Title : Mapping of O-GlcNAc sites of 20 S proteasome proteasome subunits and Hsp90 by a novel biotin-cystamine tag.
Abstract :
The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport
This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress
O-Glycosylation of the 26 S proteasome proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation
In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome proteasome , the 20 S proteasome proteasome , and the impact on proteasome activity is very limited
This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes
Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies
Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag.
The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine
The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified
The method was optimized using bovine α -crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver
Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90 β, of which one corresponds to a previously described phosphorylation site