Development a hydrazide-functionalized thermosensitive polymer based homogeneous system for highly efficient N-glycoprotein/glycopeptide enrichment from human plasma exosome

As one of the most common post-translational modifications, protein N-glycosylation precipitates in many important biological processes and has closely correlations with the occurrence and progression of multiple diseases. Plasma exosomes secreted by cells contain various bioactive N-glycoproteins which may serve as potential biomarkers for early disease diagnosis and treatment. However, the protein N-glycosylation profile in human plasma exosome is largely unknown, due to the technical challenges in glycoprotein identification. Signals of the rare N-glycoproteins/N-glycopeptides are severely suppressed by the abundant coexisting non-glycosylated counterparts in mass spectrometry analysis. Therefore, specific enrichment of N-glycoprotein/glycopeptide is a prerequisite for large scale N-glycosylation profiling. In this work, we developed a hydrazide functionalized thermosensitive polymer for efficient enrichment and in-depth identification of protein N-glycosylation in human plasma exosome by mass spectrometry. The polymer chains completely dissolve in the enrichment system to form a homogeneous solution. Therefore, efficient covalent coupling between the N-glycoprotein/glycopeptide and the polymer chain is achieved, due to the reduced interfacial mass transfer resistance and the densely packed accessible functional groups on the polymer chains. Furthermore, the thermosensitive polymer can be easily precipitated and recovered by simply rising the system temperature to above 34 degrees C. As a result, 329 N-glycosylation sites corresponding to 180 N-glycoproteins were enriched and identified from plasma exosomes of glioma patients and healthy subjects using the thermosensitive polymer. By quantitative comparison, we found 26 N-glycoproteins significantly changed between the glioma patients and the healthy subjects, demonstrating the potential of this new strategy for N-glycoproteome research of plasma exosome and biomarker discovery.