Detection of O -GlcNAc modifications on cardiac myofilament proteins

Genaro A. Ramirez-Correa, Isabel Martinez Ferrando, Gerald Hart, Anne Murphy

Research output: Chapter in Book/Report/Conference proceedingChapter

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) b -Elimination and Michael Addition (BEMAD) for O -GlcNAc Site-Mapping.

Original languageEnglish (US)
Title of host publicationHeart Proteomics
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages157-168
Number of pages12
ISBN (Print)9781627033855
DOIs
StatePublished - 2013

Publication series

NameMethods in Molecular Biology
Volume1005
ISSN (Print)1064-3745

Keywords

  • Cardiac myofilaments
  • Chemo-enzymatic labeling
  • Click chemistry
  • Michael addition
  • O -GlcNAcylation
  • O-linked- β - D -N-acetylglucosamine
  • Posttranslational modification
  • β -Elimination

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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