title: Lectin-array blotting: profiling protein glycosylation in complex mixtures.
abstract: 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.
authors.affiliation: Biofunctional Nanomaterials Unit, San Sebastian, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Etxebarria
authors.forename: Javier
authors.initials: J
authors.lastname: Calvo
authors.forename: Manuel
authors.initials: M
authors.lastname: Martin-Lomas
authors.forename: Niels-Christian
authors.initials: NC
authors.lastname: Reichardt
mesh_terms: D002241:Carbohydrates / Q000737:chemistry; D045424:Complex Mixtures; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D037102:Lectins / Q000737:chemistry*; D046228:Microarray Analysis; D011134:Polysaccharides / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics; D013050:Spectrometry, Fluorescence
chemical_list: D002241:Carbohydrates; D045424:Complex Mixtures; D000939:Epitopes; D037102:Lectins; D011134:Polysaccharides