Glycoproteomics Analysis to Identify Potential Biomarkers and Investigate CD14⁺ Monocyte Activation Mechanisms via...

Cerebral infarction (CI) is a leading cause of disability and mortality, with activated plasma monocytes and altered protein glycosylation identified as critical contributors to its pathology. However, the mechanisms linking peripheral monocyte activation and glycoproteins in CI remain inadequately understood. In this study, we developed a glycogen-functionalized nanoprobe (mSiO₂@Fe₃O₄@Glycogen) to profile plasma N-glycosylated proteins in healthy controls, CI patients, and patients in the rehabilitation phase (CIR). We identified 13 plasma N-glycoproteins with increased expression in CI patients and reduced levels in CIR patients. These N-glycoproteins, enriched in extracellular exosomes and associated with processes such as blood coagulation, fibrin clot formation, and complement activation, were validated as potential biomarkers for distinguishing CI and CIR patients through machine learning models. Flow cytometry analysis of peripheral blood mononuclear cells (PBMCs) revealed a higher proportion of inflammatory CD14⁺ monocytes and a lower proportion of anti-inflammatory CD16⁺ monocytes in CI patients compared with CIR patients. To further investigate whether exosomal proteins contribute to peripheral monocyte inflammation and could exacerbate CI pathogenesis, we developed a two-dimensional size-exclusion chromatography (SEC) method to isolate pure plasma exosomes for proteomic analysis. Four specific N-glycoproteins carried by CI-derived exosomes were identified and demonstrated the ability to directly activate human monocytes, potentially amplifying inflammation at injury sites. Our study highlights the role of exosomal N-glycoproteins in CI pathogenesis and positions them as promising diagnostic biomarkers and therapeutic targets for cerebral infarction.