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site mapping and quantitation of O‐GlcNAcylated proteins in diabetic erythrocytes
Author(s) -
Park Kyoungsook,
Wang Zihao,
Saudek Chtistopher D.,
Hart Gerald W.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.614.9
Subject(s) - chemistry , carbonic anhydrase , mass spectrometry , biochemistry , diabetes mellitus , carbonic anhydrase ii , enzyme , biology , endocrinology , chromatography
O‐linked N‐acetylglucosamine (O‐GlcNAc) is a ubiquitous post‐translational modification found on nucleocytoplasmic proteins. This modification is very dynamic and sensitive to cellular nutrients and stress. Cycling of O‐GlcNAc on many proteins can modulate diverse events in cells. Elevated global O‐GlcNAcylation is associated with development of many diseases including neurodegenerative diseases, cancer and diabetes. In diabetic patients, prolonged exposure to high glucose may result in global changes of O‐GlcNAcylation and altered occupancy of O‐GlcNAc on certain proteins. We examined the changes of O‐GlcNAcylation in diabetic red blood cells and site‐mapped O‐GlcNAc on proteins using semi‐quantitative mass spectrometry method. We modified beta‐elimination/Michael addition detection (BEMAD)/mass spectrometry method by adding enrichment steps for O‐GlcNAcylated peptides. This step improved the efficiency of O‐GlcNAc detection. We have identified multiple O‐GlcNAc sites on erythrocytic proteins such as catalase and carbonic anhydrase I and observed site‐specific differential occupancy of O‐GlcNAc. Significant changes of O‐GlcNAcylation of certain proteins/sites will be used for further study to develop a diagnostic tool for diabetes.