Glycan-selective in-situ growth of thermoresponsive polymers for thermoprecipitation and enrichment of N-glycoprotein/glycopeptides

In view of the biological significance and micro-heterogeneity of protein glycosylation for human health, specific enrichment of N-glycosylated proteins/peptides from complex biological samples is a prerequisite for the discovery of disease biomarkers and clinical diagnosis. In this work, we propose a grafting-from N-glycoprotein enriching method based on the in-situ growth of thermoresponsive polymer brushes from the N-glycosylated site of proteins. The initiator was first attached to the pre-oxidized glycan moieties by hydrazide chemistry, from which the thermoresponsive polymers can be grown to form giant protein-polymer conjugates (PPC). The thermosensitive PPC can be precipitated and separated by raising the temperature to above its lower critical solubility temperature (LCST). Mass spectrometry verified 210 N-glycopeptides corresponding to 136 N-glycoproteins in the rabbit serum. These results demonstrate the capability of the tandem thermoprecipitation strategy to enrich and separate N-glycoprotein/glycopeptide. Due to its simplicity and efficiency specifically, this method holds the potential for identifying biomarkers from biological samples in N-glycoproteome analysis.

Automated summary

This study proposes a method for enriching N-glycosylated proteins based on the in-situ growth of thermoresponsive polymer brushes from the N-glycosylated site. The method demonstrates high efficiency in enrichment and separation, and verifies the presence of N-glycopeptides in the enriched samples through mass spectrometry.