PMID: PMC2628603-1-3

 

    Legend: Gene, Sites

Title : Mapping O-GlcNAc sites and site-specific quantitation

Abstract :
  1. We first attempted to evaluate the overall dynamics of O-GlcNAc on whole protein levels by immunoprecipitation and imunoblotting, but no conclusive result was observed
  2. This may not be surprising because the extent of GlcNAcylation is site-specifically regulated and because O-GlcNAc antibody may not be sensitive enough to recognize significant change of O-GlcNAc on one specific site when the protein is modified on multiple sites
  3. Furthermore, one of the eventual goals of this study is to develop site specific O-GlcNAc antibodies
  4. Thus, we wanted to map O-GlcNAc sites and quantitate O-GlcNAc site specifically
  5. A recently developed chemoenzymatic tagging method was used to enrich O-GlcNAc at peptide levels (17,20)
  6. Y289L GalT1 was similarly used to label the GlcNAc moieties as described previously but on trypsin-digested peptides instead of whole proteins
  7. With increased accessibility of the enzyme and donor substrate, the labeling with UDP-GalNAz (an analog of UDP-galactose with azide function group) is nearly 100% after overnight incubation at 4°C (21)
  8. Biotinylation was performed with the highly efficient copper-catalyzed cycloaddition reaction or “click” chemistry under mild conditions (22) (Fig. 3)
  9. For this chemoenzymatic approach using UDP-GalNAz and biotin- PEG-alkyne, the tags added to the O-GlcNAc peptide exceed 772 Da in mass
  10. Although the biotin- PEG-alkyne tags allow for highly selective enrichment of GlcNAcylated peptides , they are problematic; not only do they negatively affect the ionization efficiency, but the heavy tags also impose other challenges for mass spectrometric analysis
  11. For example, fragmentation of the biotin moiety and the PEG linker arm on CAD makes the MS/MS spectra noisy and difficult to interpret
  12. In addition, the tagging does not change the extremely labile nature of the β-O-linkage, which undergoes neutral loss before peptide backbone fragmentation in CAD (Fig. 3)
  13. To resolve these issues, we modified and combined a previously developed chemical derivatization method called BEMAD (β-elimination followed by Michael addition with DTT) (23) with the chemoenzymatic enrichment method
  14. The BEMAD chemical derivatization was performed directly on the solid phase after the tagged peptides were captured by avidin beads (Fig. 3, inset)
  15. The derivatized peptides were released from the solid phase with the O-GlcNAc , and tags were replaced by a DTT via Michael addition
  16. The resulting DTT modification is stable and can be easily identified by mass spectrometry
  17. This approach also circumvents the need to break the strong biotin-avidin interaction with harsh conditions
  18. Mass spectrometric quantitation of O-GlcNAc peptides is also readily enabled by isotopic labeling with deuterated DTT (DTT-d6), which introduces a 6-Da mass difference between the peptide pairs (e.g., normal vs. diabetic)
  19. The overall approach is shown as a flow chart in Fig. 3 and described in detail in research design and methods
  20. Of course, it is possible that the apparent changes in GlcNAcylation may arise from different dynamics of protein expression or turnover
  21. To address this factor, we labeled the flow-through of avidin chromatography, containing mostly unmodified peptides , with iTRAQ reagents and used it to quantitate relative changes of protein expression levels
  22. With relative abundance of both O-GlcNAc peptides and corresponding protein levels, RORs of O-GlcNAc could then been calculated using a simple equation (see research design and methods)
  23. Erythrocyte lysates from normal and diabetic blood donors (10 each; Table 2) were pooled separately and used as the starting materials after partial depletion of abundant hemoglobins
  24. Three independent experiments were performed according to the flow chart shown in Fig. 3
  25. Using the standard of at least one unique peptide with a >99% confidence level, 206 erythrocyte proteins were identified and quantified (supplemental data, available in an online appendix at http ://dx.doi.org/10.2337/db08-0994)
  26. Although most proteins were equally abundant, changes were observed for a few proteins between normal and diabetic samples (Fig. 3)
  27. Thirty-five O-GlcNAc sites originating from 17 proteins were identified
  28. The relative occupancy rates of O-GlcNAc at these sites between diabetic and normal states were calculated (Table 3)
  29. A negative control sample was first treated with hexosaminidase (an enzyme that removes GlcNAc) before enrichment and yielded no identification of a GlcNAcylated protein (Fig. 3), indicating the specificity of the overall approach
  30. Differentially regulated GlcNAcylation was observed on multiple sites originating from several proteins (Table 3; Fig. 4)
  31. This regulation is clearly site specific, as observed in the cases of ankyrin-1 , hemoglobin α, and catalase (Table 3)
Output (sent_index, trigger, protein, sugar, site):
  • 30. GlcNAcylation, , -, -, sites
Output(Part-Of) (sent_index, protein, site):
  • 10. GlcNAcylated, peptides
  • 11. CAD, -
  • 11. PEG, -
  • 6. proteins, peptides
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

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