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

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.

International consensus guidelines for phosphoglucomutase 1 deficiency (PGM1‐CDG): Diagnosis, follow‐up, and management

“…Increased sensitivity and specificity is offered by different mass‐spectrometry (MS) techniques. Methods for N‐glycan analysis in CDG include MALDI‐TOF, 14,34 ESI‐MS 35 and ESI‐QTOF 36 . In particular, high‐resolution mass spectrometry techniques are capable of diagnosing PGM1‐CDG, 3 based on the combined identification of glycan loss and loss of galactose.…”

Clinical and biochemical improvement with galactose supplementation in SLC35A2-CDG

“…These were all patients younger than 3 years of age. Pre-treatment samples from 8 patients (patients 1-3, 5-7 and 9-10) were available for a recently described quantitative N-glycan analysis with demonstrated increased clinical sensitivity and specificity 12 . All 8 patients demonstrated pre-treatment abnormalities in galactosylation with the quantitative N-glycan assay.…”

“…From 8 patients (patients 1-3, 5-7 and 9-10) samples were collected before and after the galactose trial for semi-quantitative N-glycan mass spectrometry analysis (ESI-QTOF) 12 .…”

Expanding the phenotype, genotype and biochemical knowledge of ALG3‐CDG

“…Eight individuals were evaluated using the Nijmegen Pediatric CDG Rating Scale at their last clinical assessment (NPCRS), 27 which scales subjects into mild (0-14), moderate (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), or severe (26-76/82 [0-2 years/ >2 years]) categories. Most of the individuals scored in the severe range with an average score of 31 (Table 1).…”