Abstract:

As a clinical sample， serum is widely used in disease research due to the minimally invasive nature of its collection and easy accessibility. Exosomes in serum not only carry various bioactive substances such as proteins and nucleic acids but also participate in intercellular communication， which is closely associated with the development of multiple diseases. As one of the most critical post-translational modifications， protein phosphorylation dynamically regulates cellular signaling networks and participates in disease pathogenesis. However， due to the low abundance of exosomes in serum and the fact that phosphoproteins constitute only 1%–2% of the total proteome， direct analysis of exosomal phosphoproteins from serum faces significant challenges. The specific expression of phosphoproteins in exosomes provides novel perspectives for disease diagnosis. In this study， magnetic bifunctional nanoparticles （MagphTi⁴⁺） based on 2-trifluoromethyl-4-aminobenzoic acid and Ti⁴⁺ were synthesized， which anchor the exosome bilayer membrane through the hydrophobic interactions of 2-trifluoromethyl-4-aminobenzoic acid and the electrostatic interactions of Ti⁴⁺， and the magnetic responsiveness ensures the isolation of exosomes. Simultaneously， the enrichment of phosphopeptides was achieved by the chelation between Ti⁴⁺ and phosphate groups. MagphTi⁴⁺ was characterized by cold field scanning electron microscopy， thermogravimetric analysis， Fourier transform infrared spectroscopy， X-ray photoelectron spectroscopy and zeta potential measurement， demonstrating the successful synthesis and functional characteristics of MagphTi⁴⁺. The precise recognition and rapid separation of exosomes were validated by cold field scanning electron microscopy， transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using β-casein as a standard phosphoprotein model， a magnetic solid-phase extraction （MSPE）-matrix-assisted laser desorption ionization-time of flight mass spectrometry platform was established， which demonstrated high selectivity （mass ratio of β-casein to bovine serum albumin was 1∶5 000） and sensitivity （detection limit was 0.4 fmol/μL） for phosphopeptide detection. Subsequently， the method was applied to saliva and nonfat milk samples； 11 phosphopeptides were identified in saliva and 20 were identified in nonfat milk， which demonstrates excellent enrichment capability for phosphopeptide detection in real samples. When applied to clinical serum samples， the method successfully achieved the extraction of exosomes in serum and enrichment of phosphopeptides with low abundance. This integrated approach provides a powerful tool for exosome capture and phosphopeptide enrichment from complex biological samples， showing significant potential for high-throughput phosphoproteomic analysis of exosomes from clinical serum and the screening of disease biomarkers. Further application of the method developed in this study holds promise for elucidating the mechanisms of exosome-mediated intercellular transmission of phosphorylation signaling， thereby identifying novel targets related to tumor metastasis and immune escape.

Key words: serum exosomes, titanium ion, magnetic solid-phase extraction, phosphopeptides, mass spectrometric detection