PMID: PMC2628603-1-2

 

    Legend: Gene, Sites

Title : Selective enrichment and identification of putative GlcNAcylated proteins

Abstract :
  1. 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)
  2. Immunoisolation by pan-specific antibodies suffers from low efficiency due to relatively low binding affinity of the antibodies
  3. 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)
  4. In this study, a highly selective tagging method was used (18)
  5. 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
  6. GalT1 was used to enzymatically tag GlcNAc modifications on erythrocytic proteins with Gal-ketone
  7. PNGase F was used to remove N-glycans
  8. After enzymatic labeling, the ketone group was chemically tagged with an aminooxy biotin , which allowed capturing of O- GlcNAcylated proteins with strept avidin beads
  9. Enriched proteins were then eluted, separated by SDS-PAGE, and identified by an ion trap mass spectrometer after in-gel digestion (Fig. 2)
  10. By using this method, 25 erythrocyte proteins were identified as putatively GlcNAcylated (Table 1)
  11. 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)
  12. We further confirmed some of the putative GlcNAc proteins by first immunoprecipitating the proteins and then Western blotting with O-GlcNAc antibody (Fig. 2)
  13. 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):

 

 

Protein NCBI ID SENTENCE INDEX