Lectin-array blotting: profiling protein glycosylation in complex mixtures.

By combining electrophoretic protein separation with lectin-array-based glycan profiling into a single experiment, we have developed a high-throughput method for the rapid analysis of protein glycosylation in biofluids. Fluorescently tagged proteins are separated by SDS-PAGE and transferred by diffusion to a microscope slide covered with multiple copies of 20 different lectins, where they are trapped by specific carbohydrate protein interactions while retaining their relative locations on the gel. A fluorescence scan of the slide then provides an affinity profile with each of the 20 lectins containing a wealth of structural information regarding the present glycans. The affinity of the employed lectins toward N-glycans was verified on a glycan array of 76 structures. While current lectin-based methods for glycan analysis provide only a picture of the bulk glycosylation in complex protein mixtures or are focused on a few specific known biomarkers, our array-based glycoproteomics method can be used as a biomarker discovery tool for the qualitative exploration of protein glycosylation in an unbiased fashion.