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.
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.
Müller
R
R
Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna, Austria.
Marchetti
M
M
Kratzmeier
M
M
Elgass
H
H
Kuschel
M
M
Zenker
A
A
Allmaier
G
G
D000925:Anticoagulants / Q000032:analysis* / Q000378:metabolism; D000990:Antithrombin III / Q000032:analysis* / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005164:Factor IX / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013997:Time Factors
D000925:Anticoagulants; D004187:Disaccharides; D009844:Oligosaccharides; D011134:Polysaccharides; C032438:chitobiose; D001216:Asparagine; D000990:Antithrombin III; D005164:Factor IX; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase