Characterization of the N-Linked Glycan of Recombinantly-Expressed Corticotropin Releasing Factor Binding Protein
| Autor: | Mateen Akhtar, A. Grey Craig, Steve Sutton, Wylie Vale, Wolfgang H. Fischer, Karsten Schmidt |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
chemistry.chemical_classification
Glycan Glycosylation biology Electrospray ionization 010401 analytical chemistry General Medicine 010402 general chemistry Tandem mass spectrometry Mass spectrometry 01 natural sciences Atomic and Molecular Physics and Optics 0104 chemical sciences chemistry.chemical_compound chemistry Biochemistry Exoglycosidase Neuraminic acid biology.protein Glycoprotein Spectroscopy |
| Zdroj: | European Journal of Mass Spectrometry. 6:335-346 |
| ISSN: | 1751-6838 1469-0667 |
| DOI: | 10.1255/ejms.353 |
| Popis: | The N-linked glycan present on corticotropin releasing factor binding protein (CRFBP) recombinantly expressed in CHO cells has been characterized using a variety of techniques including sequential hydrolysis with specific exoglycosidases monitored with reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ES-MS). Determining the intact glycan mass and comparing possible glycan compositions with entries in the Complex Carbohydrate Database facilitated the choice of enzymes. Tandem mass spectrometry (MS/MS) was used to confirm the structure of the glycan. Using this approach, complementary information could be obtained through specific glycosidase reactions monitored with MS and MS n fragmentation of the intact or enzymatically-modified glycopeptides. No evidence was found for the presence of sites of O-linked glycosylation. In addition, to understand better the role of the glycan on CRFBP, we characterized the in vitro binding affinity of a mutant non-glycosylated [Gln180] CRFBP and an enzymatically deglycosylated form of CRFBP to human corticotropin releasing factor (hCRF). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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