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 1:791-804 Xianhui Li 03-23- 2004

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

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)

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

ß - ELIMINATION MICHAEL ADDITION dehydroalanine Serine-O-GlcNAc

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

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

Synthetic O-GlcNAc- and phosphate-modified peptides

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

Peptide Identification and DTT-modified residue

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

Controls for O-GlcNAc Specificity in BEMAD Identification

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

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

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