Title : Mapping
O-GlcNAc sites and site-specific quantitation
Abstract :
- 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
- 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
- Furthermore, one of the eventual goals of this study is to develop site specific O-GlcNAc antibodies
- Thus, we wanted to map O-GlcNAc sites and quantitate O-GlcNAc site specifically
- A recently developed chemoenzymatic tagging method was used to enrich O-GlcNAc at peptide levels (17,20)
- Y289L GalT1 was similarly used to label the GlcNAc moieties as described previously but on trypsin-digested peptides instead of whole proteins
- 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)
- Biotinylation was performed with the highly efficient copper-catalyzed cycloaddition reaction or “click” chemistry under mild conditions (22) (Fig. 3)
- For this chemoenzymatic approach using UDP-GalNAz and biotin- PEG-alkyne, the tags added to the O-GlcNAc peptide exceed 772 Da in mass
- 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
- For example, fragmentation of the biotin moiety and the PEG linker arm on CAD makes the MS/MS spectra noisy and difficult to interpret
- 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)
- 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
- The BEMAD chemical derivatization was performed directly on the solid phase after the tagged peptides were captured by avidin beads (Fig. 3, inset)
- The derivatized peptides were released from the solid phase with the O-GlcNAc , and tags were replaced by a DTT via Michael addition
- The resulting DTT modification is stable and can be easily identified by mass spectrometry
- This approach also circumvents the need to break the strong biotin-avidin interaction with harsh conditions
- 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)
- The overall approach is shown as a flow chart in Fig. 3 and described in detail in research design and methods
- Of course, it is possible that the apparent changes in GlcNAcylation may arise from different dynamics of protein expression or turnover
- 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
- 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)
- 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
- Three independent experiments were performed according to the flow chart shown in Fig. 3
- 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)
- Although most proteins were equally abundant, changes were observed for a few proteins between normal and diabetic samples (Fig. 3)
- Thirty-five O-GlcNAc sites originating from 17 proteins were identified
- The relative occupancy rates of O-GlcNAc at these sites between diabetic and normal states were calculated (Table 3)
- 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
- Differentially regulated GlcNAcylation was observed on multiple sites originating from several proteins (Table 3; Fig. 4)
- 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):