Electrospray ionization-mass spectrometric analysis of serum transferrin isoforms in patients with carbohydrate-deficient glycoprotein syndrome.

We previously reported that the carbohydrate-deficient glycoprotein (CDG) syndrome is an asparagine-N-linked sugar chain transfer deficiency [Yamashita et al. (1993) J. Biol. 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 S0, 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 S0 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 S0 were both in accordance with the molecular mass of a disialylated biantennary sugar chain [Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc] (2,206 Da), showing that S0 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 chromatography-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.