Mass Spectrometric Characterization of the β-Subunit of Human Chorionic Gonadotropin

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris

“…The majority of the Nglycans contained 9 to 10 Man residues, independent on the chosen culturing process, whereby Man 9 GlcNAc 2 nearly only represents the typical yeast isomer. However, culturing conditions did influence the molar ratio within Man [8][9][10][11][12] GlcNAc 2 . It should be noted that previous NMR structural studies of yeast glycoprotein N-glycans were always carried out on Man x (P y )GlcNAc 1 compounds; in the present investigation NMR data of Man x (P y )GlcNAc 2 compounds are presented.…”

“…Indications for the occurrence of trisialylated tri-and tri′-antennary glycans on β-hCG have also been reported [5]. Structural data for the O-glycosylation comprise mainly the conventional mono-and disialylated core 1-type structures, the disialyl-hexasaccharide (including core 2 type), and the sialylated disaccharide [6][7][8]. A random distribution of these O-glycans among the attachment sites has been suggested [9].…”

“…Expression of hCG in CHO cells shows the same mono-, di-, tri-, and tri′-antennary N-glycans as found for urinary hCG; Neu5Ac occurs in (α2-3) linkage to Gal. The O-glycans comprise mainly the convential saccharides (see above) [8,11,12]. CHO cells have also been used for generating 13 C/ 15 Nlabeled hCG for NMR studies [13].…”

Synthesis and Characterization of Insulin-like Growth Factor (IGF)-1 Photoprobes Selective for the IGF-binding Proteins (IGFBPs)

“…The sample was then placed in ice water and irradiated for 2 h with a prewarmed Fotodyne handheld, single-wavelength UV lamp (2 ϫ 4-watt 300-nm bulbs) at a distance of 2 cm. The mixture was dried in vacuo, and the proteins were reduced and alkylated using tris(2-carboxyethylyl)phosphine (32) and 4-vinylpyridine (33). abG 1 IGF-1-and bedG 1 IGF-1-photolabeled IGF-BP-2 proteins were separated from unreacted IGFBP-2 by reversedphase HPLC and trypsinized as described below.…”

Tandem Mass Spectrometry for Simultaneous Qualitative and Quantitative Analysis of Protein

“…The high signal to noise ratio reveals that this method would be able to detect a much lower concentrations of hCG. This detection limit of hCG (5 mIU/mL) using our current method is superior than SIM method where a detection limit of 25 mIU/mL was reported (Liu & Bowers 1997). At 5 mIU/ml hCG, the parent ions of the peptide marker was indistinguishable from the background noise in full scan MS.…”