Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.
We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.
Clark
Peter M
PM
Division of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Dweck
Jessica F
JF
Mason
Daniel E
DE
Hart
Courtenay R
CR
Buck
Suzanne B
SB
Peters
Eric C
EC
Agnew
Brian J
BJ
Hsieh-Wilson
Linda C
LC
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002478:Cells, Cultured; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D051381:Rats
D005456:Fluorescent Dyes; D011506:Proteins; D001710:Biotin; D005700:Galactosyltransferases; D000117:Acetylglucosamine