Selective capture of glycoproteins using lectin-modified nanoporous gold monolith

Abstract

The surface of nanoporous gold (np-Au) monoliths was modified via a flow method with the lectin Concanavalin A (Con A) to develop a substrate for separation and extraction of glycoproteins. Self-assembled monolayers (SAMs) of α-lipoic acid (LA) on the np-Au monoliths were prepared followed by activation of the terminal carboxyl groups to create amine reactive esters that were utilized in the immobilization of Con A. Thermogravimetric analysis (TGA) was used to determine the surface coverages of LA and Con A on np-Au monoliths which were found to be 1.31×10(18) and 1.85×10(15)moleculesm(-2), respectively. An in situ solution depletion method was developed that enabled surface coverage characterization without damaging the substrate and suggesting the possibility of regeneration. Using this method, the surface coverages of LA and Con A were found to be 0.989×10(18) and 1.32×10(15)moleculesm(-2), respectively. The selectivity of the Con A-modified np-Au monolith for the high mannose-containing glycoprotein ovalbumin (OVA) versus negative control non-glycosylated bovine serum albumin (BSA) was demonstrated by the difference in the ratio of the captured molecules to the immobilized Con A molecules, with OVA:Con A=2.3 and BSA:Con A=0.33. Extraction of OVA from a 1:3 mole ratio mixture with BSA was demonstrated by the greater amount of depletion of OVA concentration during the circulation with the developed substrate. A significant amount of captured OVA was eluted using α-methyl mannopyranoside as a competitive ligand. This work is motivated by the need to develop new materials for chromatographic separation and extraction substrates for use in preparative and analytical procedures in glycomics.

Keywords: Chromatography; Glycomics; Glycoprotein; Lectin; Monolith; Nanoporous gold.

Fig. 1

(a) Preparation of np-Au monolith from a precursor alloy plate (42% Au, 20% Ag, 38% Cu) through dealloying in nitric acid solution for 48 h at room temperature (RT). SEM images of the top and side views of the exterior and the interior portion of an 8 mm × 8 mm × 0.50 mm np-Au monolith. All scale bars are 0.5 μm except for the side view (left image) which is 500 μm. (b) Pore size distribution obtained by Barrett–Joyner–Halenda (BJH) analysis of the adsorption branch of the isotherm. (c) EDS spectra of np-Au monolith (at 15 kV). (d) Schematic diagram of the preparation of Con A-modified np-Au monolith.

Fig. 2

(a) TGA thermograms for determination of LA loading and stability on np-Au monolith. (b) TGA thermograms for determination of Con A loading and stability on SAM-modified np-Au monolith. The temperature was ramped at 20 °C min⁻¹.

Fig. 3

(a) UV-vis scan of LA solution. (b) UV-vis scans of protein solutions. (c) Loading curve of LA on np-Au monolith and (d) of Con A on SAM-modified np-Au monolith as recorded by a UV detector at 330 nm and 280 nm, respectively.

Fig. 4

(a) TGA thermograms showing the capture of OVA and BSA using Con A-modified np-Au monolith without the capping procedure. (b) TGA thermograms showing the effect of adding the capping procedure i.e., immobilization of Con A to the esters of SAM modified np-Au monolith was restricted by the capping procedure. (c) TGA thermograms showing the capture of OVA and BSA using Con A-modified np-Au monolith with capping procedure. The temperature was ramped at 20 °C min⁻¹.

Fig. 5

(a) Schematic diagram of the procedure used in characterizing extraction of OVA from a mixture with BSA using Con A-modified np-Au monoliths. (b) SDS-PAGE of 20 μM BSA (66.4 kDa) and 20 μM OVA (44.3 kDa) and of the aliquots obtained at the end of each 30 min cycle of circulation of 1:3 molar mixture of OVA and BSA through an increasing number of Con A-modified np-Au monoliths.

Fig. 6

(a) Chromatogram generated by flowing 2 mL 1 μM OVA solution through the flow cell without the np-Au monolith. (b) Chromatogram generated by flowing 2 mL 1 μM OVA solution through the Con A-modified np-Au monolith followed by elution using 2 mL 0.1 M α-methyl mannopyranoside (AMMP). AUCs were calculated using GraphPad Prism 6.07.