Fabry Disease Biomarkers: Analysis of Urinary Lyso‐Gb3 and Seven Related Analogs Using Tandem Mass Spectrometry

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

1 Division of Medical Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.22
DOI:  10.1002/cphg.1
Online Posting Date:  July, 2016
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Abstract

Fabry disease is an X‐linked lysosomal storage disorder caused by the absence or reduction of the enzyme α‐galactosidase A activity. Currently, globotriaosylsphingosine (lyso‐Gb3) and globotriaosylceramide (Gb3) are used as biomarkers to diagnose and monitor Fabry patients. However, recent metabolomic studies have shown that several glycosphingolipids are also elevated in biological fluids of affected patients and may be related to disease manifestations. This unit describes a multiplex methodology targeting the analysis of urinary lyso‐Gb3 and seven structurally related analogs. A solid‐phase extraction process is performed, then lyso‐Gb3 and its analogs are analyzed simultaneously with an internal standard by ultra‐performance liquid chromatography (UPLC) coupled to a tandem mass spectrometry (MS/MS) system. This methodology can be useful for the diagnosis of Fabry patients, including patients with cardiac variant mutations, but also to monitor the efficacy of therapeutic interventions, considering that lyso‐Gb3 analogs are more elevated than lyso‐Gb3 itself in urine. © 2016 by John Wiley & Sons, Inc.

Keywords: analogs; biomarkers; Fabry disease; lyso‐Gb3; mass spectrometry; urine; UPLC‐MS/MS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Lyso‐Gb3 and Related Analogs in Urine by Tandem Mass Spectrometry
  • Support Protocol 1: Preparation of the N‐Glycinated Lyso‐Ceramide Trihexoside Internal Standard Working Solution at 10 nM
  • Support Protocol 2: Preparation of Lyso‐Ceramide Trihexoside (lyso‐Gb3) Standard Solutions
  • Support Protocol 3: Preparation of Positive Quality Controls (QCs)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Lyso‐Gb3 and Related Analogs in Urine by Tandem Mass Spectrometry

  Materials
  • Methanol (MeOH), LC‐MS grade (EMD Millipore, cat. no. MX0486‐1)
  • Hydrochloric acid (HCl) ACS (VWR, cat. no. CA11020‐886)
  • Water, LC‐MS grade (EMD Millipore, cat. no. WX0001‐1)
  • Patient urine samples to be analyzed
  • Healthy control urine sample for calibration curve
  • N‐glycinated lyso‐ceramide trihexoside (lyso‐Gb 3‐Gly) internal standard working solution (see protocol 2)
  • Acetonitrile, LC‐MS grade (VWR, cat. no. CAAX0156‐1)
  • 99% formic acid (Fisher, cat. no. AC27048‐0010)
  • Standard curve working solutions (see protocol 3)
  • Quality control samples (see protocol 4)
  • Ammonium hydroxide (NH 4OH), ACS+ (Fisher, cat. no. A669‐500)
  • Nitrogen source
  • 13 × 100–mm disposable culture tubes (Fisher Scientific, cat. no. 14‐961‐27)
  • 16 × 150–mm disposable culture tubes (Fisher Scientific, cat. no. 14‐961‐31)
  • 2‐ml borosilicate vials, screw tread, 12 × 32–mm (Chromspec, cat. no.C779100WM)
  • 250‐μl inserts, 6 × 29–mm (Chromspec, cat. no. C221020M)
  • 30‐mg Oasis MCX cartridges, 60‐μm LP (Waters, cat. no. 186000782)
  • Vortex
  • Pasteur pipets
  • Evaporator
  • 9‐mm screw‐cap, preslit PTFE/silicone septum (Chromspec, cat. no. C779200XBBM)
  • Centrifuge
  • Atlantis HILIC Silica, 3‐μm, 2.1 × 50–mm column (Waters, cat. no. 186002011)
  • Xevo TQ‐S tandem quadrupole mass spectrometer coupled to an Acquity I‐Class UPLC system with autosampler (Waters)
  • MassLynx ver 4.1

Support Protocol 1: Preparation of the N‐Glycinated Lyso‐Ceramide Trihexoside Internal Standard Working Solution at 10 nM

  Materials
  • Methanol, LC‐MS‐grade (EMD Millipore, cat. no. MX0486‐1)
  • 1 M triethylammonium bicarbonate (Sigma, cat. no. T7408)
  • Chloroform (VWR, cat. no. CACX1054‐6)
  • Boc‐glycine N‐hydroxysuccinimide ester (Boc‐Gly‐NHS) (Chem Impex, cat. no. 3793)
  • Lyso‐ceramide trihexoside (Lyso‐Gb 3; Matreya, cat. no. 1520)
  • 99% trifluoroacetic acid (Sigma, cat. no. T62200)
  • Acetonitrile, LC‐MS‐grade (VWR, cat. no. CAAX0156‐1)
  • Isopropyl alcohol (EMD Millipore, cat. no. PX1834‐1)
  • 99% formic acid (Fisher, cat. no. AC27048‐0010)
  • Water, LC‐MS‐grade (EMD Millipore, cat. no. WX0001‐1)
  • Orbital shaker
  • Nitrogen evaporator
  • 2‐ml vials, screw tread, 12 × 32–mm, clear glass (Chromspec, cat. no. C779100WM)
  • Inserts, 250‐μl, 6 × 29–mm (Chromspec, cat. no. C221020M)
  • 9‐mm screw caps, preslit PTFE/silicone septum (Chromspec, cat. no. C779200XBBM)
  • SunFire C8, 3.5‐μm, 4.6 × 100–mm column (Waters, cat. no. 186002731)
  • Quattro Micro tandem quadrupole mass spectrometer equipped with an Alliance 2795 HPLC system with autosampler (Waters)
  • Rotary evaporator
  • Lyophilizer
  • Amber vials

Support Protocol 2: Preparation of Lyso‐Ceramide Trihexoside (lyso‐Gb3) Standard Solutions

  Materials
  • Lyso‐ceramide trihexoside (lyso‐Gb 3, Matreya, cat. no. 1520)
  • Acetonitrile (ACN) LC‐MS grade (VWR, cat. no. CAAX0156‐1)
  • Water, LC‐MS‐grade (EMD Millipore, cat. no. WX0001‐1)
  • 99% formic acid (Fisher, cat. no. AC27048‐0010)
  • Sonicator
  • 10‐ml volumetric flask
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Figures

Videos

Literature Cited

Literature Cited
  Abaoui, M., Boutin, M., Lavoie, P., and Auray‐Blais, C. 2016. Tandem mass spectrometry multiplex analysis of methylated and non‐methylated urinary Gb3 isoforms in Fabry disease patients. Clin. Chim. Acta. 452:191‐198. doi: 10.1016/j.cca.2015.11.018.
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  Boutin, M. and Auray‐Blais, C. 2015. Metabolomic discovery of novel urinary galabiosylceramide analogs as Fabry disease biomarkers. J. Am. Soc. Mass. Spectrom. 26:499‐510. doi: 10.1007/s13361‐014‐1060‐3.
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