Analytical and Bioanalytical Chemistry

, Volume 394, Issue 3, pp 811–820 | Cite as

HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum

  • Luciano Callipo
  • Patrizia Foglia
  • Riccardo Gubbiotti
  • Roberto Samperi
  • Aldo LaganàEmail author
Original Paper


A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 ± 12%, while analytical recovery was 81 ± 10%. For the eight samples analyzed, the matrix effect was found to be only −14 ± 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%).


Liquid chromatography-tandem mass spectrometry Chip Triple quadrupole Peptides Protein absolute quantification 



The authors are grateful to Dr. Fiorella Guadagni (Scientific Institute for Research, Hospitalization and Health Care (SIRHHC or IRCCS in the Italian acronym) San Raffaele-Roma) for supplying the serum samples.

A special acknowledgment is given to Agilent Technologies and Agilent staff members, for their invaluable help and technical assistance. In particular, the authors wish to thank Alberto Stocco (Agilent Technologies) for his technical assistance and his infinite helpfulness.


  1. 1.
    Hardouin J, Joubert-Caron R, Caron M (2007) J Sep Sci 30:1482CrossRefGoogle Scholar
  2. 2.
    Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Nat Biotechnol 17:994CrossRefGoogle Scholar
  3. 3.
    Ross RL, 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 3:1154CrossRefGoogle Scholar
  4. 4.
    Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Mol Cell Proteomics 1:376CrossRefGoogle Scholar
  5. 5.
    Shevchenko A, Chernushevich I, Ens W, Standing KG, Thomas B, Wilm M, Mann M (1997) Rapid Commun Mass Spectrom 11:1015CrossRefGoogle Scholar
  6. 6.
    Yao X, Freas A, Ramirez J, Demirev PA, Fensalau C (2001) Anal Chem 73:2836CrossRefGoogle Scholar
  7. 7.
    Anderson NL, Anderson NG, Haines LR, Hardie DB, Olafson RW, Pearson TW (2004) J Proteome Res 3:235CrossRefGoogle Scholar
  8. 8.
    Whiteaker JR, Zhao L, Zhang HY, Feng L, Piening BD, Anderson L, Paulovich AG (2007) Anal Biochem 362:44CrossRefGoogle Scholar
  9. 9.
    Silva JC, Gorenstein MV, Li G, Vissers JPC, Geromanos SJ (2006) Mol Cell Proteomics 5:144CrossRefGoogle Scholar
  10. 10.
    Yin H, Killeen K, Brennen R, Sobek D, Werlich M, van de Goor T (2005) Anal Chem 77:527CrossRefGoogle Scholar
  11. 11.
    Hardouin J, Duchateau M, Joubert-Caron R, Caron M (2006) Rapid Commun Mass Spectrom 20:3236CrossRefGoogle Scholar
  12. 12.
    Palminiello S, Kida E, Kaur K, Walus M, Wisniewski KE, Wierzba-Bobrowicz T, Rabe A, Albertini G, Golabek AA (2008) Brain Res 1190:193CrossRefGoogle Scholar
  13. 13.
    Wu-Hsien K, Shun-Fa Y, Yih-Shou H, Chiou-Sheng T, Wen-Li H, Shu-Chen C (2005) Clinica Chimica Acta 351:79CrossRefGoogle Scholar
  14. 14.
    Nishita T, Takahasi M, Kasuya T, Matsui K, Ichihara N, Murakami M, Asari M (2005) J Vet Med Sci 67(1):63CrossRefGoogle Scholar
  15. 15.
    Demür N, Demür Y, Coþkun F (2001) Turk J Med Sci 31:477Google Scholar
  16. 16.
    Lund SG, Dyment P, Gervais MR, Moyes CD, Tufts BL (2002) J Comp Physiol B 172:467CrossRefGoogle Scholar
  17. 17.
    Li X, Alvarez B, Casey JR, Reithmeier RA, Fliegel L (2002) J Biol Chem 277:36085CrossRefGoogle Scholar
  18. 18.
    Alver A, Mentese A, Karahan SC, Erem C, Keha EE, Arikan MK, Eminagaoglu MS, Deger O (2007) Exp Clin Endocrinol Diabetes 115:287CrossRefGoogle Scholar
  19. 19.
    Sly WS, Hu PY (1995) Annu Rev Biochem 64:375CrossRefGoogle Scholar
  20. 20.
    Supirpan CT, Briganti F, Tilli S, Chegwidden WR, Scozzafava A (2001) Bioorgan Med Chem 9:703CrossRefGoogle Scholar
  21. 21.
    Vlkolinský R, Cairns N, Fountoulakis M, Lubec G (2001) Neurobiol Aging 22:547CrossRefGoogle Scholar
  22. 22.
    Sly WS, Hewett-Emmett D, Whyte MP, Yu YL, Tashian RE (1983) Proc Natl Acad Sci USA 80:2752CrossRefGoogle Scholar
  23. 23.
    Tashian RE, Hewett-Emmett D, Dodgson SJ, Forster RE, Sly WS (1984) Ann N Y Acad Sci 429:263Google Scholar
  24. 24.
    Morris CS, Esiri MM, Sprinkle TJ, Gregson N (1994) Neuropathol Appl Neurobiol 20:272CrossRefGoogle Scholar
  25. 25.
    DeLuca GC, Nagy Z, Esiri MM, Davey P (2002) Acta Neuropathol 103:590CrossRefGoogle Scholar
  26. 26.
    Ozensoy O, Arslan O, Oznur Sinan S (2004) Biochemistry 69(2):216PubMedGoogle Scholar
  27. 27.
    Yoshiura K, Nakaoka T, Nishishita T, Sato K, Yamamoto A, Shimada S, Saida T, Kawakami Y, Takahashi TA, Fukuda H, Imajoh-Ohmi S, Oyaizu N, Yamashita N (2005) Clin Cancer Res 11(22):8201CrossRefGoogle Scholar
  28. 28.
    Cavaliere C, Cucci F, Guarino C, Gubbiotti R, Samperi, Laganà A (2008) Rapid Commun Mass Spectrom 22:1159CrossRefGoogle Scholar
  29. 29.
    Barnidge DR, Dratz EA, Martin T, Bonilla LE, Moran LB, Lindall A (2003) Anal Chem 75:445CrossRefGoogle Scholar
  30. 30.
    Barnidge DR, Goodmanson MK, Klee GC, Muddiman DC (2004) J Proteome Res 3:644CrossRefGoogle Scholar
  31. 31.
    Kuhn E, Wu J, Karl J, Liao H, Werner Z, Guild B (2004) Proteomics 4:1175CrossRefGoogle Scholar
  32. 32.
    Bizzarri M, Cavaliere C, Foglia P, Guarino C, Samperi R, Laganà A (2008) Anal Bioanal Chem 391:1969CrossRefGoogle Scholar
  33. 33.
    Yin H, Killeen K (2007) J Sep Sci 30:1427CrossRefGoogle Scholar
  34. 34.
    Vollmer M, Miller C, Gauthier GL (2005) Agilent Technologies Application Note 5989-3538ENGoogle Scholar
  35. 35.
    Tamvakopoulos C (2007) Mass Spectrom Rev 26:389CrossRefGoogle Scholar
  36. 36.
    Bacaloni A, Cavaliere C, Faberi A, Pastorini E, Samperi R, Laganà A (2005) J Agric Food Chem 53:5518CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Luciano Callipo
    • 1
  • Patrizia Foglia
    • 1
  • Riccardo Gubbiotti
    • 1
  • Roberto Samperi
    • 1
  • Aldo Laganà
    • 1
    Email author
  1. 1.Department of ChemistrySapienza University of RomeRomeItaly

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