Tandem Mass Spectrometry Quantitation of Lyso‐Gb3 and Six Related Analogs in Plasma for Fabry Disease Patients

Michel Boutin1, Pamela Lavoie1, Mona Abaoui1, Christiane Auray‐Blais1

1 Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.23
DOI:  10.1002/cphg.4
Online Posting Date:  July, 2016
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Fabry disease is an X‐linked lysosomal storage disorder, caused by a deficit in α‐galactosidase A enzyme activity, leading to the storage of sphingolipids such as globotriaosylsphingosine (lyso‐Gb3), globotriaosylceramide (Gb3), and galabiosylceramide (Ga2) in organs, tissues and biological fluids. A recent metabolomic study performed in plasma revealed lyso‐Gb3 analogs as novel Fabry disease biomarkers. These molecules correspond to lyso‐Gb3 with different chemical modifications on the sphingosine chain (−C2H4, −H2, +O, +H2O, +H2O2, and +H2O3). An ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the multiplex analysis of lyso‐Gb3 and its 6 analogs in plasma. The samples are prepared by solid phase extraction using mixed‐mode strong cation exchange (MCX) cartridges. An in‐house synthesized N‐glycinated lyso‐Gb3 derivative was used for the internal standard. The limits of detection (LODs) measured for lyso‐Gb3 and its analogs ranged from 0.06 to 0.29 nM. © 2016 by John Wiley & Sons, Inc.

Keywords: analogs; biomarkers; Fabry disease; Lyso‐Gb3; MS/MS; plasma; tandem mass spectrometry; ultra‐performance liquid chromatography (UPLC)

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Table of Contents

  • Introduction
  • Basic Protocol 1: Tandem Mass Spectrometry Analysis of Lyso‐Gb3 and Related Analogs in Plasma
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Tandem Mass Spectrometry Analysis of Lyso‐Gb3 and Related Analogs in Plasma

  • Methanol, LC/MS grade (EMD Millipore, cat. no. MX0486‐1)
  • 85% o‐phosphoric acid (Fisher Scientific, cat. no. A 242 500)
  • Plasma samples in lavender (K 2‐EDTA) vacutainer tubes
  • Control plasma (K 2‐EDTA), 2× charcoal stripped (BioreclamationIVT, cat. no. HMPLEDTA2‐STRPD)
  • Water, LC/MS grade (EMD Millipore, cat. no. WX0001‐1)
  • Standard curve working solutions (see recipe)
  • 10 nM N‐glycinated globotriaosylsphingosine (lyso‐Gb 3‐Gly) (synthesized in‐house, Lavoie et al., ; commercially available from Matreya LLC, cat. no. 1530) in methanol
  • 99% formic acid (Fisher, cat. no. AC27048‐0010)
  • 29% ammonium hydroxide, ACS+ (Fisher, cat. no. A669‐500), prepare fresh
  • Acetonitrile, LC/MS grade (VWR, cat. no. CAAX0156‐1)
  • Disposable culture tubes, 13 × 100–mm (Fisher Scientific, cat. no. 14‐961‐27)
  • Disposable culture tubes, 16 × 150–mm (Fisher Scientific, cat. no. 14‐961‐31)
  • 2‐ml vials, screw tread, 12 × 32–mm, clear glass (Chromspec, cat. no. C779100WM)
  • 30‐mg Oasis MCX cartridges, 60‐μm LP (Waters, cat. cat. 186000782)
  • Repeater (Eppendorf)
  • 200‐µl pipet tips (VWR, cat. no. 89079‐478)
  • 1000‐µl pipet tips (VWR, cat. no. 89079‐470)
  • Vortex
  • Pasteur pipets
  • Nitrogen evaporator
  • 250‐µl glass inserts, 6 × 29–mm (Chromspec, cat. no. C221020M)
  • 9‐mm screw caps, preslit PTFE/silicone septum (Chromspec, cat. no. C779200XBBM)
  • Centrifuge
  • UPLC system with autosampler (Acquity I‐Class, Waters)
  • BEH C18, 1.7‐µm, 2.1 × 50–mm columns (Waters, cat. no. 186002350)
  • Xevo TQ‐S tandem quadrupole mass spectrometer (Waters)
  • MassLynx software version 4.1 (Waters)
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Literature Cited

Literature Cited
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