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Identification of Proteins O‐GlcNAc Modified During Osteoblastogenesis
Author(s) -
Ball Lauren E,
Schilling Michael,
Waller Lashanda N,
Nagel Alexis
Publication year - 2012
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.26.1_supplement.776.14
Subject(s) - chemistry , transcription factor , adipogenesis , phosphorylation , trypsin , osteoblast , biochemistry , microbiology and biotechnology , protein phosphorylation , biology , in vitro , gene , protein kinase a , enzyme
O‐GlcNAcylation of key transcription factors critical to adipogenesis and osteoblastogenesis alters the rate of differentiation. To assess the extent of O‐GlcNAcylation during osteoblast differentiation, global protein O‐GlcNAc modification was measured in murine MC3T3‐E1 and primary human osteoblasts (NHOst) 0, 1, 3, 7, 14, and 21 days after induction in osteogenic media. The extent of global protein O‐GlcNAc modification peaked between days 3–7. To identify these proteins, O‐GlcNAcylated peptides were enriched from trypsin digested MC3T3‐E1 protein lysate at day 7 by WGA lectin weak affinity chromatography. Peptides were analyzed by C18 nLC‐MS/MS with CID and ETD fragmentation (LTQ‐XL). ETD MS/MS spectra were searched with Protein Prospector 2 and positive hits were manually confirmed. To date, 23 O‐GlcNAcylated peptides were identified primarily from proteins known to regulate transcription. Ten novel sites of O‐GlcNAc modification and six sites of O‐GlcNAc modification at known phosphorylation sites were identified. New sites of O‐GlcNAcylation were identified in Tab‐1 and Tab‐2, signaling proteins hypothesized to contribute to cell fate determination. Current studies are measuring changes in OGT and OGA expression during osteoblastogenesis and further characterizing the role of O‐GlcNAc modification of proteins important to osteoblastogenesis. Supported by RO1DE020925 and T32DE017551.