Mass spectrometric characterization of glycosylated interferon-gamma variants separated by gel electrophoresis.
Glycosylated proteins in polyacrylamide gels were characterized by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and glycosidase digestion. Sodium dodecyl sulfate-polyacrylamide get electrophoresis (SDS-PAGE) of natural, human interferon-gamma (IFN-gamma) showed two glycosylated variants with apparent molecular masses of 20 and 24 kDa. MALDI-MS of the intact IFN-gamma, electroeluted from the two bands, confirmed that these correspond to IFN-gamma molecules glycosylated at one or both of the two potential glycosylation sites, respectively. The peptide map obtained by MALDI-MS after digestion in the gel covers 92% of the IFN-gamma sequence and revealed an N-terminal pyroglutamate residue and one oxidized methionine residue. One glycosylated peptide was detected after treatment of the peptide mixture with neuraminidase, and the carbohydrate structure partially elucidated by sequential glycosidase digestion monitored by MALDI-MS. A second glycosylated peptide, due to a very heterogeneous glycan structure, could only be observed after separation of the peptides by high performance liquid chromatography (HPLC).
Mørtz
E
E
Department of Molecular Biology, Odense University, Denmark.
Sareneva
T
T
Haebel
S
S
Julkunen
I
I
Roepstorff
P
P
D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis*; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D007371:Interferon-gamma; D014357:Trypsin