Advertisement

Analytical and Bioanalytical Chemistry

, Volume 398, Issue 3, pp 1305–1312 | Cite as

Mass spectrometric detection of siRNA in plasma samples for doping control purposes

  • Maxie Kohler
  • Andreas Thomas
  • Katja Walpurgis
  • Wilhelm Schänzer
  • Mario ThevisEmail author
Original Paper

Abstract

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25–1 nmol mL−1) and performance (precision 11–21%, recovery 23–67%) for typical antisense oligonucleotides currently used in clinical studies.

Online abstract figure

A method for the mass spectrometric detection of siRNA molecules in doping control is described. siRNA, which blocks the translation of genes, could be used by athletes for illicit performance enhancement by e.g. down-regulating the myostatin gene for enhanced muscle growth.

Keywords

Doping Sport Exactive mass spectrometry Oligonucleotide Duplex 

Notes

Acknowledgements

The study was carried out with the support of Antidoping Switzerland (Berne, Switzerland), the Manfred Donike Institute of Doping Analysis, and the Federal Ministry of the Interior of the Federal Republic of Germany.

References

  1. 1.
    Whitehead KA, Langer R, Anderson DG (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov 8:129–138CrossRefGoogle Scholar
  2. 2.
    Kurreck J (2009) RNA interference: from basic research to therapeutic applications. Angew Chem Int Ed Engl 48:1378–1398CrossRefGoogle Scholar
  3. 3.
    Rana TM (2007) Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev Mol Cell Biol 8:23–36CrossRefGoogle Scholar
  4. 4.
    Corey DR (2007) Chemical modification: the key to clinical application of RNA interference? J Clin Invest 117:3615–3622CrossRefGoogle Scholar
  5. 5.
    Kurreck J (2003) Antisense technologies. Improvement through novel chemical modifications. Eur J Biochem 270:1628–1644CrossRefGoogle Scholar
  6. 6.
    Juliano R, Bauman J, Kang H, Ming X (2009) Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm 6:686–695CrossRefGoogle Scholar
  7. 7.
    Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C, Langkjaer N, Babu BR, Hojland T, Abramov M, Van Aerschot A et al (2009) A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res 37:2867–2881CrossRefGoogle Scholar
  8. 8.
    Gao S, Dagnaes-Hansen F, Nielsen EJ, Wengel J, Besenbacher F, Howard KA, Kjems J (2009) The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice. Mol Ther 17:1225–1233CrossRefGoogle Scholar
  9. 9.
    Cho IS, Kim J, Lim do H, Ahn HC, Kim H, Lee KB, Lee YS (2008) Improved serum stability and biophysical properties of siRNAs following chemical modifications. Biotechnol Lett 30:1901–1908CrossRefGoogle Scholar
  10. 10.
    Behlke MA (2008) Chemical modification of siRNAs for in vivo use. Oligonucleotides 18:305–319CrossRefGoogle Scholar
  11. 11.
    Bumcrot D, Manoharan M, Koteliansky V, Sah DW (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol 2:711–719CrossRefGoogle Scholar
  12. 12.
    Haussecker D (2008) The business of RNAi therapeutics. Hum Gene Ther 19:451–462CrossRefGoogle Scholar
  13. 13.
    Schuelke M, Wagner KR, Stolz LE, Hubner C, Riebel T, Komen W, Braun T, Tobin JF, Lee SJ (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350:2682–2688CrossRefGoogle Scholar
  14. 14.
    Liu CM, Yang Z, Liu CW, Wang R, Tien P, Dale R, Sun LQ (2008) Myostatin antisense RNA-mediated muscle growth in normal and cancer cachexia mice. Gene Ther 15:155–160CrossRefGoogle Scholar
  15. 15.
    Acosta J, Carpio Y, Borroto I, Gonzalez O, Estrada MP (2005) Myostatin gene silenced by RNAi show a zebrafish giant phenotype. J Biotechnol 119:324–331CrossRefGoogle Scholar
  16. 16.
    Tsuchida K (2008) Targeting myostatin for therapies against muscle-wasting disorders. Curr Opin Drug Discov Dev 11:487–494Google Scholar
  17. 17.
    Huang TY, Liu J, Liang X, Hodges BD, McLuckey SA (2008) Collision-induced dissociation of intact duplex and single-stranded siRNA anions. Anal Chem 80:8501–8508CrossRefGoogle Scholar
  18. 18.
    Bahr U, Aygun H, Karas M (2008) Detection and relative quantification of siRNA double strands by MALDI mass spectrometry. Anal Chem 80:6280–6285CrossRefGoogle Scholar
  19. 19.
    Huber CG, Buchmeiser MR (1998) On-line cation exchange for suppression of adduct formation in negative-ion electrospray mass spectrometry of nucleic acids. Anal Chem 70:5288–5295CrossRefGoogle Scholar
  20. 20.
    Zou Y, Tiller P, Chen IW, Beverly M, Hochman J (2008) Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry. Rapid Commun Mass Spectrom 22:1871–1881CrossRefGoogle Scholar
  21. 21.
    Zhang G, Lin J, Srinivasan K, Kavetskaia O, Duncan JN (2007) Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography–tandem mass spectrometry. Anal Chem 79:3416–3424CrossRefGoogle Scholar
  22. 22.
    McCarthy SM, Gilar M, Gebler J (2009) Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA. Anal Biochem 390:181–188CrossRefGoogle Scholar
  23. 23.
    Thevis M, Schänzer W (2007) Current role of LC–MS(/MS) in doping control. Anal Bioanal Chem 388:1351–1358CrossRefGoogle Scholar
  24. 24.
    Thevis M, Schänzer W (2007) Mass spectrometry in sports drug testing: Structure characterization and analytical assays. Mass Spectrom Rev 26:79–107CrossRefGoogle Scholar
  25. 25.
    Greig M, Griffey RH (1995) Utility of organic bases for improved electrospray mass spectrometry of oligonucleotides. Rapid Commun Mass Spectrom 9:97–102CrossRefGoogle Scholar
  26. 26.
    Andreasen D, Fog JU, Biggs W, Salomon J, Dahslveen IK, Baker A, Mouritzen P (2010) Improved microRNA quantification in total RNA from clinical samples. Methods 50:S6–S9CrossRefGoogle Scholar
  27. 27.
    Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z, Hood LE, Galas DJ (2009) Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci USA 106:4402–4407CrossRefGoogle Scholar
  28. 28.
    Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105:10513–10518CrossRefGoogle Scholar
  29. 29.
    Mandel J (1964) The statistical analyses of experimental data. John Wiley & Sons, New YorkGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Maxie Kohler
    • 1
  • Andreas Thomas
    • 1
  • Katja Walpurgis
    • 1
  • Wilhelm Schänzer
    • 1
  • Mario Thevis
    • 1
    Email author
  1. 1.Institute of Biochemistry/Center for Preventive Doping ResearchGerman Sport University CologneCologneGermany

Personalised recommendations