Laboratory Seminars and Journal Club

29. 10. 2012 The University of Cape Town

Moore et al., (2012). Evolution of an HIV glycan–dependent broadly neutralizing antibody epitope through immune escape. Nature Medicine.

16. 05. 2013 The University of Cape Town

Go et la., (2013) Characterization of Host-Cell Line Specific Glycosylation Profiles of Early Transmitted/Founder HIV-1 gp120 Envelope Proteins

Glycosylation modulates

• Biological properties
• Structural properties
• Therapeutical properties

Difficulties of protein glycosylation analysis

• Heterogeneity
• Diversity

ETD (electron transfer dissociation) - to complement CID (collision induced dissociation)

Ability to identify O-linked amino acids

Only CHO were phosphorylated. No HEK 293T cells were phosphorylated. Phosphorylation crucial for crossing the blood brain barrier.

O-linked CHO core1 type and HEK 293T had core 1, 2, 3 -type

Expression system crucial for pharmaco-activity, protein folding and post-translational modifications.

N-linked - NXT/S while O-linked - Threonine and Serine

Glycosylation Analysis

• Enzyme based glycans released
• Chemical based glycans released
• Glycopeptide based MS

Determine glycan motif, extent of glycosylation

Typical Workflow

Glycopeptides are digested with a specific or nonspecific protease to generate a peptide/glycopeptide mixture that is subjected to separation or enrichment step prior to MS and tandem MS, Data analysis.

ETD combines the ability to fragment the peptide backbone while leaving the glycan modification intact. CID produces conventional glycosidic cleavages.

Deglycosylation - PNGase or EndoH

Parrish et al., (2013). Phenotypic properties of transmitted founder HIV-1