In view of the biological significa

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 prerequisite for the discovery of disease biomarkers and clinical diagnosis. In this work, we propose a “grafting-from” N-glycoprotein enriching method based on 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 thermoresponsive polymer can be grown to form a giant protein-polymer conjugates (PPC). The thremosensitive PPC can be precipitated and separated by rising 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 specific enrich and separate N-glycoprotein/glycopeptide. Due to the simplicity and efficiency, this method holds the potential for the identification of biomarkers from biological samples in N-glycoproteome analysis.