Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans

Abstract

In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of "robust enrichment" afforded by covalent-labeling techniques and "specificity for glycoproteins" typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.

Circulating Avian Influenza Viruses Closely Related to the 1918 Virus Have Pandemic Potential

“…Virus samples were directly labeled with biotin (Ramya et al, 2013) and then applied to the slide array; slide scanning to detect virus bound to glycans was conducted as described previously (Watanabe et al, 2013) and in the Supplemental Experimental Procedures. A complete list of glycans present on the array is provided in Table S6.…”

Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein

“…Virus supernatant was laid over a cushion of 30% sucrose in PBS, ultracentrifuged at 76,755 ϫ g for 2 h at 4°C, and then resuspended in PBS for storage at Ϫ80°C. Virus samples were directly labeled with biotin (26) and then applied to the slide array. Slide scanning to detect virus bound to glycans was conducted as described previously (27).…”

Oxidation with galactose oxidase: Multifunctional enzymatic catalysis

“…GAO-based biosensors have also facilitated investigations of protein glycosylation. Immobilized GAO oxidized and thus tagged galactose-terminated glycans, which could then be reduced by NaBD 4 and analyzed with LC-MS [87] or labelled with aminoxybiotin and analyzed with MS/MS [88]. A similar approach has been used in chemiluminescent imaging of glycan expression [89].…”