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Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.

Overath T, Kuckelkorn U, Henklein P, Strehl B, Bonar D, Kloss A, Siele D, Kloetzel PM, Janek K - Mol. Cell Proteomics (2012)

Bottom Line: O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation.Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies.Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Charité-Universitätsmedizin Berlin, 13347 Berlin, Germany.

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 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, the 20 S 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 core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.

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Strategy for identifying O-GlcNAc sites by chemical tagging with the biotin-cystamine tag BiCy and relative quantification.
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Figure 4: Strategy for identifying O-GlcNAc sites by chemical tagging with the biotin-cystamine tag BiCy and relative quantification.

Mentions: To avoid unspecific tagging of cysteine residues, the proteins were first oxidized by performic acid and then proteolytically digested (Fig. 4). The samples were split in halves and treated first with or without OGA and then with phosphatase. After enzyme incubation, the samples were derivatized with equivalent amounts of BiCy-d0 or BiCy-d4 and then combined. Excess of free BiCy tag was removed by size exclusion chromatography, followed by enrichment of BiCy-tagged peptides by affinity chromatography on streptavidin (supplemental Fig. 2) and LC-MS/MS analysis including relative quantification. In principle, the specificity of the procedure could be also changed to the analysis of phosphorylation sites, when the split samples are treated with or without phosphatase and with OGA as performed by Vosseller et al. (30).


Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.

Overath T, Kuckelkorn U, Henklein P, Strehl B, Bonar D, Kloss A, Siele D, Kloetzel PM, Janek K - Mol. Cell Proteomics (2012)

Strategy for identifying O-GlcNAc sites by chemical tagging with the biotin-cystamine tag BiCy and relative quantification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412975&req=5

Figure 4: Strategy for identifying O-GlcNAc sites by chemical tagging with the biotin-cystamine tag BiCy and relative quantification.
Mentions: To avoid unspecific tagging of cysteine residues, the proteins were first oxidized by performic acid and then proteolytically digested (Fig. 4). The samples were split in halves and treated first with or without OGA and then with phosphatase. After enzyme incubation, the samples were derivatized with equivalent amounts of BiCy-d0 or BiCy-d4 and then combined. Excess of free BiCy tag was removed by size exclusion chromatography, followed by enrichment of BiCy-tagged peptides by affinity chromatography on streptavidin (supplemental Fig. 2) and LC-MS/MS analysis including relative quantification. In principle, the specificity of the procedure could be also changed to the analysis of phosphorylation sites, when the split samples are treated with or without phosphatase and with OGA as performed by Vosseller et al. (30).

Bottom Line: O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation.Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies.Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Charité-Universitätsmedizin Berlin, 13347 Berlin, Germany.

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 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, the 20 S 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 core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.

Show MeSH
Related in: MedlinePlus