title: Detection of O-GlcNAc modifications on cardiac myofilament proteins.
abstract: In this chapter it is described a general method that has been used successfully by more than one laboratory interested in detecting O-GlcNAc in myofilament proteins. Alternative reagents for chemo-enzymatic or metabolic labeling will be indicated, as well as references for more details in alternative methods. The outline is divided into (1) Enrichment of O-GlcNAc Stoichiometry, (2) Cardiac Myofilament Protein Isolation, (3) SDS-PAGE, (4) "Reduction and Alkylation," (5) In-Gel Protein Digestion, (6) Chemo-enzymatic Labeling of O-GlcNAc Moieties (Click Chemistry), (7) Biotin Alkyne Tagging, (8) Strong Cation Exchange (SCX) and Streptavidin, and (9) β-Elimination and Michael Addition (BEMAD) for O-GlcNAc Site-Mapping.
authors.affiliation: Division of Cardiology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Genaro A
authors.initials: GA
authors.lastname: Ramirez-Correa
authors.forename: Isabel Martinez
authors.initials: IM
authors.lastname: Ferrando
authors.forename: Gerald
authors.initials: G
authors.lastname: Hart
authors.forename: Anne
authors.initials: A
authors.lastname: Murphy
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001710:Biotin; D057930:Click Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D013058:Mass Spectrometry; D051379:Mice; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D009210:Myofibrils / Q000737:chemistry* / Q000378:metabolism; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D011506:Proteins; D001710:Biotin; D019809:Streptavidin; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine