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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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Derivatization of a synthetic O-GlcNAc-modified peptide by alkali-mediated β-elimination and Michael addition with a novel biotin-cystamine tag.A, shown is the strategy for replacement of the O-GlcNAc moiety on serine or threonine residues by the stable affinity tag BiCy. To discriminate between specific and unspecific reactions, differential isotopic tags (BiCy-d0 and BiCy-d4) were synthesized. B, MALDI-TOF/TOF-MS spectra of a synthetic O-GlcNAc-modified peptide before (upper lane) and after derivatization with BiCy-d0 (lower lane). The mass shift of 82 Da corresponds to the loss of O-GlcNAc (−203 Da) and water (−18 Da) and the addition of BiCy-d0 (303 Da). C, the MALDI-TOF/TOF-MS/MS spectrum of the BiCy-tagged O-GlcNAc peptide with precursor mass m/z 1927.0 is shown. Diagnostic ions at m/z 227.1 and 304.1 and the neutral loss of [M-303+H]+ confirm the tagging of the peptide. The site of modification is localized to Ser5.
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Figure 3: Derivatization of a synthetic O-GlcNAc-modified peptide by alkali-mediated β-elimination and Michael addition with a novel biotin-cystamine tag.A, shown is the strategy for replacement of the O-GlcNAc moiety on serine or threonine residues by the stable affinity tag BiCy. To discriminate between specific and unspecific reactions, differential isotopic tags (BiCy-d0 and BiCy-d4) were synthesized. B, MALDI-TOF/TOF-MS spectra of a synthetic O-GlcNAc-modified peptide before (upper lane) and after derivatization with BiCy-d0 (lower lane). The mass shift of 82 Da corresponds to the loss of O-GlcNAc (−203 Da) and water (−18 Da) and the addition of BiCy-d0 (303 Da). C, the MALDI-TOF/TOF-MS/MS spectrum of the BiCy-tagged O-GlcNAc peptide with precursor mass m/z 1927.0 is shown. Diagnostic ions at m/z 227.1 and 304.1 and the neutral loss of [M-303+H]+ confirm the tagging of the peptide. The site of modification is localized to Ser5.

Mentions: Based on above data, we set out to identify the sites of O-GlcNAc modification on 20 S proteasome subunits. For chemical modification of O-GlcNAc peptides by alkali-mediated β-elimination and Michael addition, initial experiments were performed with the tag dithiothreitol as described by Wells et al. (25) and Vosseller et al. (30). Although the derivatization and thiol-Sepharose enrichment of samples with low complexity (β-O-GlcNAc model peptide and α-crystallin) allowed the detection of the expected O-GlcNAc sites, for the analysis of complex 20 S proteasomes the procedure was unsatisfactory (data not shown). Because of the often observed extremely low stoichiometry of the O-GlcNAc modification on proteins, it is essential that the tag allows an efficient affinity purification of the labeled peptides. Therefore, we synthesized a new tag with a biotin moiety (Fig. 3A), which allows the usage of streptavidin instead of thiol-Sepharose. The thiol group of the cystamine moiety of the tag imparts the S-nucleophilic attack of β-eliminated serine and threonine residues. It is known, however, that the β-elimination/Michael addition reaction is not only specific for O-GlcNAc-modified serine and threonine residues. Unspecific derivatization of cysteine, alkylated cysteine, serine, and threonine residues that are unmodified or phosphorylated can occur (25, 30, 33, 35). To discriminate between specific derivatization of O-GlcNAc sites and unspecific modified residues, we synthesized a “light” BiCy-d0 and a deuterated “heavy” BiCy-d4 tag that allow relative quantification. Fig. 3B shows the MS spectra of the synthetic O-GlcNAc-modified peptide AIPVgSREEKPSSAPSS, which corresponds to the C-terminal, tryptic peptide 158–173 of bovine α-crystallin chain A before and after derivatization with BiCy-d0. The tagging results in a mass shift of m/z +82 compared with the O-GlcNAc-modified peptide. Under MS/MS conditions the tag generates indicator ions that are diagnostic for the presence of BiCy-derivatized peptides and therefore allow a sensitive screening for modified peptides. The signal at m/z 304.1 corresponds to the protonated BiCy-d0, at m/z 227.1 to the biotinylium ion (36) and the neutral loss of 303 mass units at m/z 1624.8 to the fragment [M-BiCy-d0+H]+ (Fig. 3C). The O-GlcNAc site can be localized to Ser5 because of the difference of 372 mass units between the y11 and y12 ion corresponding to the BiCy-d0 serine residue.


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)

Derivatization of a synthetic O-GlcNAc-modified peptide by alkali-mediated β-elimination and Michael addition with a novel biotin-cystamine tag.A, shown is the strategy for replacement of the O-GlcNAc moiety on serine or threonine residues by the stable affinity tag BiCy. To discriminate between specific and unspecific reactions, differential isotopic tags (BiCy-d0 and BiCy-d4) were synthesized. B, MALDI-TOF/TOF-MS spectra of a synthetic O-GlcNAc-modified peptide before (upper lane) and after derivatization with BiCy-d0 (lower lane). The mass shift of 82 Da corresponds to the loss of O-GlcNAc (−203 Da) and water (−18 Da) and the addition of BiCy-d0 (303 Da). C, the MALDI-TOF/TOF-MS/MS spectrum of the BiCy-tagged O-GlcNAc peptide with precursor mass m/z 1927.0 is shown. Diagnostic ions at m/z 227.1 and 304.1 and the neutral loss of [M-303+H]+ confirm the tagging of the peptide. The site of modification is localized to Ser5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Derivatization of a synthetic O-GlcNAc-modified peptide by alkali-mediated β-elimination and Michael addition with a novel biotin-cystamine tag.A, shown is the strategy for replacement of the O-GlcNAc moiety on serine or threonine residues by the stable affinity tag BiCy. To discriminate between specific and unspecific reactions, differential isotopic tags (BiCy-d0 and BiCy-d4) were synthesized. B, MALDI-TOF/TOF-MS spectra of a synthetic O-GlcNAc-modified peptide before (upper lane) and after derivatization with BiCy-d0 (lower lane). The mass shift of 82 Da corresponds to the loss of O-GlcNAc (−203 Da) and water (−18 Da) and the addition of BiCy-d0 (303 Da). C, the MALDI-TOF/TOF-MS/MS spectrum of the BiCy-tagged O-GlcNAc peptide with precursor mass m/z 1927.0 is shown. Diagnostic ions at m/z 227.1 and 304.1 and the neutral loss of [M-303+H]+ confirm the tagging of the peptide. The site of modification is localized to Ser5.
Mentions: Based on above data, we set out to identify the sites of O-GlcNAc modification on 20 S proteasome subunits. For chemical modification of O-GlcNAc peptides by alkali-mediated β-elimination and Michael addition, initial experiments were performed with the tag dithiothreitol as described by Wells et al. (25) and Vosseller et al. (30). Although the derivatization and thiol-Sepharose enrichment of samples with low complexity (β-O-GlcNAc model peptide and α-crystallin) allowed the detection of the expected O-GlcNAc sites, for the analysis of complex 20 S proteasomes the procedure was unsatisfactory (data not shown). Because of the often observed extremely low stoichiometry of the O-GlcNAc modification on proteins, it is essential that the tag allows an efficient affinity purification of the labeled peptides. Therefore, we synthesized a new tag with a biotin moiety (Fig. 3A), which allows the usage of streptavidin instead of thiol-Sepharose. The thiol group of the cystamine moiety of the tag imparts the S-nucleophilic attack of β-eliminated serine and threonine residues. It is known, however, that the β-elimination/Michael addition reaction is not only specific for O-GlcNAc-modified serine and threonine residues. Unspecific derivatization of cysteine, alkylated cysteine, serine, and threonine residues that are unmodified or phosphorylated can occur (25, 30, 33, 35). To discriminate between specific derivatization of O-GlcNAc sites and unspecific modified residues, we synthesized a “light” BiCy-d0 and a deuterated “heavy” BiCy-d4 tag that allow relative quantification. Fig. 3B shows the MS spectra of the synthetic O-GlcNAc-modified peptide AIPVgSREEKPSSAPSS, which corresponds to the C-terminal, tryptic peptide 158–173 of bovine α-crystallin chain A before and after derivatization with BiCy-d0. The tagging results in a mass shift of m/z +82 compared with the O-GlcNAc-modified peptide. Under MS/MS conditions the tag generates indicator ions that are diagnostic for the presence of BiCy-derivatized peptides and therefore allow a sensitive screening for modified peptides. The signal at m/z 304.1 corresponds to the protonated BiCy-d0, at m/z 227.1 to the biotinylium ion (36) and the neutral loss of 303 mass units at m/z 1624.8 to the fragment [M-BiCy-d0+H]+ (Fig. 3C). The O-GlcNAc site can be localized to Ser5 because of the difference of 372 mass units between the y11 and y12 ion corresponding to the BiCy-d0 serine residue.

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