Abstract
Publisher Summary Characterization of glycoproteins requires application of several chromatographic and mass spectrometric analytical techniques. This chapter discusses carbohydrate microheterogeneity using high pH anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and electrospray ionization mass spectrometry (ES-MS) after isolation and identification of the peptides containing glycosylation sites. MALDI-TOF mass spectrometry generally gives good results for the glycopeptides at sub-picomolar to low picomolar levels, and it is possible to analyze nearly all of the peptides using this technique. The purified glycopeptides are analyzed by ES-MS and in several cases produced some different ion populations than what is observed in the MALDI-TOF spectra. Based on the mass spectrometric and high pH anion exchange chromatographic results, the major difference between the two r-HuB61 150 forms secreted from CHO cells is in the type of N-linked carbohydrate glycosylation at Asn 8 . The partial glycosylation pattern observed in this study is also common to other recombinant glycoproteins produced in CHO cells, such as stem cell factor.
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