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Generation of O‐GlcNAc specific monoclonal antibodies using a novel synthetic immunogen
Author(s) -
Teo ChinFen,
Ingale Sampat,
Wolfert Margreet A.,
Elsayed Galal A.,
Not Laszlo G.,
Chatham John C.,
Wells Lance,
Boons GeertJan
Publication year - 2010
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.24.1_supplement.904.7
Subject(s) - immunogen , monoclonal antibody , antibody , phosphorylation , chemistry , threonine , serine , epitope , biochemistry , microbiology and biotechnology , biology , immunology
O‐GlcNAc modification is a ubiquitous and dynamic post‐translational modification that has been implicated in many cellular processes, frequently via interplay with phosphorylation that can occur on the same serine and threonine residues. Unlike phosphorylation for which a wide range of pan‐ and site‐specific phospho‐antibodies are available, only two pan‐O‐GlcNAc specific antibodies have been described: an IgM pan‐O‐GlcNAc antibody and an IgG antibody raised against O‐GlcNAc modified components of the nuclear pore complex. In fact, it is well known that O‐GlcNAc modified glycoconjugates do not elicit relevant IgG isotype antibodies. Here, we report a novel method combining a fully synthetic three‐component immunogen with classical hybridoma technology to obtain a series of O‐GlcNAc‐specific IgG monoclonal antibodies. Large‐scale shotgun proteomics approach utilizing these antibodies led to the identification of more than 200 O‐GlcNAc modified proteins, of which approximately two‐thirds were novel. Application of this tool allowed us to delineate differentially modified proteins of the liver in response to hemorrhagic trauma and resuscitation in a rat model. This work was supported by grants from the NIDDK/NIH RO1 DK075069 (LW), NIH P41‐RR‐5351 (GJB), NIH RO1 CA88986 (GJB), NIH HL067464 (JCC) and NIH HL079364 (JCC). CFT is an AHA pre‐doctoral fellow (0715377B).