, Volume 66, Issue 9–10, pp 709–715 | Cite as

The Behaviour of Reduced, Alkylated and Native Proteins in a pH-Gradient LC System

  • Bao Q. TranEmail author
  • Miliam Pepaj
  • Elsa Lundanes
  • Tyge Greibrokk


Two dimensional (2D) liquid chromatography (LC) separations of proteins can be obtained faster and more automated than traditional 2D gel electrophoresis. Previously we have described a 2D LC method for separation of native proteins with separation according to pI by pH-gradient strong anion exchange (SAX) chromatography in the first dimension, and according to hydrophobicity by reversed phase chromatography in the second dimension. Since there are few literature reports on the combination of reduced/alkylated proteins and modern LC, a basic study of the chromatographic properties of a few reduced /alkylated proteins was undertaken with a pH-gradient SAX chromatographic system. Proteins where the disulfide groups were reduced, but not alkylated, were also included. The conditions that separated native proteins according to pI could not be used for neither reduced nor reduced/alkylated proteins. High concentrations of urea (4–8 M) were needed in the mobile phase in order to obtain good peak shapes. Addition of urea had an undesired impact on both the retention of the proteins and the pH gradient profile, with the effect that little correlation between reported pI values and elution pH was found. The conclusion was that proteins should be separated in the native state if good pI–pH correlations are important, and in the alkylated state with urea if other considerations are more important.


Ion-exchange chromatography pH-Gradient Urea Reduction and alkylation Plasma 



BQT is supported by the Government of Vietnam and the research is partly supported by the EU research training network HPRN-CT-2001-00180 (COM-CHROM). The Q-TOF2 technical support by John Ø Vedde is greatly appreciated.


  1. 1.
    Bunai K, Yamane K (2005) J Chromatogr B 815:227–236CrossRefGoogle Scholar
  2. 2.
    Cooper JW, Wang Y, Lee CS (2004) Electrophoresis 25:3913–3926CrossRefGoogle Scholar
  3. 3.
    Righetti PG (2006) J Chromatogr B 841:14–22CrossRefGoogle Scholar
  4. 4.
    Herbert B, Galvani M, Hamdan M, Olivieri E, MacCarthy J, Perdersen S, Righetti PG (2001) Electrophoresis 22:2046–2057CrossRefGoogle Scholar
  5. 5.
    Magnus EM, Khan K (1992) Cereal Chem 69(6):607–612Google Scholar
  6. 6.
    Hearn MTW, Nguyen T, Fridman M (1988) J Chromatogr 435(2):271–284CrossRefGoogle Scholar
  7. 7.
    Burnouf T, Bietz JA (1984) J Chromatogr 299(1):185–199CrossRefGoogle Scholar
  8. 8.
    Hedström M, Andersson M, Galaev IY, Mattiasson B (2005) J Chromatogr A 1080:117–123CrossRefGoogle Scholar
  9. 9.
    Duan J, Sun L, Liang Z, Zhang J, Wang H, Zhang L, Zhang W, Zang Y (2006) J Chromatogr A 1106:165–174CrossRefGoogle Scholar
  10. 10.
    Andersen T, Pepaj M, Trones R, Lundanes E, Greibrokk T (2004) J Chromatogr A 1025:217–226CrossRefGoogle Scholar
  11. 11.
    Yan JX, Kett WC, Herbert BR, Gooley AA, Packer NH, Williams KL (1998) J Chromatogr A 813:187–200CrossRefGoogle Scholar
  12. 12.
    Nadler T, Blackburn C, Mark J, Gordon N, Regnier FE, Vella G (1996) J Chromatogr A 743:91–98CrossRefGoogle Scholar
  13. 13.
    Romijn EP, Krijgsveld J, Heck AJR (2001) Anal Chem 73:3576–3582CrossRefGoogle Scholar
  14. 14.
    Bosma JC, Wesselingh JA (1998) AIChE J 44:2399–2409CrossRefGoogle Scholar
  15. 15.
    Wait R, Gegum S, Brambilla D, Carabelli AM, Conserva F, Guerini AR, Eberini I, Ballerio R, Gemeiner M, Miller I, Gianazza E (2005) Amino Acids 28:239–272CrossRefGoogle Scholar
  16. 16.
    Chaudhuri AR, Khan IA, Ludueña RF (2001) Biochememistry 40:8834–8841CrossRefGoogle Scholar
  17. 17.
    Eyles SJ, Radford SE, Robinson CV, Dobson CM (1994) Biochemistry 33:13038–13048CrossRefGoogle Scholar
  18. 18.
    Nandel FS, Verma R, Singh B, Jain DVS (1998) Pure Appl Chem 70:659–664CrossRefGoogle Scholar
  19. 19.
    Kadoya T, Okuyama T (1983) Bunseki Kagaku 32:664–669Google Scholar

Copyright information

© Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH 2007

Authors and Affiliations

  • Bao Q. Tran
    • 1
    Email author
  • Miliam Pepaj
    • 1
  • Elsa Lundanes
    • 1
  • Tyge Greibrokk
    • 1
  1. 1.Department of ChemistryUniversity of OsloOsloNorway

Personalised recommendations