Detection of Hypo‐N‐Glycosylation Using Mass Spectrometry of Transferrin

John F. O'Brien1, Jean M. Lacey1, H. Robert Bergen1

1 Mayo Clinic College of Medicine, Rochester, Minnesota
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.4
DOI:  10.1002/0471142905.hg1704s54
Online Posting Date:  July, 2007
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Many congenital disorders of glycosylation (CDG) can be diagnosed by observing the extent of glycosylation of the abundant serum glycoprotein transferrin (Trf). Trf is an N‐glycosylated protein with two asparagine glycation sites. CDG types I are those genetic defects which occur prior to transfer of the complex oligosaccharide to the acceptor asparagine in the cotranslated polypeptide chain. CDG Ia constitutes by far the most frequent form of CDG and is the result of mutations in the phosphomannomutase gene. CDG Ia and the Ib subtype (Phosphomannoisomerase deficiency) result in low cellular mannose‐1‐phosphate levels, a required precursor for oligosaccharide assembly in the endoplasmic reticulum. The deficiency in oligosaccharides with branched mannose structures is thereafter expressed by the appearance of glycoproteins with unoccupied N‐glycosylation sites (hypoglycosylation). Currently, there have been at least 11 Type I defects, type Ia being by far the most frequently occurring. Most, if not all type I defects result in unoccupied N‐glycation sites. Hypoglycosylated Trf, also known as carbohydrate‐deficient Trf (CDT), can be detected using mass spectrometry (MS) to measure the masses of the serum Trf. The methods for sample preparation using affinity chromatography and MS analysis are described in this unit. Curr. Protoc. Hum. Genet. 54:17.4.1‐17.4.9 © 2007 by John Wiley & Sons, Inc.

Keywords: congenital disorders of glycosylation (CDG); carbohydrate deficient Transferrin (CDT) or hypoglycosylated tranferrin; transferrin (Trf); glycoforms; glycoproteins; oligosaccharides

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Table of Contents

  • Basic Protocol 1: Preparation of Affinity Columns
  • Basic Protocol 2: Analysis of Serum Samples for Hypoglycosylatation of Transferrin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Affinity Columns

  • 14 mg/ml rabbit anti–human Trf (Dako, no. A0061)
  • PBS (see recipe)
  • Poros AL Self Pack Media (PE Biosystems)
  • High‐salt buffer (see recipe)
  • 100 mg/ml sodium cyanoborohydride (NaCNBH 3; see recipe)
  • Capping buffer: 0.2 M Tris·Cl, pH 7.2 ( appendix 2D)
  • Sodium chloride (NaCl; Sigma)
  • 10 K MWCO Slide‐A‐Lyzer Dialysis Cassettes and 18‐G needle accessories (Pierce)
  • 15‐ml polypropylene screw cap tube
  • Platform rocker
  • 1‐mm × 2‐cm C‐128 guard column (Upchurch)
  • 5 µm SS Frit (black) (Upchurch)

Basic Protocol 2: Analysis of Serum Samples for Hypoglycosylatation of Transferrin

  • Reverse osmosis (RO) H 2O
  • Normal pooled serum
  • Abnormal pooled serum
  • Patient serum
  • Elution buffer (see recipe)
  • Aqueous mobile phase (see recipe)
  • Organic mobile phase (see recipe)
  • apo‐Trf standard (Sigma)
  • Trf standard (use pooled waste serum from normal patients)
  • 96‐well sterile assay plates, round bottom (Fisher Chemical, no. 087727)
  • Micromat well cover
  • Autosampler (Perkin Elmer Series 200)
  • HPLC system including:
    • PE Sciex API 150 LC/MS/MS with TurboIonSpray ion source
    • Micropump (Perkin Elmer Series 200)
    • System Controller, (Shimadzu, SCL‐10Avp)
    • Liquid Chromatography pump (Shimadzu, LC‐10Advp)
    • Two Position Actuator Control Module, (Valco Instruments)
    • Microfiter (Upchurch Scientific, M‐560)
    • Security Guard Kit KJO‐4282 (Phenomenex)
    • Nitrogen, purified, use in‐house supply and filter using hydrocarbon trap (Chromtech, BHT1)
    • Air compressed, use in‐house supply
  • Affinity column (see protocol 1)
  • Widepore C4 (Butyl) 4 mm × 2.0–mm cartridge (Phenomenex, no. AJO‐4329)
  • Additional reagents and equipment for preparation of the affinity columns ( protocol 1)
NOTE: All pump flow rates are 200 µl per min. Wash and elution times are determined by flow rate and switch dwell times.NOTE: The affinity purification procedure was adapted from that previously used prior to IEF analysis. Using IEF, only Trf was seen after Coomassie staining, so the immuno‐capture is assumed to be specific for Trf. Moreover, mass selection provides another level of specificity and peaks at masses other than that shown in the Figure are not experienced even after 500+ injections on a column.
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Literature Cited

Literature Cited
   Jaeken, J. and Matthijs, G. 2001. Congenital disorders of glycosylation. Annu. Rev. Genomics. Hum. Genet. 2:129‐151.
   Lacey, J.M., Bergen, H.R., Magera, M.J., Naylor, S., and O'Brien, J.F. 2001. Rapid determination of transferrin isoforms by immunoaffinity liquid chromatography and electrospray mass spectrometry. Clin. Chem. 47:513‐518.
   O'Brien, J.F. 2005. Methods for detection of carbohydrate‐deficient glycoprotein syndromes. Semin. Pediatr. Neurol. 12:159‐162.
   Sparks, S.E. 2006. Inherited disorders of glycosylation. Molecular Genetics and Metabolism 87:1‐7.
   Wada, Y., Nishikawa, A., Okamoto, N., Inui, K., Tsukamoto, H., Okada, S., and Taniguchi, N. 1992. Structure of serum transferrin in carbohydrate‐deficient glycoprotein syndrome. Biochem. Biophys. Res. Commun. 189:832‐836.
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