TITLE

Comparison of planar SDS-PAGE, CGE-on-a-chip, and MALDI-TOF mass spectrometry for analysis of the enzymatic de- N-glycosylation of antithrombin III and coagulation factor IX with PNGase F

AUTHOR(S)
Müller, R.; Marchetti, M.; Kratzmeier, M.; Elgass, H.; Kuschel, M.; Zenker, A.; Allmaier, G.
PUB. DATE
November 2007
SOURCE
Analytical & Bioanalytical Chemistry;Nov2007, Vol. 389 Issue 6, p1859
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Three different analytical techniques (planar SDS-PAGE, CGE-on-a-chip and MALDI-TOF-MS) applied for determination of the molecular weight of intact and partly and completely de- N-glycosylated human serum glycoproteins (antithrombin III and coagulation factor IX) have been compared. N-Glycans were removed from the protein backbone of both complex glycoproteins using PNGase F, which cleaves all types of asparagine-attached N-glycan provided the oligosaccharide has at least the length of a chitobiose core unit. Two of the applied techniques were based on gel electrophoretic separation in the liquid phase while the third technique was the gas-phase technique mass spectrometry. It was demonstrated that the enzymatic de- N-glycosylation generally worked well (completely or partially) with both glycoproteins (one containing only N-glycans and the second N- and O-glycans). All three methods were suitable for monitoring the de- N-glycosylation progress. While the molecular weights determined with MALDI-TOF-MS were most accurate, both gel electrophoretic methods provided molecular weights that were too high because of the attached glycan structures. [Figure not available: see fulltext.]
ACCESSION #
27733990

 

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