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Mass spectrometric characterization of glycosylated interferon‐γ variants separated by gel electrophoresis
Author(s) -
Mørtz Ejvind,
Sareneva Timo,
Haebel Sophie,
Julkunen Ilkka,
Roepstorff Peter
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170514
Subject(s) - chemistry , chromatography , glycosylation , peptide , gel electrophoresis , glycan , polyacrylamide gel electrophoresis , mass spectrometry , matrix assisted laser desorption/ionization , residue (chemistry) , biochemistry , glycoprotein , enzyme , desorption , organic chemistry , adsorption
Glycosylated proteins in polyacrylamide gels were characterized by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and glycosidase digestion. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of natural, human interferon‐γ (IFN‐γ) showed two glycosylated variants with apparent molecular masses of 20 and 24 kDa. MALDI‐MS of the intact IFN‐γ, electroeluted from the two bands, confirmed that these correspond to IFN‐γ 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‐γ 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).