Methods and kits for detection of O-GlcNAc and N-GlcNAc modification of peptides and proteins

摘要

Methods and kits for detecting the presence of GlcNAc modification of a peptide or protein. The steps of detection may include combining the peptide or protein with PAP35S and a sulfotransferase to produce a reaction product including 35S labeled GlcNAc modified protein or peptide, removing background labeling with a glycosidase, separating the 35S labeled GlcNAc modified protein or peptide from PAP35S, free S-35 sulfate and labeled oligosaccharides if present, and detecting isotopic emissions from the 35S labeled GlcNAc modified peptide or protein. The detection may further include, prior to detection, the step of combining the peptide or protein with a GlcNAc transferase and UDP-GlcNAc under appropriate conditions for the GlcNAc transferase to transfer a GlcNAc to the peptide or protein.

主权项

1. A method of detecting the presence of O-GlcNAc (O-linked β-N-acetvlglucosamine) modification of a peptide or protein comprising sequential steps of:
a) combining the peptide or protein with EOGT (extracellular O-GlcNAc transferase) or OGT (O-GlcNAc transferase) and UDP-GlcNAc (Uridine 5′-diphospho-N-acetylglucosamine) to produce a first reaction product; b) combining the first reaction product with PAP35S (3′-Phosphoadenosine-5′-phosphate including 35S) and a sulfotransferase to produce a second reaction product including 35S labeled O-GlcNAc modified peptide or protein; c) removing background labeling with a glycosidase; d) separating the 35S labeled O-GlcNAc modified peptide or protein from PAP35S, free 35S sulfate and labeled oligosaccharides if present; and e) detecting isotopic emissions from the 35S labeled O-GlcNAc modified peptide or protein.