Electrospray Ionization-Mass Spectrometric Analysis of Serum Transferrin Isoforms in Patients with Carbohydrate-Deficient Glysoprotein Syndrome1

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Abstract
We previously reported that the carbohydrate-deficient glycoprotein (CDG) syndrome is an asparagine-iV-linked sugar chain transfer deficiency [Yamashita et al. (1993) J. BioI. Chem 268, 5783–5789]. In order to confirm this hypothesis, we applied electrospray ionization-mass spectrometric analysis to transferrin isoforms purified from patients with the.CDG syndrome. Transferrin isoforms containing 4, 2, and 0 sialic acid residues, S4, -S2, and So, were separated by Mono Q anion exchange column chromatography from serum of a patient with the CDG syndrome. The molecular masses of S4, S2, and So were determined to be 79, 570±5, 77, 364±6, and 75, 157±6 Da by electrospray ionization mass spectrometry (ESI/MS). The differences between S4 and S2, and between S2 and So were both in accordance with the molecular mass of a disialylated biantennary sugar chain {Neu5Acα2→6Galβ1→4GlcNAcβ1→2Manα 1 → 6(Neu5Ac α 2 → 6Gal β1 → 4GlcNAc β1 → 2Man α 1 → 3)Man β1 → 4GlcNAcβ1→4GlcNAc} (2, 206 Da), showing that So is nonglycosylated, and that S4 and S2 carry 2 and 1 mol of asparagine-N-linked sugar chains, respectively. The nonglycosylated asparagine site of S2 was elucidated to be random by high performance liquid chromatog-raphy-ESI/MS of a tryptic peptide of reduced and pyridylethylated S2. ESI/MS analysis of transferrin purified through one step from serum is applicable for a definite diagnosis of the CDG syndrome.

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