μLC coupled to ICP–SFMS with post-column isotope dilution analysis of sulfur for absolute protein quantification

  • Nico Zinn
  • Ralf Krüger
  • Peter Leonhard
  • Jörg BettmerEmail author
Original Paper


Absolute protein quantification has become an important challenge in modern bioanalytical chemistry. Among several approaches based on mass spectrometric techniques, inductively coupled plasma (ICP) as ionisation source provides element-selective and sensitive detection of heteroatoms, and thus, a potentially emerging tool in protein analysis. In this work we applied coupling of capillary liquid chromatography (μLC) and inductively coupled plasma-sector field mass spectrometry (ICP–SFMS) to the separation and determination of standard proteins. For quantification purposes, post-column isotope dilution of sulfur was applied and optimised for this type of hyphenated technique. Provided that the protein sequence is known (number of sulfur-containing amino acids, i.e. cysteines and methionines) the protein amount can then be directly calculated from the determined sulfur content in a certain protein fraction. In order to prove the reliability of the presented method, two different certified reference materials were analysed: CRM 393 (human apolipoprotein A-I) and CRM 486 (α-fetoprotein). For CRM 393 excellent agreement (37.0 ± 1.4 μmol L−1) was obtained with the certificate (37.7 ± 1.8 μmol L−1). However, the recovery rate for α-fetoprotein in CRM 486 was found to be about 60% indicating incomplete elution of the protein during the chromatographic separation.


μLC ICP–MS Isotope dilution Protein quantification CRM 393 CRM 486 



The authors are grateful for support from Thermo Fisher Scientific, Bremen. Furthermore, financial support by the DFG within the graduate program GRK 826 “Analysis of element species: development of methods and applications” is gratefully acknowledged. J. Bettmer acknowledges the Spanish ministry for education and science (MEC) for the contract within the Ramón y Cajal program.


  1. 1.
    Ong SE, Mann M (2005) Nat Chem Biol 1:252–262CrossRefGoogle Scholar
  2. 2.
    Aebersold R, Mann M (2003) Nature 422:198–207CrossRefGoogle Scholar
  3. 3.
    Linscheid MW (2005) Anal Bioanal Chem 381:64–66CrossRefGoogle Scholar
  4. 4.
    Domon B, Aebersold R (2006) Science 312:212–217CrossRefGoogle Scholar
  5. 5.
    Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Nat Biotechnol 17(10):994–999CrossRefGoogle Scholar
  6. 6.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Mol Cell Proteomics 1:376–386CrossRefGoogle Scholar
  7. 7.
    Unwin RD, Pierce A, Watson RB, Sternberg DW, Whetton AD (2005) Mol Cell Proteomics 4:924–935CrossRefGoogle Scholar
  8. 8.
    Gerber SA, Rush J, Stemman O, Krschner MW, Gygi SP (2003) Proc Nat Acad Sci USA 100:6940–6945CrossRefGoogle Scholar
  9. 9.
    Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Mol Cell Proteomics 3:1154–1169CrossRefGoogle Scholar
  10. 10.
    Leitner A, Lindner W (2004) J Chromatogr B 813:1–26CrossRefGoogle Scholar
  11. 11.
    Leitner A, Lindner W (2006) Proteomics 6:5418–5434CrossRefGoogle Scholar
  12. 12.
    Unwin RD, Evans CA, Whetton AD (2006) Trend Biochem Sci 31:473–484CrossRefGoogle Scholar
  13. 13.
    Sanz-Medel A, Montes-Bayón M, Fernández Sánchez ML (2003) Anal Bioanal Chem 377:236–247CrossRefGoogle Scholar
  14. 14.
    Lobinski R, Schaumlöffel D, Szpunar J (2006) Mass Spectrom Rev 25:255–289CrossRefGoogle Scholar
  15. 15.
    Szpunar J (2005) Analyst 130:442–465CrossRefGoogle Scholar
  16. 16.
    Bettmer J, Jakubowski N, Prange A (2006) Anal Bioanal Chem 386:7–11CrossRefGoogle Scholar
  17. 17.
    Larsen EH, Stürup S (1994) J Anal At Spectrom 9:1099–1105CrossRefGoogle Scholar
  18. 18.
    Helfrich A, Bettmer J (2004) J Anal At Spectrom 19:1330–1334CrossRefGoogle Scholar
  19. 19.
    Hu Z, Hu S, Gao S, Liu Y, Lin S (2004) Spectrochim Acta B 59:1463–1470CrossRefGoogle Scholar
  20. 20.
    Kovačevič M, Goessler W, Mikac N, Veber M (2005) Anal Bioanal Chem 383:145–151CrossRefGoogle Scholar
  21. 21.
    Heumann KG (1992) Mass Spectrom Rev 11:41–67CrossRefGoogle Scholar
  22. 22.
    Heumann KG (2004) Anal Bioanal Chem 378:318–329CrossRefGoogle Scholar
  23. 23.
    Rodríguez-González, Marchante-Gayón JM, García Alonso JI, Sanz-Medel A (2005) Spectrochim Acta B 60:151–207CrossRefGoogle Scholar
  24. 24.
    Wind M, Wegener A, Eisenmenger A, Kellner R, Lehmann WD (2003) Angew Chem Int Ed 42:3425–3427CrossRefGoogle Scholar
  25. 25.
    Prange A, Schaumlöffel D, Brätter P, Richarz AN, Wolf C (2001) Fresenius J Anal Chem 371:764–774CrossRefGoogle Scholar
  26. 26.
    Schaumlöffel D, Prange A, Marx G, Heumann KG, Brätter P (2002) Anal Bioanal Chem 372:155–163CrossRefGoogle Scholar
  27. 27.
    Giusti P, Schaumlöffel D, Encinar JR, Szpunar J (2005) J Anal At Spectrom 20:1101–1107CrossRefGoogle Scholar
  28. 28.
    Schaumlöffel D, Giusti P, Preud’Homme H, Szpunar J, Lobinski R (2007) Anal Chem 79:2859–2868CrossRefGoogle Scholar
  29. 29.
    Del Castillo Busto ME, Montes-Bayón M, Sanz-Medel A (2006) Anal Chem 78:8218–8226CrossRefGoogle Scholar
  30. 30.
    Heilmann J, Boulyga SF, Heumann KG (2004) Anal Bioanal Chem 380:190–197CrossRefGoogle Scholar
  31. 31.
    Prange A, Schaumlöffel D (1999) J Anal At Spectrom 14:1329–1332CrossRefGoogle Scholar
  32. 32.
    Schaumlöffel D, Encinar JR, Lobinski R (2003) Anal Chem 75:6837–6842CrossRefGoogle Scholar
  33. 33.
    Pröfrock D, Leonhard P, Ruck W, Prange A (2005) Anal Bioanal Chem 381:194–204CrossRefGoogle Scholar
  34. 34.
    Rottmann L, Heumann KG (1994) Fresenius J Anal Chem 350:221–227CrossRefGoogle Scholar
  35. 35.
    Profilis C, Colinet E, Parfait RG, Terrana B. (1996) European Commission Report EUR 16862Google Scholar
  36. 36.
    Anderson NL, Anderson NG (2002) Mol Cell Proteomics 1:845–867CrossRefGoogle Scholar
  37. 37.
    Meija J, Mester Z (2008) Anal Chim Acta 607:115–125CrossRefGoogle Scholar
  38. 38.
    Sanz-Medel A, Montes-Bayón M, de la Campa MRF, Encinar JR, Bettmer J (2008) Anal Bioanal Chem 390:3–16CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Nico Zinn
    • 1
    • 3
  • Ralf Krüger
    • 2
    • 3
  • Peter Leonhard
    • 4
  • Jörg Bettmer
    • 5
    Email author
  1. 1.Molecular Structure AnalysisGerman Cancer Research CenterHeidelbergGermany
  2. 2.University Hospital MainzInstitute of Clinical Chemistry and Laboratory MedicineMainzGermany
  3. 3.Johannes Gutenberg-University MainzInstitute of Inorganic Chemistry and Analytical ChemistryMainzGermany
  4. 4.Merck KGaACentral Analytical Research, ZD-A/ZFA 4.1DarmstadtGermany
  5. 5.Department of Physical and Analytical ChemistryFaculty of Chemistry, University of OviedoOviedoSpain

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