Carbohydrate structure characterization of two soluble forms of a ligand for the ECK receptor tyrosine kinase

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.