SmartEnzymes™

 

Enrichment of O-glycoproteins

Specific Enrichment of O-glycosylated Proteins and Peptides

O-linked glycans are involved in several biological processes, including inhibiting infections caused by pathogens, inflammatory responses, hematopoiesis and cell-signaling. GlycOCATCH®is an affinity resin that specifically binds to O-glycosylated (mucin-type) proteins and peptides, allowing an easy workflow to enrich and purify proteins and peptides carrying O-glycans.

GlycOCATCH workflow - O-glycoproteins

Selectively Binds O-glycoproteins

To test the selectivity, a selection of O-glycosylated, N-glycosylated and non-glycosylated proteins were incubated with GlycOCATCH. The spin columns were washed and the bound proteins were eluted with 8 M urea. The analysis of the eluted material demonstrates an O-glycan-dependent affinity purification, since only O-glycosylated proteins are eluted and the resin shows negligible binding to non- and N-glycosylated proteins (Fig. 1).

 
Figure 1. Demonstration of the selectivity of GlycOCATCH. All samples were desialylated using SialEXO before they were incubated with GlycOCATCH, washed using PBS and eluted with 8 M urea. a) As a model substrate, the O-glycosylated TNFR was enriched using GlycOCATCH. b) Specific enrichment of O-glycosylated proteins from the following mix: aflibercept, TNFR, bovine serum albumin (BSA), alpha-1-acid glycoprotein (AGP), apolipoprotein E (ApoE), and the Fc domain of IgG. c) Cetuximab, aflibercept, BSA, AGP, carbonic anhydrase showed no binding to GlycOCATCH.

Figure 1. Demonstration of the selectivity of GlycOCATCH.

Enrichment of O-glycopeptides

To study the O-glycan specificity of GlycOCATCH on the peptide level, an O-glcyosylated peptide (glycodrosocin) was mixed with a selection of unmodifed peptides. The mixture was loaded onto GlycOCATCH, the resin was washed and the bound peptides were eluted with 8 M urea. After purification, the samples were separated on RP-LC-MS. The data shows selective purification of the glycodrosocin peptide, carrying a core 1 O-glycan (Fig. 2). 

 

 

Enrichment of O-glycoproteins

 

Figure 2. Enrichment of an O-GalNAcGal peptide from a mix of peptides. A peptide mix containing H2686, H4062, H8390, insulin oxidized beta chain (IOB) and glycodrosocin together with SialEXO was loaded on the GlycOCATCH resin. Glycodrosocin, the only peptide in the mix containing an O-GalNAcGal, was predominantly found in the eluted fraction and the remaining peptides in the flow-through fraction. Separation was performed on a RP-LC C18 column (Advance BioPeptide Map 2.1x100 2.7µm from Agilent) and detected with ESI-Q-TOF Bruker Impact II mass spectrometer.

Specific O-glycopeptide Enrichment from IgA

Specific enrichment of O-glycoproteins

Figure 3. Specific enrichment of O-glycosylated peptides from IgA.

IgA antibodies contain both N-linked glycosylation and a heavily O-glycosylated hinge region. After trypsin digestion of IgA, the mix of peptides and SialEXO was loaded onto the GlycOCATCH resin and washed using PBS and centrifugation. Elution with 8 M urea showed an enrichment of the expected O-glycosylated hinge peptide with various degrees of glycosylation.

Figure 3. Specific enrichment of O-glycosylated peptides from IgA. IgA was trypsinated and incubated with the GlycOCATCH resin. The resin was washed and the bound peptides were eluted using 8 M urea. Separation was performed on a RP-LC C18 column (Advance BioPeptide Map 2.1x100 2.7µm from Agilent) and detected with ESI-Q-TOF Bruker Impact II mass spectrometer.

Enrichment of O-glycoproteins from Human Serum

Enrichment of O-glycoproteins from Human Serum

Figure 4. Enrichment of O-glycoproteins from human serum.

GlycOCATCH can also be used to selectively enrich O-glycosylated proteins from a complex sample mixture, such as human serum. Briefly, serum was treated with SialEXO and applied to the GlycOCATCH resin. The resin was washed, and O-glycosylated proteins were eluted with 8 M urea (Fig. 4a). The sample was digested with trypsin and analyzed using RP-LC-MS/MS, and a list of proteins was identified using the Swiss Prot database. O-glycosylated proteins are depicted in yellow (Fig. 4b). 

 

 

Figure 4. Enrichment of O-glycoproteins from human serum. Human serum (Sigma) was treated with SialEXO sialidases and incubated with the GlycOCATCH resin. a) Load, flow-through and eluted fractions were analysed on SDS-PAGE. b) The eluted proteins were denatured, reduced, alkylated and trypsin digested. Peptides were analyzed using RP-LC-MS/MS on a C18 column (Advance BioPeptide Plus 2.1x150mm 2.7µm from Agilent Technologies) in a 0.1%FA in MQ: 0.1%FA in 95% ACN gradient at 45°C and a flow of 0.2ml/min. Detection was on an ESI-Q-TOF Bruker Impact II instrument. Data was converted to mgf format files in Data Analysis Software v 4.4 and searched against the Swiss Prot database using BioPharma Compass v 2.0.1. Identified O-glycosylated proteins and other proteins with >12 matching peptides and a MASCOT score >200 are listed.

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