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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Abstract

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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Materials

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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
  Auray‐Blais, C. and Boutin, M. 2012. Novel Gb3 isoforms detected in urine of Fabry disease patients: A metabolomic study. Curr. Med. Chem. 19:3241‐3252. doi: 10.2174/092986712800784739.
  Auray‐Blais, C., Boutin, M., Gagnon, R., Dupont, F.O., Lavoie, P., and Clarke, J.T.R. 2012. Urinary globotriaosylsphingosine‐related biomarkers for Fabry disease targeted by metabolomics. Anal. Chem. 84:2745‐2753. doi: 10.1021/ac203433e.
  Auray‐Blais, C., Blais, C.‐M., Ramaswami, U., Boutin, M., Germain, D.P., Dyack, S., Bodamer, O., Pintos‐Morell, G., Clarke, J.T., Bichet, D.G., Warnock, D.G., Echevarria, L., West, M.L., and Lavoie, P. 2015. Urinary biomarker investigation in children with Fabry disease using tandem mass spectrometry. Clin. Chim. Acta 438:195‐204. doi: 10.1016/j.cca.2014.08.002.
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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.
  Boutin, M., Gagnon, R., Lavoie, P., and Auray‐Blais, C. 2012. LC‐MS/MS analysis of plasma lyso‐Gb3 in Fabry disease. Clin. Chim. Acta 414:273‐280. doi: 10.1016/j.cca.2012.09.026.
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  Dupont, F.O., Gagnon, R., Lavoie, P., and Auray‐Blais, C. 2012. A metabolomic study reveals novel plasma lyso‐Gb3 analogs as Fabry disease biomarkers. Curr. Med. Chem. 20:280‐288. doi: 10.2174/0929867311320020008.
  Ferreira, S., Auray‐Blais, C., Boutin, M., Lavoie, P., Nunes, J.P., Martins, E., Garman, S., and Oliveira, J.P. 2015. Variations in the GLA gene correlate with globotriaosylceramide and globotriaosylsphingosine analog levels in urine and plasma. Clin. Chim. Acta. 447:96‐104. doi: 10.1016/j.cca.2015.06.003.
  Lavoie, P., Boutin, M., and Auray‐Blais, C. 2013. Multiplex analysis of novel urinary Lyso‐Gb3‐related biomarkers for fabry disease by tandem mass spectrometry. Anal. Chem. 85:1743‐1752. doi: 10.1021/ac303033v.
  Manwaring, V., Boutin, M., and Auray‐Blais, C. 2013. A metabolomic study to identify new globotriaosylceramide‐related biomarkers in the plasma of Fabry disease patients. Anal. Chem. 85:9039‐9048. doi: 10.1021/ac401542k.
  Meikle, P.J., Hopwood, J.J., Clague, A.E., and Carey, W.F. 1999. Prevalence of lysosomal storage disorders. JAMA 281:249‐254. doi: 10.1001/jama.281.3.249.
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  Spada, M., Pagliardini, S., Yasuda, M., Tukel, T., Thiagarajan, G., Sakuraba, H., Ponzone, A., and Desnick, R.J. 2006. High incidence of later‐onset Fabry disease revealed by newborn screening. Am. J. Hum. Genet. 79:31‐40. doi: 10.1086/504601.
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