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.