1. Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications
Wells, Vosseller, Cole, Cronshaw, Matunis and Hart
(2002) Molecular and Cellular Proteomics 1:
Xianhui Li

2. Outline Introduction Methods Results Conclusions
Postranslational modifications
Mass spectrometry
Methods
Chemistry (BEMAD)
Mass spectrometry analysis
Results
Synthetic peptides
Known proteins
New protein
Conclusions

3. Introduction
O-GlcNAc modification is widespread but its functional significance is poorly understood.
Direct detection of O-GlcNAc by mass spectrometry is difficult because the linkage is labile and easily cleaved.
Prior replacement of the O-GlcNAc group with a more stable group allows MS detection.
Method is compatible with various types of MS analysis and also analysis of Ser/Thr phosphorylation (MS/MS, MALDI TOF)

4. BEMAD method followed by affinity chromatography and MS Analysis
Protein Digestion, isolation of peptide
β-elimination of O-GlcNAc
Michael addition with DTT or BAP to tag former O-GlcNAc site
Purification by affinity chromatography
MS/MS or MALDI-TOF

6. ß - ELIMINATION
MICHAEL ADDITION
dehydroalanine
Serine-O-GlcNAc

7. Method Testing and Application
Synthetic O-GlcNAc and O-phosphate-modified peptides
Conditions
GlcNac vs. Phosphate Detection
Affinity purification
Known O-GlcNAc-modified protein
Site mapping on purified synapsin I
Identification of novel O-GlcNAc sites
Site mapping from a NPC preparation

8. MALDI-TOF Analysis of a Synthetic O-GlcNAc Peptide
DTT
(136.2 D)
BAP
(310.5 D)

9. Synthetic O-GlcNAc- and phosphate-modified peptides

10. Affinity Enrichment of DTT-or BAP-modified peptides
PSVPVS(DTT/BAP)GSAPGR

11. Peptide Identification and DTT-modified residue

12. Identification of a Novel O-GlcNAc-modified site in the Lamin B receptor at serine 96 by BEMAD

13. Controls for O-GlcNAc Specificity in BEMAD Identification

14. Identification of a Novel O-GlcNAc in Soluble NPC Protein Nup155 at serine 525

15. Conclusion
BEMAD modification occurs under mild conditions for O-GlcNAc and PO4* modified proteins
Affinity purification enhances sensitivity and decreases complexity.
Allows confirmation of O-GlcNAc-modified residues in synthetic peptide and known proteins
Allows the identification of O-GlcNAc modified residues in unknown proteins
*Incomplete reaction

16. Thank you!