Apolipoprotein C-III (apoCIII) is a small glycoprotein with a single mucin-type core-1 oligosaccharide and is analyzed by isoelectric focusing (IEF) for the diagnosis of genetic defects in O-glycan biosynthesis such as congenital disorders of glycosylation. In the present study, mass spectrometry of apoCIII, after a simple procedure for sample preparation using a small amount of serum, was demonstrated to be a reliable alternative to IEF. It allows reproducible glycan profiling and detection of unglycosylated species. This method was applied to an autosomal recessive cutis laxa type-2 patient and demonstrated decreased site occupancy by O-glycosylation.

Congenital disorders of glycosylation (CDG) form a group of metabolic disorders caused by deficient glycosylation of proteins and/or lipids. Isoelectric focusing (IEF) of serum transferrin is the most common screening method to detect abnormalities of protein N-glycosylation. On the basis of the IEF profile, patients can be grouped into CDG type I or CDG type II. Several protein variants of transferrin are known that result in a shift in isoelectric point (pI). In some cases, these protein variants co-migrate with transferrin glycoforms, which complicates interpretation. In two patients with abnormal serum transferrin IEF profiles, neuraminidase digestion and subsequent IEF showed profiles suggestive of the diagnosis of CDG type I. Mass spectrometry of tryptic peptides of immunopurified transferrin, however, revealed a novel mutation at the N-glycan attachment site. In case 1, a peptide with mutation p.Asn630Thr in the 2nd glycosylation site was identified, resulting in an additional band at disialotransferrin position on IEF. After neuraminidase digestion, a single band was found at the asialotransferrin position, indistinguishable from CDG type I patients. In case 2, a peptide with mutation p.Asn432His was found. These results show the use of mass spectrometry of transferrin peptides in the diagnostic track of CDG type I.

Mass spectrometry of glycopeptides is an efficient strategy for profiling glycans at specific sites in glycoproteins. To assess the reliability of this method for determining the fucosylation levels of glycoproteins, we conducted mass spectrometry of fucosylated glycopeptides from transferrin and haptoglobin. The biantennary glycans containing antenna alpha1,3/4 fucose or alpha1,6 core fucose showed different fragmentation behaviors in collision-induced dissociation of protonated glycopeptides. Stability was dependent on peptide backbone sequences. The major dissociation, cleavage of the GlcNAcbeta1-->2Man linkage of antenna, was evident at a slightly lower activation energy for the core fucosylated species, while the linkage of alpha1,6 core fucose was more stable than that of antenna alpha1,3/4 fucose. However, these fragmentations were induced only with sufficient loading of activation energy. The quantitation of fucosylated glycans by mass spectrometry of glycopeptides, without collisional activation, was thus justified. The fucosylation levels calculated from the signal intensities in electrospray (nanospray) ionization and ultraviolet matrix-assisted laser desorption/ionization mass spectra were essentially the same. The mass spectrometric profiling of glycopeptides from transferrin of congenital disorders of glycosylation (CDG-Ia and CDG-IIc) patients demonstrated that the elevation or reduction of fucosylation in pathological conditions can be reliably determined by MS of glycopeptides.

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