexity and ionization-suppression effects. Herein, we designed a novel magnetic zwitterionic material, namely glutathione-functionalized thioether covalent organic frameworks (Fe₃O₄@Thio-COF@Au@GSH), possessing fast magnetic responsiveness, regular porosity, and a suitable surface area. Thanks to the hydrophilicity and charge-switchable feature of GSH, for the first time, both the capture of exosomes from biological fluids and enrichment of the inherent glycoproteins/phosphoproteins in the exosomes were achieved with the same material. Furthermore, the high enrichment capacity was validated by theoretical calculations. The low detection limits (0.2/0.4 fmol for HRP/β-casein), high selectivity (1 : 1000 for HRP/β-casein : BSA molar ratio), and high exosomal glycoproteomics/phosphoproteomics profiling capability proved the feasibility of the developed method. This work provides a new heuristic strategy to solve the problems of exosomal capture and glycoproteins/phosphoproteins pretreatment in exosomal proteomics.