Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins

Abstract

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

Glycosaminoglycan and Proteoglycan Biotherapeutics in Articular Cartilage Protection and Repair Strategies: Novel Approaches to Visco‐supplementation in Orthobiologics

“…Routine analysis of the GAG chains in PGs typically involves compositional and disaccharide analysis, mapping of 4-8 oligosaccharide segments and occasionally domain analysis; however, extensive sequencing is not normally undertaken. [55,88,90,91] [87,88] Embedded sulphation motifs such as CS-D and CS-E internally in the CS chains have growth factor interactive properties, the surrounding regions of the CS chain serve a carrier function for these biological motifs.…”

“…Glycan sequencing methodology is steadily improving and it is now possible to sequence small GAG samples; however, this still cannot be considered a routine procedure. Technological advances in mass spectroscopy are also improving such determinations …”

Rapid On‐Chip Synthesis of Complex Glycomimetics from N‐Glycan Scaffolds for Improved Lectin Targeting

“…We have previously studied the enzymatic on‐chip synthesis of glycan libraries using recombinant glycosyltransferases and pairs of lectins with complimentary binding specificities to monitor the conversion of the on‐chip reactions . Later, we and others developed conductive array surfaces which facilitated the analysis of on‐chip reactions by MALDI‐TOF MS and employed these for screening the substrate specificities of glycosyltransferases and glycosidases . The Paulson group has prepared sialoside mimetics for array‐based binding assays using nonnatural sialic acid derivatives to include alkyne moieties that were further diversified via copper mediated cycloaddition .…”

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies

“…Label-free methods also could provide improved measurements of certain glycan-protein interactions, because the chemical labeling of a protein could affect binding. Surface plasmon resonance offers measurements of binding affinities as well as the ability to identify low-affinity interactions (61,62). Mass spectrometry could provide solution-phase detection of the glycans bound by a protein and potentially more accurate measurements of binding strengths relative to solid-phase methods (63).…”