O-GlcNAc Detection using BEMAD

[gabevillegas]

Was wondering if anyone has any experience with BEMAD. I'm trying to map sites of O-GlcNAc modifications on my protein of interest via MS/MS but the fragmentation energy is too high and so knocks the O-GlcNAc off the peptide before it's read so you can tell it was there but not where exactly it was.

 

Using Beta elimination followed by Michaels Addition of DTT (BEMAD) you can replace the O-GlcNAc with a DTT moiety which should be able to survive fragmentation. Then by mapping DTT modified sites, you can tell where the O-GlcNAc was.

 

My Question is does anyone have experience using BEMAD and if so could you post a protocol that worked for you to use as a jumping off point for my experiment?