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Characterization of O-glycosylated precursors of insulin-like growth factor II by matrix-assisted laser desorption/ionization mass spectrometry

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Author: Jespersen, S. · Koedam, J.A. · Hoogerbrugge, C.M. · Tjaden, U.R. · Greef, J. van der · Brande, J.L. van den
Institution: TNO Voeding
Source:Journal of Mass Spectrometry, 8, 31, 893-900
Identifier: 233414
doi: doi:10.1002/(SICI)1096-9888(199608)31:8<893::AID-JMS374>3.0.CO;2-S
Keywords: Nutrition · Matrix-assisted laser desorption/ionization · O-glycosylation · Precursor insulin-like growth factor II · Somatomedin b · Amino acid sequence · Article · Drug isolation · Drug purification · Drug structure · Glycosylation · Mass spectrometry · Molecular weight · Priority journal · Protein degradation · Amino Acid Sequence · Glycoside Hydrolases · Humans · Hydrolysis · Insulin-Like Growth Factor II · Molecular Sequence Data · Molecular Weight · Neuraminidase · Oligosaccharides · Peptide Mapping · Protein Precursors · Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


High molecular weight precursors of insulin-like growth factor II (IGF-II) were isolated from Cohn fraction IV of human plasma by ultrafiltration, affinity chromatography and reversed-phase high-performance liquid chromatography. Molecular weight determination by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of two high molecular weight IGF-II preparations revealed heterogeneous glycosylation. A combination of enzymatic degradation and MALDI-MS were applied for further structural characterization of the glycosylated precursors of IGF-II. The first step was molecular weight determination of intact high molecular weight IGF-IIs prior to and after treatment with neuraminidase and O-glycosidase. This, together with a comparison of molecular weight information available from the cDNA, revealed that both high molecular weight IGF-II species contain an identical C-terminal extension of 20 residues but different degrees of glycosylation. Second, comparative Endo Glu-C digestion of the preparations prior to and after enzymatic release of carbohydrates and subsequent remeasurement of the molecular weight by MALDI-MS confirmed the primary structure of precursor IGF-II1-87. The O-linked carbohydrates were found to be associated with the C-terminal extension and the heterogeneity was identified as varied sialylated forms of one and two HexNAc-Hex groups.