Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry
Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains.
KeywordsMass spectrometry Human galectin-3 Glycine max lectin Blood group oligosaccharides CRD Recognition sites Proteolytic excision, proteolytic extraction Ligand epitope peptides SAW-biosensor analysis
Carbohydrate recognition domain
Surface acoustic waves
We thank Drs. Stefan Maeser and Elisa Peroni for the valuable discussions and critical reading of the manuscript.
This work has been partially supported by the European Union through the Marie-Curies IRSES grant “Integrating high performance mass spectrometry with applications in life science” (MSLife). Partial support is also acknowledged from the Bundesministerium für Wirtschaft (BMWi; SPR-MS).
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