Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation

Glycoproteins have vast structural
diversity that plays an important
role in many biological processes and have great potential as disease
biomarkers. Here, we report a novel functionalized reverse phase protein
array (RPPA), termed polymer-based reverse phase glycoprotein array
(polyGPA), to capture and profile glycoproteomes specifically, and
validate glycoproteins. Nitrocellulose membrane functionalized with
globular hydroxyaminodendrimers was used to covalently capture preoxidized
glycans on glycoproteins from complex protein samples such as biofluids.
The captured glycoproteins were subsequently detected using the same
validated antibodies as in RPPA. We demonstrated the outstanding specificity,
sensitivity, and quantitative capabilities of polyGPA by capturing
and detecting purified as well as endogenous α-1-acid glycoprotein
(AGP) in human plasma. We further applied quantitative N-glycoproteomics
and the strategy to validate a panel of glycoproteins identified as
potential biomarkers for bladder cancer by analyzing urine glycoproteins
from bladder cancer patients or matched healthy individuals.