Glycosylation Analysis for Congenital Disorders of Glycosylation

Xueli Li1, Mohd A. Raihan1, Francis Jeshira Reynoso2, Miao He3

1 Palmieri Metabolic Disease Laboratory, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, 2 Department of Genetics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, 3 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.18
DOI:  10.1002/0471142905.hg1718s86
Online Posting Date:  July, 2015
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Abstract

Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an increasing number are defects of glycolipid or proteoglycan biosynthesis. A group of biochemical markers has been used to characterize protein glycosylation abnormalities in CDG. This unit describes three protocols that can be used to measure plasma or serum carbohydrate deficient transferrin (CDT) profile, N‐glycan profile, and O‐glycan profile by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) or liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS). The quantification of particular biomarkers, such as T antigens or sialylated T antigens, could also be achieved by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). These techniques can be used to identify a majority of patients with defects in protein glycosylation, although different techniques, such as flow cytometry with immunostaining, are necessary to detect defects in glycolipid or proteoglycan biosynthesis which is not included in this unit. © 2015 by John Wiley & Sons, Inc.

Keywords: transferrin; N‐linked glycan; O‐linked glycan; congenital disorders of glycosylation; MALDI‐TOF‐MS; LC‐MS/MS; Thomsen‐Friedenreich antigen; N‐acetyl‐galactosamine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of CDT by MALDI‐TOF
  • Support Protocol 1: Regeneration of Columns
  • Support Protocol 2: Preparation of the Anti‐Transferrin Affinity Column
  • Support Protocol 3: Preparation of Monoglycosylated Transferrin Protein
  • Basic Protocol 2: Plasma/Serum N‐Glycan Profile Analysis
  • Basic Protocol 3: Plasma/Serum O‐Glycan Qualitative Profiling by MALDI‐TOF‐MS and O‐Glycan Quantification by LC‐MS/MS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of CDT by MALDI‐TOF

  Materials
  • Normal control plasma (see recipe)
  • Positive control plasma (see recipe)
  • Patient samples (i.e., plasma or serum in tubes containing heparin or EDTA)
  • ProteoSeek Antibody‐Based Albumin/IgG Removal Kit (e.g., Pierce)
  • Polyclonal rabbit anti‐human transferrin antibody‐coupling affinity column (see protocol 3)
  • Washing solution (resin storage buffer; see recipe)
  • Elution buffer (see recipe)
  • Protein determination reagent (for BCA assay; e.g., Bio‐Rad)
  • MALDI matrix solution (α‐cyano‐4‐hydroxycinnamic acid; see recipe)
  • Benchtop shaker
  • Centrifuge
  • YM‐10 Microcon filter (e.g., Millipore)
  • 2‐ml microcentrifuge tubes
  • Lyophilizer
  • MALDI plate (384 well; e.g., ABSciex)
  • MALDI‐TOF mass spectrometer (e.g., ABSciex 4800 plus)
  • Computer running spreadsheet program (e.g., Microsoft Excel)

Support Protocol 1: Regeneration of Columns

  Materials
  • Albumin/IgG antibody‐coupling gel column (from protocol 1)
  • Transferrin affinity column (from protocol 1)
  • Washing solution (see recipe)
  • Elution buffer (see recipe)
  • Centrifuge

Support Protocol 2: Preparation of the Anti‐Transferrin Affinity Column

  Materials
  • Polyclonal rabbit anti‐human transferrin antibody (e.g., Dako or Bio‐Rad)
  • 10 mM MOPS‐NaOH buffer (pH 7.5; see recipe)
  • Affi‐Gel 10 resin (e.g., Bio‐Rad)
  • 0.1 M MOPS‐NaOH buffer (pH 7.5; see recipe)
  • 1 M ethanolamine‐HCl buffer (pH 8.0, see recipe)
  • Washing buffer (see recipe)
  • Slide‐A‐Lyzer 10K dialysis cassettes (e.g., Pierce)
  • 5‐ml syringe
  • 15‐ml plastic centrifuge tubes (e.g., Corning)
  • Lyophilizer
  • Nalgene Rapid‐Flow filter unit, alpha PES membrane, 0.2 μm (e.g., Thermo Scientific)
  • Benchtop shaker
  • Centrifuge
  • Empty mini spin column

Support Protocol 3: Preparation of Monoglycosylated Transferrin Protein

  Materials
  • Pure transferrin protein (e.g., Sigma‐Aldrich)
  • PNGase F enzyme and digestion buffer (e.g., New England Biolabs)
  • Anti‐human transferrin affinity column (see protocol 1 and protocol 3)
  • Protein assay kit
  • YM‐10 Microcon filter (e.g., Millipore)
  • MALDI plate (384 well; e.g., ABSciex)
  • MALDI‐TOF mass spectrometer (e.g., ABSciex 4800 plus)

Basic Protocol 2: Plasma/Serum N‐Glycan Profile Analysis

  Materials
  • Plasma or serum samples, including normal control and positive control (see recipe)
  • 10 μM mannopentose (internal standard)
  • PNGase F enzyme with associated digestion buffers (e.g., New England Biolabs)
  • Sep‐Pak C18 3 cc Vac cartridges (e.g., Waters)
  • Methanol
  • Extract‐Clean SPE Carbograph column (e.g., UCT)
  • 80% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid (TFA) in water (Optima LC/MS; e.g., Fischer Scientific)
  • 30% (v/v) acetonitrile with 0.1% (v/v) TFA in water (Optima LC/MS; e.g., Fischer Scientific)
  • Nitrogen source
  • Anhydrous dimethyl sulfoxide (DMSO)
  • NaOH beads (20‐40 mesh)
  • Iodomethane
  • Chloroform
  • MALDI matrix solution (2,5‐dihydroxybenzoic acid, DHB; see recipe)
  • Water bath
  • 15‐ml plastic centrifuge tubes (e.g., Corning)
  • Centrifuge
  • Lyophilizer
  • 5‐ml syringe and needle
  • Benchtop shaker
  • 2‐ml microcentrifuge tube
  • MALDI‐TOF plate (384 well; e.g., ABSciex)
  • MALDI‐TOF mass spectrometer (e.g., ABSciex 4800 plus)

Basic Protocol 3: Plasma/Serum O‐Glycan Qualitative Profiling by MALDI‐TOF‐MS and O‐Glycan Quantification by LC‐MS/MS

  Materials
  • Plasma or serum samples, including normal control and positive control (see recipe)
  • 50 μM galacto‐N‐biose (T antigen)
  • 500 μM 3′‐N‐acetylneuraminyl‐N‐acetyllactosamine sodium salt (ST antigen)
  • 10 μM D‐(+)‐raffinose pentahydrate (internal standard)
  • 2 M sodium borohydrate in 0.1 M sodium hydroxide solution (NaBH 4‐NaOH; make fresh; see recipe)
  • 0.25 M acetic acid in methanol (see recipe)
  • Nitrogen source
  • Methanol
  • AG 50W‐X8 (see recipe; e.g., Bio‐Rad)
  • Anhydrous DMSO
  • NaOH beads
  • Iodomethane
  • Chloroform
  • MALDI matrix solution (DHB; see recipe)
  • 15‐ml plastic centrifuge tube (e.g., Corning)
  • Oven
  • 9‐inch disposable Pasteur pipets
  • Glass beads
  • Lyophilizer
  • 5‐ml syringe and needle
  • Empty ultra microspin column
  • Centrifuge
  • MALDI plate (384 well; e.g., ABSciex)
  • MALDI‐TOF mass spectrometer (e.g., ABSciex 4800 plus)
  • High‐performance liquid chromatography (HPLC) vial
  • LC‐MS/MS system (e.g., ABSciex 5500 system)
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Figures

Videos

Literature Cited

Literature Cited
  Freeze, H.H. 2006. Genetic defects in the human glycome. Nat. Rev. 7:537‐551.
  Freeze, H.H., Chong, J.X., Bamshad, M.J., and Ng, B.G. 2014. Solving glycosylation disorders: Fundamental approaches reveal complicated pathways. Am. J. Hum. Genet. 94:161‐175
  Guillard, M., Morava, E., van Delft, F.L., Hague, R., Körner, C., Adamowicz, M., Wevers, R.A., and Lefeber, D.J. 2011. Plasma N‐glycan profiling by mass spectrometry for congenital disorders of glycosylation type II. Clin. Chem. 57:593‐602.
  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.
  Lauc, G., Huffman, J.E., Pučić, M., Zgaga, L., Adamczyk, B., Mužinić, A., Novokmet, M., Polašek, O., Gornik, O., Krištić, J., Keser, T., Vitart, V., Scheijen, B., Uh, H.W., Molokhia, M., Patrick, A.L., McKeigue, P., Kolčić, I., Lukić, I.K., Swann, O., van Leeuwen, F.N., Ruhaak, L.R., Houwing‐Duistermaat, J.J., Slagboom, P.E., Beekman, M., de Craen, A.J., Deelder, A.M., Zeng, Q., Wang, W., Hastie, N.D., Gyllensten, U., Wilson, J.F., Wuhrer, M., Wright, A.F., Rudd, P.M., Hayward, C., Aulchenko, Y., Campbell, H., and Rudan, I. 2013. Loci associated with N‐glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers. PLoS Genet. 9:e1003225.
  Leoyklang, P., Malicdan, M.C., Yardeni, T., Celeste, F., Ciccone, C., Li, X., Jiang, R., Gahl, W.A., Carrillo‐Carrasco, N., He, M., and Huizing, M. 2014. Sialylation of Thomsen‐Friedenreich antigen is a noninvasive blood‐based biomarker for GNE myopathy. Biomark. Med. 8:641‐652.
  Maenhout, T.M., Poll, A., Vermassen, T., De Buyzere, M.L., Delanghe, J.R.; ROAD Study Group. 2014. Usefulness of indirect alcohol biomarkers for predicting recidivism of drunk‐driving among previously convicted drunk‐driving offenders: Results from the Recidivism Of Alcohol‐impaired Driving (ROAD) study. Addiction 109:71‐78.
  Thiel, C., Schwarz, M., Peng, J., Grzmil, M., Hasilik, M., Braulke, T., Kohlschütter, A., von Figura, K., Lehle, L., and Körner, C. 2003. A new type of congenital disorders of glycosylation (CDG‐Ii) provides new insights into the early steps of dolichol‐linked oligosaccharide biosynthesis. J. Biol. Chem. 278:22498‐22505.
  Wopereis, S., Grünewald, S., Huijben, K.M., Morava, E., Mollicone, R., van Engelen, B.G., Lefeber, D.J., and Wevers, R.A. 2007. Transferrin and apolipoprotein C‐III isofocusing are complementary in the diagnosis of N‐ and O‐glycan biosynthesis defects. Clin. Chem. 53:180‐187.
  Xia, B., Zhang, W., Li, X., Jiang, R., Harper, T., Liu, R., Cummings, R.D., and He, M. 2013. Serum N‐glycan and O‐glycan analysis by mass spectrometry for diagnosis of congenital disorders of glycosylation. Anal. Biochem. 442:178‐185.
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