Characterization of O-glycosylated precursors of insulin-like growth factor II by matrix-assisted laser desorption/ionization mass spectrometry.
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.
Jespersen
S
S
Division of Analytical Chemistry, University of Leiden, The Netherlands.
Koedam
J A
JA
Hoogerbrugge
C M
CM
Tjaden
U R
UR
van der Greef
J
J
Van den Brande
J L
JL
D000595:Amino Acid Sequence; D006026:Glycoside Hydrolases; D006801:Humans; D006868:Hydrolysis; D007335:Insulin-Like Growth Factor II / Q000032:analysis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009439:Neuraminidase; D009844:Oligosaccharides / Q000032:analysis; D010449:Peptide Mapping; D011498:Protein Precursors / Q000032:analysis* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009844:Oligosaccharides; D011498:Protein Precursors; D007335:Insulin-Like Growth Factor II; D006026:Glycoside Hydrolases; D009439:Neuraminidase