Increased Clinical Sensitivity and Specificity of Plasma Protein -Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry.

BACKGROUND

Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of -linked glycosylation, in which carbohydrate residues, namely, -glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma -glycan assay using flow injection-electrospray ionization-quadrupole time-of-flight mass spectrometry.

METHODS

After PNGase F digestion of plasma glycoproteins, -glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard.

RESULTS

This assay differed from other -glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary -glycan. The majority of -glycans in normal plasma had <1% abundance. Abnormal -glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal -linked protein glycosylation by carbohydrate-deficient transferrin analysis.

CONCLUSIONS

The clinical specificity and sensitivity of -glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.