Developing novel method and strategy for comprehensive glycoprotein analysis
2010
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.
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