Original Article| Volume 166, ISSUE 6, P639-649.e1, December 2015

High-resolution mass spectrometry glycoprofiling of intact transferrin for diagnosis and subtype identification in the congenital disorders of glycosylation

  • Monique van Scherpenzeel
    Reprint requests: Monique van Scherpenzeel, Department of Neurology, Laboratory for Genetic, Endocrine and Metabolic Diseases, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands
    Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands

    Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Gerry Steenbergen
    Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Eva Morava
    Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands

    Department of Pediatrics, Hayward Genetics Center, Tulane University Medical School, New Orleans, La
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  • Ron A. Wevers
    Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Dirk J. Lefeber
    Translational Metabolic Laboratory, Radboud University Medical Center, Nijmegen, The Netherlands

    Department of Neurology, Radboud University Medical Center, Nijmegen, The Netherlands
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Published:August 07, 2015DOI:
      Diagnostic screening of the congenital disorders of glycosylation (CDG) generally involves isoelectric focusing of plasma transferrin, a robust method easily integrated in medical laboratories. Structural information is needed as the next step, as required for the challenging classification of Golgi glycosylation defects (CDG-II). Here, we present the use of high-resolution nano liquid chromatography-chip (C8)-quadrupole time of flight mass spectrometry (nanoLC-chip [C8]-QTOF MS) for protein-specific glycoprofiling of intact transferrin, which allows screening and direct diagnosis of a number of CDG-II defects. Transferrin was immunopurified from 10 μL of plasma and analyzed by nanoLC-chip-QTOF MS. Charge distribution raw data were deconvoluted by Mass Hunter software to reconstructed mass spectra. Plasma samples were processed from controls (n = 56), patients with known defects (n = 30), and patients with secondary (n = 6) or unsolved (n = 3) cause of abnormal glycosylation. This fast and robust method, established for CDG diagnostics, requires only 2 hours analysis time, including sample preparation and analysis. For CDG-I patients, the characteristic loss of complete N-glycans could be detected with high sensitivity. Known CDG-II defects (phosphoglucomutase 1 [PGM1-CDG], mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase [MGAT2-CDG], β-1,4-galactosyltransferase 1 [B4GALT1-CDG], CMP-sialic acid transporter [SLC35A1-CDG], UDP-galactose transporter [SLC35A2-CDG] and mannosyl-oligosaccharide 1,2-alpha-mannosidase [MAN1B1-CDG]) resulted in characteristic diagnostic profiles. Moreover, in the group of Golgi trafficking defects and unsolved CDG-II patients, distinct profiles were observed, which facilitate identification of the specific CDG subtype. The established QTOF method affords high sensitivity and resolution for the detection of complete glycan loss and structural assignment of truncated glycans in a single assay. The speed and robustness allow its clinical diagnostic application as a first step in the diagnostic procedure for CDG defects.


      ApoCIII (apolipoprotein CIII), ATP6V0A2 (ATPase, H+ transporting, lysosomal V0 subunit a2), B4GALT1 (β-1,4-galactosyltransferase 1), CDG (Congenital Disorders of Glycosylation), CE (Capillary electrophoresis), CMP (cytidine monophosphate), COG1 (component of oligomeric Golgi complex 1), CV (Coefficient of variation), DPAGT1 (dolichyl-phosphate (UDP-N-acetylglucosamine) N-acetylglucosaminephosphotransferase 1), EDTA (Ethylenediaminetetraacetic acid), ESI (Electron spray ionization), GDP (guanosine diphosphate), HPLC (High pressure liquid chromatography), HUS (Hemolytic uremic syndrome), IEF (Isoelectric focusing), LC (Liquid Chromatography), MALDI (Matrix assisted laser desorption ionization), MAN1B1 (mannosyl-oligosaccharide 1,2-alpha-mannosidase), MGAT2 (mannosyl(α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase), MS (Mass spectrometry), NHS (N-Hydroxysuccinimidyl), PGM1 (Phosphoglucomutase 1), PMM2 (phosphomannomutase 2), QTOF (Quadrupole Time Of Flight), SLC35A1 (CMP-sialic acid transporter), SLC35A2 (UDP-galactose transporter), SLC35C1 (GDP-fucose transporter), Tf (transferrin), TIEF (Transferrin Isoelectric focusing), TMEM165 (transmembrane protein 165), Tris (2-Amino-2-(hydroxymethyl)-1,3-propanediol), UDP (uridine disphosphate), VPS13B (vacuolar protein sorting 13 homolog B)
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