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Eur. J. Mass Spectrom. 6, 335 - 346 (2000)

Characterization of the N-linked glycan of recombinantly-expressed corticotropin releasing factor binding protein

Steve W. Sutton, Mateen Akhtar, Karsten Schmidt, Wolfgang H. Fischer, Wylie W. Vale and A. Grey Craig*
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA 92037, USA

ABSTRACT:
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 MSn 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).

Keywords: corticotropin releasing factor, glycoprotein, neuraminic acid, mass spectrometry, exoglycosidase, chromatography

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