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Clin Chem. 2019 May;65(5):653-663. doi: 10.1373/clinchem.2018.296780. Epub 2019 Feb 15.

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

Author information

1
Division of Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA.
2
Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA.
3
Division of Endocrinology and Diabetes, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA.
4
Department of Clinical Genomics, Mayo Clinic, Rochester, MN.
5
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA.
6
Division of Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA; hem@email.chop.edu.

Abstract

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 N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection-electrospray ionization-quadrupole time-of-flight mass spectrometry.

METHODS:

After PNGase F digestion of plasma glycoproteins, N-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 N-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 N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-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 N-linked protein glycosylation by carbohydrate-deficient transferrin analysis.

CONCLUSIONS:

The clinical specificity and sensitivity of N-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.

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