Title : Selective enrichment and identification of putative
GlcNAcylated proteins
Abstract :
- Enrichment is key for mass spectrometric identification of GlcNAcylated proteins because of low stoichiometry and ion suppression by unmodified peptide ions in the mass spectrometer (21)
- Immunoisolation by pan-specific antibodies suffers from low efficiency due to relatively low binding affinity of the antibodies
- Using lectins to enrich O-GlcNAc proteins suffers from low specificity because lectins may bind strongly to proteins with other forms of glycosylation (rev. in 21)
- In this study, a highly selective tagging method was used (18)
- This method takes advantage of the mutant UDP-galactose transferase (Y289L GalT1) (19), which has an enlarged donor-substrate binding pocket and can accommodate UDP-galactose analogs, in this case, UDP-Gal-ketone
- GalT1 was used to enzymatically tag GlcNAc modifications on erythrocytic proteins with Gal-ketone
- PNGase F was used to remove N-glycans
- After enzymatic labeling, the ketone group was chemically tagged with an aminooxy biotin , which allowed capturing of O- GlcNAcylated proteins with strept avidin beads
- Enriched proteins were then eluted, separated by SDS-PAGE, and identified by an ion trap mass spectrometer after in-gel digestion (Fig. 2)
- By using this method, 25 erythrocyte proteins were identified as putatively GlcNAcylated (Table 1)
- A mock experiment with no UDP-Gal-ketone added yielded no signal when blotted with horseradish peroxidase–conjugated avidin , indicating the specificity of the approach (Fig. 2)
- We further confirmed some of the putative GlcNAc proteins by first immunoprecipitating the proteins and then Western blotting with O-GlcNAc antibody (Fig. 2)
- O-GlcNAc antibody competition with excess free GlcNAc was routinely performed and eliminated the signals in immunoblotting, documenting the antibody specificity (data not shown)
Output (sent_index, trigger,
protein,
sugar,
site):
- 6. proteins, , proteins, Gal-ketone, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):