Kinetic Analysis of Glycoprotein–Lectin Interactions by Label-Free Internal Reflection Ellipsometry

Abstract

Introduction Glycoproteomics is undergoing rapid development, largely as a result of advances in technologies for isolating glycoproteins and analyzing glycan structures. However, given the number and diversity of glycans, there is need for new technologies that can more rapidly provide differential carbohydrate-protein structural information on a large scale. We describe a new microarray platform based on a label-free imaging ellipsometry technique, which permits simultaneous detection of multiple glycoproteinlectin interactions without the need for reporter labels, while still providing high throughput kinetic information at much lower cost. Our results demonstrate the utility of LFIRE™ (Label-Free Internal Reflection Ellipsometry) for the rapid kinetic screening of carbohydrate-lectin recognition. The technology was also used to evaluate the benefits of the lectin immobilization format using multi-lectin affinity chromatography (M-LAC) to capture glycoproteins (with enhanced binding strength or avidity) from biological samples. Using a printed panel of lectins, singly or in combination, we examined the binding characteristics of standard glycoproteins.Results and Discussion Using kinetic measurements, it was observed that the binding strength of lectins to carbohydrates is enhanced using a multi-lectin strategy, suggesting that improved selectivity and specificity can lead to increased functional avidity. The data presented confirm that this label-free technology can be used to effectively screen single or combinations of lectins. Furthermore, the combination of LFIRE™ and M-LAC may permit more rapid and sensitive identification of novel biomarkers based on carbohydrate changes in glycoproteins, and lead to a better understanding of the connections of glycan function in cellular mechanisms of health and disease.

Automated Platform for Fractionation of Human Plasma Glycoproteome in Clinical Proteomics

“…It was for these reasons that we developed the multi-lectin affinity approach (M-LAC), which uses admixtures of lectins, and gives rise to multivalent association with plasma glycoproteins, resulting in better capture of the plasma glycoproteome. In a recent publication, we reported up to 10 fold enhancment in binding affinities with the multiple lectin format compare to the corresponding individual lectins (ConA, JAC and WGA) 10. Furthermore, we have previously reported on the combination of abundant protein depletion with M-LAC and have shown a deeper mining of the plasma glycoproteome6, 11…”

Modulation of Glycan Detection on Specific Glycoproteins by Lectin Multimerization

“…Two of the antibody-bound clusters were further joined through an anti-goat IgG antibody to form a final cluster of 12 HA proteins. Another approach used the physical admixture of separate lectins bound to either agarose or pressure resistant supports to facilitate the clustering effect and demonstrated up to a 10-fold enhancement of binding affinity by ellipsometry measurments in glycoproteins with glycan motifs complimentary to the lectin specificity 24 . This approach was used to develop the multiple lectin affinity chromatography approach (M-LAC) and used in studies of plasma glycoproteins 25–26 .…”

Selective Functionalization of the Protein N Terminus with N‐Myristoyl Transferase for Bioconjugation in Cell Lysate

“…[28,29] LFIRE measures real-time changes in elliptically polarized light caused by surface capture of proteins or other biomolecules. [30] We employed the LFIRE technique to analyze glass slides that were printed with cyclooctyne spots and incubated with the four lysate samples described above. LFIRE data corroborated our fluorescence results: significant signal indicative of protein conjugation was observed only within wells incubated with lysate containing 12-ADA-yARF-GFP, and only where cyclooctyne had been printed (Figures 4 and S4).…”