Determination of Sialylated and Neutral Oligosaccharides in Urine by Mass Spectrometry

Peter R. Clements1

1 SA Pathology/Women's and Children's Hospital, North Adelaide, South Australia
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.10
DOI:  10.1002/0471142905.hg1710s72
Online Posting Date:  January, 2012
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This protocol describes a method to allow for the detection of specific oligosaccharide fragments in urine by tandem mass spectrometry. The detection of fragments with specific masses indicates the presence of one of a number of diseases where the deficiency of lysosomal enzymes involved in the degradation of the glyco‐ moieties of glycoproteins is present in the patient. This method describes the derivatization of oligosaccharides present in urine with phenyl‐1‐methylpyrazolone, which renders them hydrophobic, thus allowing desalting with Combi cleanup columns prior to injection. This method allows the detection of storage of oligosaccharides, which may indicate the presence of one of the infantile Pompe disease, α‐mannosidosis, Gm1‐gangliosidosis, Sandhoff disease, sialidosis, galactosialidosis, I‐cell disease, and aspartylglucosaminuria. Curr. Protoc. Hum. Genet. 72:17.10.1‐17.10.14 © 2012 by John Wiley & Sons, Inc.

Keywords: lysosomal disease; oligosacchariduria; tandem mass spectrometry; lysosomal enzymes

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Urine Samples for Mass Spectrometric Analysis
  • Support Protocol 1: Mass Spectrometer Settings
  • Basic Protocol 2: Processing Oligosaccharide Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of Urine Samples for Mass Spectrometric Analysis

  • Creatinine
  • 1‐Phenyl‐3‐methyl‐5‐pyrazolone solution (PMP solution; see recipe)
  • Internal standards:
    • 2′‐O‐Methyllactose (see recipe)
    • 13C Starch hydrolysate (see recipe)
  • Solid‐phase extraction cartridges (combination 50 mg/1 ml C18/aminopropyl; UCT Technologies, cat. no. UCTCUNAX2L1)
  • Methanol (BDH)
  • 0.8 M formic acid (see recipe)
  • Chloroform with 1% (v/v) ethanol (BDH)
  • Milli‐Q water, pH 11.5 (see recipe)
  • 50% acetonitrile/Millli‐Q water at pH 11.5
  • 50% acetonitrile/Milli‐Q water at pH 11.5/0.025% formic acid
  • Quality control urine samples
  • Christ lyophilizer with SpeedVac attachment
  • 1.5‐ and 2.0‐ml microcentrifuge tubes (Eppendorf)
  • 70°C heating block or oven
  • Vacuum manifold (e.g., Supelco Visiprep 24)
  • Micropipettes
  • 96‐well, U‐bottom, heat‐resistant plates (Nunc, cat. no. 442587)
  • Perkin Elmer SCIEX 3000 triple‐quadrupole tandem mass spectrometer controlled by Analyst software (Perkin Elmer) with Gilson autosampler and Agilent HP1100 HPLC system on the front end
  • Aluminum foil
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Literature Cited

Literature Cited
   Conzelmann, E. and Sandhoff, K. 1991. Partial enzyme deficiencies: Residual activities and the development of neurological disorders. Dev. Neurosci. 13:197‐204.
   Fuller, M., Rozaklis, T., Ramsay, S.L., Hopwood, J.J., and Meikle, P.J. 2004. Disease‐specific markers for the mucopolysaccharidoses. Pediatr. Res. 56:733‐738.
   Peelen, G.O., de Jong, J.G., and Wevers, R.A. 1994. HPLC analysis of oligosaccharides in urine from oligosaccharidosis patients. Clin. Chem. 40:914‐921.
   Ramsay, S.L., Meikle, P.J., Hopwood, J.J., and Clements, P.R. 2005. Profiling oligosaccharidurias by electrospray tandem mass spectrometry: Quantifying reducing oligosaccharides. Anal. Biochem. 345:30‐46.
   Sewell, A.C. 1981. Simple laboratory determination of excess oligosacchariduria. Clin. Chem. 27:243‐245.
   Sowell, J. and Wood, T. 2011. Towards a selected reaction monitoring mass spectrometry fingerprint approach for the screening of oligosaccharidoses. Anal. Chim. Acta 686:102‐106.
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