Skip to main content
SpringerLink
Log in

Online Capillary IsoElectric Focusing-ElectroSpray Ionization Mass Spectrometry (CIEF-ESI MS) in Glycerol–Water Media for the Separation and Characterization of Hydrophilic and Hydrophobic Proteins

  • Protocol
  • First Online:
Capillary Electrophoresis of Proteins and Peptides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1466))

Abstract

Capillary isoelectric focusing (CIEF) is a high-resolution technique for the separation of ampholytes, such as proteins, according to their isoelectric point. CIEF coupled online with MS is regarded as a promising alternative to 2-D PAGE for fast proteome analysis with high-resolving capabilities and enhanced structural information without the drawbacks of conventional slab-gel electrophoresis. However, online coupling has been rarely described, as it presents some difficulties. A new methodology for the online coupling of CIEF with electrospray ionization mass spectrometry (ESI-MS) has been developed in glycerol–water media. This new integrated methodology provides a mean for the characterization of a large number of hydrophilic and hydrophobic proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Yang L, Lee CS, Hofstadler SA et al (1998) Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization. Anal Chem 70:3235–3241

    Article  CAS  PubMed  Google Scholar 

  2. Jensen PK, Pasa-Tolic L, Peden KK et al (2000) Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures. Electrophoresis 21: 1372–1380

    Article  CAS  PubMed  Google Scholar 

  3. Minarik M, Foret F, Karger BL (2000) Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing. Electrophoresis 21:247–254

    Article  CAS  PubMed  Google Scholar 

  4. Foret F, Muller O, Thoren J et al (1996) Analysis of protein fractions by micropreparative capillary isoelectric focusing and matrix-assisted laser desorption time-of-flight mass spectrometry. J Chromatogr A 716:157–166

    Article  Google Scholar 

  5. Chartogne A, Gaspari M, Jespersen S et al (2002) On-target fraction collection for the off-line coupling of capillary isoelectric focusing with matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 16:201–207

    Article  CAS  PubMed  Google Scholar 

  6. Yu W, Yan L, Deng C et al (2006) Comprehensive two-dimensional separation in coupling of reversed-phase chromatography with capillary isoelectric focusing followed by MALDI-MS identification using on-target digestion for intact protein analysis. Electrophoresis 27:2100–2110

    Article  CAS  PubMed  Google Scholar 

  7. Mokadddem M, Gareil P, Varenne A (2009) Online CIEF-ESI-MS in glycerol-water media with a view to hydrophobic protein applications. Electrophoresis 30:4040–4048

    Article  Google Scholar 

  8. Przybylski C, Mokaddem M, Prull-Janssen M et al (2015) On-line capillary isoelectric focusing hyphenated to native electrospray ionization mass spectrometry for the characterization of interferon-γ and variants. Analyst 140:543–550

    Article  CAS  PubMed  Google Scholar 

  9. Righetti PG, Simo C, Sebastiano R et al (2007) Carrier ampholytes for IEF, on their fortieth anniversary (1967–2007), brought to trial in court: the verdict. Electrophoresis 28:3799

    Article  CAS  PubMed  Google Scholar 

  10. Tang Q, Harrata AK, Lee CS (1995) Capillary isoelectric focusing-electrospray mass spectrometry for protein analysis. Anal Chem 67:3515–3519

    Article  CAS  Google Scholar 

  11. Zhou F, Johnston MV (2004) Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry. Anal Chem 76:2734–2740

    Article  CAS  PubMed  Google Scholar 

  12. Storms HF, Van der Heijden R, Tjaden UR et al (2004) Capillary isoelectric focusing-mass spectrometry for shotgun approach in proteomics. Electrophoresis 25:3461–3467

    Article  CAS  PubMed  Google Scholar 

  13. Storms HF, Van der Heijden R, Tjaden UR et al (2005) Optimization of protein identification from digests as analyzed by capillary isoelectric focusing-mass spectrometry. J Chromatogr B 824:189–200

    Article  CAS  Google Scholar 

  14. Clarke NJ, Naylor S (2002) Capillary isoelectric focusing-mass spectrometry: analysis of proteins mixtures human body fluids. Biomed Chromatogr 16:287–297

    Article  CAS  PubMed  Google Scholar 

  15. Lamoree MH, Tjaden UR, Van der Greef J (1997) Use of microdialysis for the on-line coupling of capillary isoelectric focusing with electrospray mass spectrometry. J Chromatogr A 777:31–39

    Article  CAS  Google Scholar 

  16. Yang L, Lee CS, Hofstadler SA et al (1998) Characterization of microdialysis acidification for capillary isoelectric focusing-microelectrospray ionization mass spectrometry. Anal Chem 70:4945–4950

    Article  CAS  PubMed  Google Scholar 

  17. Ullsten S, Zuberovic A, Wetterhall M et al (2004) A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides. Electrophoresis 25:2090–2099

    Article  CAS  PubMed  Google Scholar 

  18. Wei J, Yang L, Harrata AK, Lee CS (1998) High resolution analysis of protein phosphorylation using capillary isoelectric focusing-electrospray ionization-mass spectrometry. Electrophoresis 19:2356–2360

    Article  CAS  PubMed  Google Scholar 

  19. Martinovic S, Pasa-Tolic KL, Masselon C et al (2000) Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry. Electrophoresis 21:2368–2375

    Article  CAS  PubMed  Google Scholar 

  20. Jensen PK, Harrata AK, Lee CS (1998) Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry. Anal Chem 70:2044–2049

    Article  CAS  PubMed  Google Scholar 

  21. Li M, Liu L, Wu JT et al (1997) Use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on-line detection via an ion trap storage/reflectron time-of-flight mass spectrometer. Anal Chem 69:2451–2456

    Article  CAS  PubMed  Google Scholar 

  22. Kelly JF, Ramaley L, Thibault P (1997) Use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on-line detection via an ion trap storage/reflectron time-of-flight mass spectrometer. Anal Chem 69:51–60

    Article  CAS  Google Scholar 

  23. Simo C, Elvira C, Gonzalez N et al (2004) Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis. Electrophoresis 25:2056–2064

    Article  CAS  PubMed  Google Scholar 

  24. Soga T, Ueno Y, Naraoka H et al (2002) Simultaneous determination of anionic intermediates for bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry. Anal Chem 74:2233–2239

    Article  CAS  PubMed  Google Scholar 

  25. Zhang C-X, Xiang F, Pasa-Tolic L et al (2000) Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry. Anal Chem 72:1462–1468

    Article  CAS  PubMed  Google Scholar 

  26. Ohnesorge J, Sänger-van de Griend C, Wätzig H (2005) Quantification in capillary electrophoresis-mass spectrometry: long- and short-term variance components and their compensation using internal standards. Electrophoresis 26:2360–2375

    Article  CAS  PubMed  Google Scholar 

  27. Geiser L, Rudaz S, Veuthey JL (2003) Validation of capillary electrophoresis-mass spectrometry methods for the analysis of a pharmaceutical formulation. Electrophoresis 24:3049–3056

    Article  CAS  PubMed  Google Scholar 

  28. Nunez O, Moyano E, Galceran MT (2002) Capillary electrophoresis-mass spectrometry for the analysis of quaternary ammonium herbicides. J Chromatogr A 974:243–255

    Article  CAS  PubMed  Google Scholar 

  29. Sanz-Nebot V, Benavente F, Balaguer E et al (2003) Capillary electrophoresis coupled to time of flight-mass spectrometry of therapeutic peptide hormones. Electrophoresis 24:883–891

    Article  CAS  PubMed  Google Scholar 

  30. Nilsson SL, Bylund D, Jörnten-Karlsson M et al (2004) A chemometric study of active parameters and their interaction effects in a nebulized sheath-liquid electrospray interface for capillary electrophoresis-mass spectrometry. Electrophoresis 25:2100–2107

    Article  CAS  PubMed  Google Scholar 

  31. Mokaddem M, Gareil P, Belgaied JE et al (2008) A new insight into suction and dilution effects in capillary electrophoresis coupled to mass spectrometry via an electrospray ionization interface. Part I-suction effect. Electrophoresis 29:1957–1964

    Article  CAS  PubMed  Google Scholar 

  32. Iavarone AT, Williams ER (2002) Supercharging in electrospray ionization: effects on signal and charge. Int J Mass Spectrom 219:63–72

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Chimie ParisTech, PSL Research University Unité de Technologies Chimiques et Biologiques pour la Santé, Paris, 75005, France

    Meriem Mokaddem, Fanny d’Orlyé & Anne Varenne

  2. INSERM, Unité de Technologies Chimiques et Biologiques pour la Santé (n° 1022), Paris, 75005, France

    Meriem Mokaddem, Fanny d’Orlyé & Anne Varenne

  3. CNRS, Unité de Technologies Chimiques et Biologiques pour la santé UMR 8258, Paris, 75005, France

    Meriem Mokaddem, Fanny d’Orlyé & Anne Varenne

  4. Université Paris Descartes, Sorbonne Paris Cité, Unité de Technologies Chimiques et Biologiques pour la Santé, Paris, 75005, France

    Meriem Mokaddem, Fanny d’Orlyé & Anne Varenne

Authors
  1. Meriem Mokaddem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fanny d’Orlyé
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne Varenne
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anne Varenne .

Editor information

Editors and Affiliations

  1. Institut Galien Paris-Sud, UMR 8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France

    Nguyet Thuy Tran

  2. Institut Galien Paris-Sud, UMR 8612, Protein and Nanotechnology in Analytical Science (PNAS), CNRS, Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France

    Myriam Taverna

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Mokaddem, M., d’Orlyé, F., Varenne, A. (2016). Online Capillary IsoElectric Focusing-ElectroSpray Ionization Mass Spectrometry (CIEF-ESI MS) in Glycerol–Water Media for the Separation and Characterization of Hydrophilic and Hydrophobic Proteins. In: Tran, N., Taverna, M. (eds) Capillary Electrophoresis of Proteins and Peptides. Methods in Molecular Biology, vol 1466. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-4014-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-4014-1_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-4012-7

  • Online ISBN: 978-1-4939-4014-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation