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

The N-linked glycan present on corticotropin releasing factor binding prote in (CRFBP) recombinantly expressed in CHO cells has been characterized usin g a variety of techniques including sequential hydrolysis with specific exo glycosidases monitored with reversed-phase high performance liquid chromato graphy (RP-HPLC), matrix-assisted laser desorption/ionization mass spectrom etry (MALDI-MS) and electrospray ionization mass spectrometry (ES-MS), Dete rmining the intact glycan mass and comparing possible glycan compositions w ith 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 obt ained through specific glycosidase reactions monitored with MS and MSn frag mentation of the intact or enzymatically-modified glycopeptides. No evidenc e was found for the presence of sites of O-linked glycosylation, In additio n, to understand better the role of the glycan on CRFBP, we characterized t he in vitro binding affinity of a mutant non-glycosylated [Gln(180)] CRFBP and an enzymatically deglycosylated form of CRFBP to human corticotropin re leasing factor (hCRF).