Title : Development and application of mass spectrometric methods for the analysis of
progranulin N-glycosylation
Abstract :
- PGRN is a modular protein with 7 1/2 repeats of the granulin domain separated by short spacer sequences
- Elevated expression of PGRN is associated with cancer growth, while mutations of PGRN cause frontotemporal lobar degeneration (FTLD), an early onset form of dementia
- PGRN is a glycoprotein , containing five N-glycosylation consensus sequons, three of which fall within granulin domains
- A method tailored to enable detailed analysis of the PGRN oligosaccharides and glycopeptides has been developed
- The approach involves in-gel deglycosylation using peptide-N-glycosidase F ( PNGase F) followed by permethylation of the released oligosaccharides
- Permethylation was applied for rapid sample clean-up and to improve sensitivity of MS detection and mass spectrometric fragmentation
- Reversed-phase monolithic LC- ESI-MS/MS was used for analysis of permethylated oligosaccharides, enabling structural characterization of released N-linked glycans in one chromatographic run
- In-gel tryptic digestion was further applied to the gel pieces containing deglycosylated protein , for N-glycosylation site determination
- In addition, glycopeptides were produced using in-solution pronase digestion to identify species of N-glycan attached at particular sites
- The method developed was applied to progranulin ( PGRN ) to characterize the structures of the released glycans and to identify the sites of glycosylation
- Glycosylation of four out of five potential PGRN N-glycosylation consensus sites was demonstrated (the final one remains undetermined), with one of the four observed to be partially occupied
- Two of the observed glycosylation sites occur within granulin domains , which may have important implications for understanding the structural basis of PGRN action
Output (sent_index, trigger,
protein,
sugar,
site):
- 1. domain, , -, 7 1/2 repeats, domain
- 1. protein, , protein, 7 1/2 repeats, -
- 10. glycosylation, , -, -, sites
- 11. Glycosylation, , -, -, sites
- 11. N-glycosylation, , -, -, sites
- 12. glycosylation, , -, -, sites
- 3. N-glycosylation, , -, -, -
- 3. glycoprotein, , PGRN, -, -
- 3. glycoprotein, , glycoprotein, -, -
- 4. glycopeptides, , -, -, glycopeptides
- 8. deglycosylated, , protein, -, -
- 9. attached, , -, N-glycan, sites
- 9. glycopeptides, , -, -, glycopeptides
Output(Part-Of) (sent_index,
protein,
site):
- 1. granulin, domain
- 11. PGRN, sites
- 12. granulin, domains
- 3. PGRN, -
- 3. glycoprotein, -
- 3. granulin, domains
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):