Shotgun glycomics: a microarray strategy for functional glycomics

Abstract

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.

Figure 1

Schematic for Shotgun glycomics Glycans are released chemically or enzymatically from glycoproteins, GSLs are modified directly; the fluorescently labeled products are separated, quantified, and printed to create microarrays available for interrogation with GBPs.

Figure 2

Fluorescent derivatization of GSLs for shotgun glycomics: (a) The derivatization of GSLs with a bifunctional linker; (b) The C18-HPLC profiles of AOAB derivatization of GM1, GD1a, GT1b and BBG mixture detected by fluorescence; (c) The MALDI-TOF spectra of GM1-AOAB, GD1a-AOAB and GT1b-AOAB purified by HPLC as shown in (b). The spectra were acquired in the reflective negative mode; (d) The normal phase HPLC profiles of crude ODA treatment of BBG-PNPA conjugates without precipitation, the precipitate and the filtrate of BBG-PNPA mixture after addition of acetonitrile.

Figure 3

Binding assay on the BBG GSL-AOAB microarray prepared from 2D HPLC separation: (a) cholera toxin subunit B (CTSB) at 0.1 μg ml⁻¹ and (b) anti-GD1a antibody at 1:20 dilution of ascites fluid on the BBG-AOAB microarray. Average RFU of 4 replicates is reported, error bars = standard deviation. Structural characterization of bound fraction (c) #9 and (d) #24 by MS and MS/MS.

Figure 4

The binding of Lyme disease and control sera on the BBG microarray: (a) comparison of IgG binding of sera from Lyme disease patients and control sera (tested at 1:100 dilution). The average RFUs are normalized in each serum sample by setting the binding of fraction 33 in control and patient serum to 100. (b) Distribution of binding by patient and control sera over six selected GSL-AOAB fractions (#12, 17, 26, 33, 39, and 40). Blue squares = individual control sera values, Red circles = individual patient sera values. P values, calculated with Student’s t-test, are given for the comparison of control to patient for the selected 6 glycans. * = P < 0.05. (c) Proposed structural characterization of bound fraction #12 by MS and MS/MS.

Figure 5

The GSL microarray from human erythrocytes and its interrogation with lectins and antibodies: (a) C18-HPLC profiles of O-blood type erythrocyte GSL-AOAB, 23 fractions; (b) C18-HPLC profiles of A-blood type erythrocyte GSL-AOAB, 25 fractions; The binding of plant lectins (c) AAL (1 μg ml⁻¹), (d) UEA-1 (10 μg ml⁻¹), and (e) HPA (10μg ml⁻¹), and (f) anti-blood group A antibody (10 μg ml⁻¹) where GSL-AOAB #1-23 were from human blood group O erythrocytes, #24-48 were from human blood group A erythrocytes, and #49–52 were controls, including the AEAB derivatives of LNnT, LNFIII, Le^yLe^x and biotin. Average RFU of 4 replicates is reported, error bars = standard deviation.