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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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Representative mass spectra of specific and unspecific BiCy-tagged proteasomal peptides.A, the isotope pattern of the peptide 4GBiCySSAGFDR11 (α1, spleen) shows an L/H ratio of ≈1.7, which indicates a specific labeling of the O-GlcNAcylated serine residue. B, a typical spectrum of an unspecific tagged peptide with a BiCy-d0/d4 ratio of 1:1 (29G BiCySTAVGVR36, α4 spleen) is depicted.
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Figure 5: Representative mass spectra of specific and unspecific BiCy-tagged proteasomal peptides.A, the isotope pattern of the peptide 4GBiCySSAGFDR11 (α1, spleen) shows an L/H ratio of ≈1.7, which indicates a specific labeling of the O-GlcNAcylated serine residue. B, a typical spectrum of an unspecific tagged peptide with a BiCy-d0/d4 ratio of 1:1 (29G BiCySTAVGVR36, α4 spleen) is depicted.

Mentions: The enrichment strategy described above was applied to the analysis of 20 S proteasomes, as a complex biological sample, and to Hsp90, a molecular chaperone protein separated during proteasome purification. To investigate tissue-specific differences of O-GlcNAcylation, we analyzed proteasomes from mouse spleen (mainly comprising immunoproteasomes) and brain (mainly comprising standard proteasomes). As summarized in Table II, we identified 17 BiCy-labeled peptides with L/H (or H/L) ratios of 0.71 to greater than 10. To define the cut-off for a specific labeling, we took the L/H (and H/L) ratios of seven technical replicates from the expected 1:1 BiCy-labeled peptides of α-crystallin and compared them in a t test with the ratios of the individual peptides of spleen proteasome measured in three replicates. A significance of p < 0.001 was found for a ratio of 1.3 (1:0.77), which corresponds to a 23% decreased intensity of the BiCy-d4-labeled peptide compared with the d0-labeled (for L/H ratios). Because the greatest deviation from the expected 1:1 ratio for α-crystallin was 29% (ratio 0.71), we increased for more confidence the cut-off ratio to 1.43 (1:0.7), which corresponds to a reduced signal intensity of the heavy peptide of >30%. With this cut-off, we identified six proteasomal O-GlcNAc sites with L/H (or H/L) ratios of 1.5 to greater than 10 (Table II). In contrast, unspecific derivatization products occurred with ratios of 0.71–1.33. Representative mass spectra of specific and unspecific products are shown in Fig. 5. Remarkably, among the unspecific labeled peptides, cysteine-labeled products were also observed. N-terminal to these residues, acidic amino acids (Glu and Asp) are localized. We suppose that these residues interfere with the cysteine oxidation resulting in BiCy tagging.


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)

Representative mass spectra of specific and unspecific BiCy-tagged proteasomal peptides.A, the isotope pattern of the peptide 4GBiCySSAGFDR11 (α1, spleen) shows an L/H ratio of ≈1.7, which indicates a specific labeling of the O-GlcNAcylated serine residue. B, a typical spectrum of an unspecific tagged peptide with a BiCy-d0/d4 ratio of 1:1 (29G BiCySTAVGVR36, α4 spleen) is depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Representative mass spectra of specific and unspecific BiCy-tagged proteasomal peptides.A, the isotope pattern of the peptide 4GBiCySSAGFDR11 (α1, spleen) shows an L/H ratio of ≈1.7, which indicates a specific labeling of the O-GlcNAcylated serine residue. B, a typical spectrum of an unspecific tagged peptide with a BiCy-d0/d4 ratio of 1:1 (29G BiCySTAVGVR36, α4 spleen) is depicted.
Mentions: The enrichment strategy described above was applied to the analysis of 20 S proteasomes, as a complex biological sample, and to Hsp90, a molecular chaperone protein separated during proteasome purification. To investigate tissue-specific differences of O-GlcNAcylation, we analyzed proteasomes from mouse spleen (mainly comprising immunoproteasomes) and brain (mainly comprising standard proteasomes). As summarized in Table II, we identified 17 BiCy-labeled peptides with L/H (or H/L) ratios of 0.71 to greater than 10. To define the cut-off for a specific labeling, we took the L/H (and H/L) ratios of seven technical replicates from the expected 1:1 BiCy-labeled peptides of α-crystallin and compared them in a t test with the ratios of the individual peptides of spleen proteasome measured in three replicates. A significance of p < 0.001 was found for a ratio of 1.3 (1:0.77), which corresponds to a 23% decreased intensity of the BiCy-d4-labeled peptide compared with the d0-labeled (for L/H ratios). Because the greatest deviation from the expected 1:1 ratio for α-crystallin was 29% (ratio 0.71), we increased for more confidence the cut-off ratio to 1.43 (1:0.7), which corresponds to a reduced signal intensity of the heavy peptide of >30%. With this cut-off, we identified six proteasomal O-GlcNAc sites with L/H (or H/L) ratios of 1.5 to greater than 10 (Table II). In contrast, unspecific derivatization products occurred with ratios of 0.71–1.33. Representative mass spectra of specific and unspecific products are shown in Fig. 5. Remarkably, among the unspecific labeled peptides, cysteine-labeled products were also observed. N-terminal to these residues, acidic amino acids (Glu and Asp) are localized. We suppose that these residues interfere with the cysteine oxidation resulting in BiCy tagging.

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