A pH-responsive soluble polymer-based homogeneous system for fast and highly efficient N-glycoprotein/glycopeptide enrichment and identification by mass spectrometry

Liquid phase homogeneous reactions using soluble polymer supports have found numerous applications in homogeneous catalysis and organic synthesis because of their advantages of no interface mass transfer limitation and a high conversion rate. However, their application in analytical separation is limited by the inefficient/inconvenient recovery of the target molecules from the extremely complex biological samples. Here, we report a stimuli-responsive polymer system for facile and efficient enrichment of trace amounts of biomolecules from complex biological samples. The soluble polymer supports provide a homogeneous reaction system with fast mass transfer and facilitate interactions between the supports and the target molecules. More importantly, the stimuli-responsive polymers exhibit reversible self-assembly and phase separation under pH variations, which leads to facial sample recovery with a high yield of the target biomolecules. The stimuli-responsive polymer is successfully applied to the enrichment of low abundant N-glycoproteins/glycopeptides, which play crucial roles in various key biological processes in mammals and are closely correlated with the occurrence, progression and metastasis of cancer. N-Glycoprotein is coupled to the stimuli-responsive polymer using the reported hydrazide chemistry with pre-oxidation of the oligosaccharide structure. Highly efficient enrichment of N-glycoproteins/N-glycopeptides with >95% conversion rate is achieved within 1 h, which is eight times faster than using solid/insoluble hydrazide enrichment materials. Mass spectrometry analysis achieves low femtomolar identification sensitivity and obtained 1317 N-glycopeptides corresponding to 458 N-glycoproteins in mouse brain, which is more than twice the amount obtained after enrichment using commercial solid/insoluble materials. These results demonstrate the capability of this "smart" polymer system to combine stimuli-responsive and target-enrichment moieties to achieve improved identification of key biological and disease related biomolecules.