Title : Site-specific characterization of glycoprotein carbohydrates by exoglycosidase digestion and laser desorption mass spectrometry
Abstract :
- A rapid and sensitive method for sequencing oligosaccharides has been developed, using matrix-assisted laser desorption mass spectrometry to monitor the digestion of glycopeptides by specific exoglycosidases
- Recombinant human tissue inhibitor of metalloproteinases ( TIMP ), which has two glycosylation sites , has been characterized to illustrate this new approach for obtaining site-specific information
- Glycopeptides which span residues Asn30 and Asn78 were generated by tryptic digestion of 1 nmol of TIMP and separated by reverse-phase high-performance liquid chromatography
- The oligosaccharide composition of the glycoforms was inferred from the observed mass shifts following digestion by peptide-N-glycosidase F. Com position and sequence were then elucidated by digestion with specific exoglycosidases, using a total of 200 pmol of each glycopeptide
- Glycopeptides from well-characterized proteins , fetuin , alpha 1-acid glycoprotein , and tissue plasminogen activator were also analyzed to confirm exoglycosidase specificity for glycopeptides and establish the quantitative significance of the relative intensities of peaks in the mass spectra
- Both TIMP glycosylation sites exhibited extensive heterogeneity comprising mainly fucosylated complex oligosaccharides, but in different proportions
- The Asn78 site also contained 4.4% nonfucosylated mannose (Man4) oligosaccharide
- The merits and limitations of this approach as a universal method for oligosaccharide analysis are discussed
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. glycoprotein, , -, glycoprotein carbohydrates, -
- 1. glycopeptides, , -, -, glycopeptides
- 2. glycosylation, , -, -, sites
- 4. glycopeptide, , -, -, glycopeptide
- 5. glycopeptides, , -, -, glycopeptides
- 5. glycoprotein, , glycoprotein, -, -
- 5. glycoprotein, , proteins, -, -
- 6. fucosylated, , -, fucosylated complex oligosaccharides, -
- 6. glycosylation, , -, -, sites
- 7. contained, , -, 4.4% nonfucosylated mannose (Man4) oligosaccharide, Asn78 site
- 7. nonfucosylated, , -, 4.4% nonfucosylated mannose (Man4) oligosaccharide, -
Output(Part-Of) (sent_index,
protein,
site):
- 2. tissue inhibitor of metalloproteinases, sites
- 5. proteins, Glycopeptides
- 6. Both TIMP, sites
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 7. -, 4.4% nonfucosylated mannose (Man4) oligosaccharide, Asn78 site