Developing novel method and strategy for comprehensive glycoprotein analysis

Glycoprotein analysis has been paid increasing attention in the field of prognosis, diagnosis, and proteomics for the discovery of biomarkers. Although multiple selective enrichment methods have been developed, the complex and heterogeneous nature of glycosylation renders none of these methods could potentially cover all of the glycosylation information in complex biological samples. Accordingly, the development of new methods and strategies for comprehensive glycoprotein analysis is still an imperative task. I n our recent study, a global strategy was developed to perform large-scale glycoprotein profiling of rat brain by the pre-fractionation of proteins into soluble and insoluble ones. Soluble proteins were denatured by traditional urea protocol, and insoluble proteins were solubillized by the addition of 1% (v/v) 1-butyl-3- methylimidazolium tetrafluoroborate, which showed superiority than urea, methanol and RapiGest. Followed by hydraizde based enrichment, a total number of 1108 unique Nglycosites, covering 626 non-redundant glycoprotein groups were identified, among which 99% glycosites were not reported. For all glycoprotein groups, 399 proteins contained at least one predicted transmembrane domain. Furthermore, a novel enrichment method of glycopeptides by the chemical modification of bifunctional fluorescent tag, 8-aminonaphthalene- 1, 3, 6-trisulfonic acid disodium salt, was developed. After captured by TiO2, 39 unique Nlinked glycoproteins, representing of 53 unique Nlinked glycosites were identified via μRPLC-ESIMS/ MS for the analysis of human serum glycoproteins. In comparison to the direct capture by TiO2 and traditional hydraizde chemistry strategy, an overlap of less than 40% for glycosites was achieved, which indicated the complementary nature of this protocol with the previously developed methods.