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

• Published in: Analytical and bioanalytical chemistry Vol. 389; no. 6; pp. 1859 - 1868
• Main Authors: Müller, R, Marchetti, M, Kratzmeier, M, Elgass, H, Kuschel, M, Zenker, A, Allmaier, G
• Format: Journal Article
• Language: English
• Published: Germany 01.11.2007
• Subjects: Anticoagulants - analysis; Anticoagulants - metabolism; Antithrombin III - analysis; Antithrombin III - metabolism; Asparagine - chemistry; Asparagine - metabolism; Carbohydrate Sequence; Disaccharides - chemistry; Disaccharides - metabolism; Electrophoresis, Capillary - methods; Electrophoresis, Polyacrylamide Gel - methods; Factor IX - analysis; Factor IX - metabolism; Glycosylation; Humans; Molecular Sequence Data; Molecular Weight; Oligosaccharides - chemistry; Oligosaccharides - metabolism; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase - metabolism; Polysaccharides - chemistry; Polysaccharides - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Time Factors
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-007-1586-3

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.