Characterization of the N-Linked Glycan of Recombinantly-Expressed Corticotropin Releasing Factor Binding Protein

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 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).