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.
Abbreviations:
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)To read this article in full you will need to make a payment
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Article info
Publication history
Published online: August 07, 2015
Accepted:
July 23,
2015
Received in revised form:
July 22,
2015
Received:
December 15,
2014
Identification
Copyright
© 2015 Elsevier Inc. Published by Elsevier Inc. All rights reserved.
