Analytical and Bioanalytical Chemistry

, Volume 392, Issue 6, pp 1209–1214 | Cite as

Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent

Original Paper
First Online:
Received:
Revised:
Accepted:

Abstract

Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin, and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides from their unmodified counterparts, even at the preparative scale required for synthetic peptides.

Keywords

Electrospray ionization (ESI) Glycation Mass spectrometry (MS) Matrix-assisted laser desorption/ionization (MALDI) Solid-phase peptide synthesis (SPPS) 
This is a preview of subscription content, log in to check access.

References

  1. 1.
    Mann M, Jensenn ON (2003) Nat Biotechnol 21:255–261CrossRefGoogle Scholar
  2. 2.
    Traverso N, Menini S, Cottalasso D, Odetti P, Marinari UM, Pronzato MA (1997) Biochim Biophys Acta 1336:409–418Google Scholar
  3. 3.
    Stadtman ER, Levine RL (2003) Amino Acids 25:207–218CrossRefGoogle Scholar
  4. 4.
    Wright HT (1991) Protein Eng 4:283–294CrossRefGoogle Scholar
  5. 5.
    Portero-Otin M, Bellmunt MJ, Requena JR, Pamplona R (2003) Biochem Soc Trans 31:1403–1405Google Scholar
  6. 6.
    Lapolla A, Fedele D, Aronica R, Garbeglio M, D’Alpaos M, Plebani M, Seraglia R, Traldi P (1997) Rapid Commun Mass Spectrom 11:613–617CrossRefGoogle Scholar
  7. 7.
    Ulrich P, Cerami A (2001) Recent Prog Horm Res 56:1–21CrossRefGoogle Scholar
  8. 8.
    Stultz CM, Edelman ER (2003) Biophys J 85:2198–2204CrossRefGoogle Scholar
  9. 9.
    Schalkwijk CG, Ligtvoet N, Twaalfhoven H, Jager A, Blaauwgeers HG, Schlingemann RO, Tarnow L, Parving HH, Stehouwer CDA, van Hinsberg VW (1999) Diabetes 48:2446–2453CrossRefGoogle Scholar
  10. 10.
    Baynes JW, Thorpe SR (1999) Diabetes 48:1–9CrossRefGoogle Scholar
  11. 11.
    Ledesma MD, Bonay P, Avila J (1995) J Neurochem 65:1658–1664CrossRefGoogle Scholar
  12. 12.
    Ge SJ, Lee TC (1996) J Agric Food Chem 44:1053–1057CrossRefGoogle Scholar
  13. 13.
    Reutter M, Eichner K (1989) Z Lebensm Unters Forsch 188:28–35CrossRefGoogle Scholar
  14. 14.
    Lapolla A, Fedele D, Reitano R, Arico NC, Seraglia R, Traldi P, Marotta E, Tonani R (2004) J Am Soc Mass Spectrom 15:496–509CrossRefGoogle Scholar
  15. 15.
    Frolov A, Hoffmann P, Hoffmann R (2006) J Mass Spec 41:1459–1469CrossRefGoogle Scholar
  16. 16.
    Zhang Q, Tang N, Brock JWC, Mottaz HM, Ames JM, Baynes JW, Smith RD, Metz TO (2007) J Proteome Res 6:2323–2330CrossRefGoogle Scholar
  17. 17.
    Lapolla A, Brancia FL, Bereszczak J, Fedele D, Baccarin L, Seraglia R, Traldi P (2007) Mol Nutr Food Res 51:456–461CrossRefGoogle Scholar
  18. 18.
    Frolov A, Singer D, Hoffmann R (2007) J Pept Sci 13:862–867CrossRefGoogle Scholar
  19. 19.
    Stefanowicz P, Kapczynska K, Kluczyc A, Szewczuk Z (2007) Tetrahedron Lett 48:967–969CrossRefGoogle Scholar
  20. 20.
    O’Harte FP, Gray AM, Flatt PR (1998) J Endocrinol 156:237–243CrossRefGoogle Scholar
  21. 21.
    Jakas A, Horvat S (2003) Biopolymers 69:421–431CrossRefGoogle Scholar
  22. 22.
    Frolov A, Singer D, Hoffmann R (2006) J Pept Sci 12:389–395CrossRefGoogle Scholar
  23. 23.
    Jakas A, Horvat S (1996) J Chem Soc Perkin Trans II 231:789–794CrossRefGoogle Scholar
  24. 24.
    Linetsky MD, Shipova EV, Legrand RD, Argirov OO (2005) Biochim Biophys Acta 1724:181–193Google Scholar
  25. 25.
    Rich DH, Singh J (1979) The carbodiimide method. In: Gross E, Meinenhofer J (eds) The peptides. Academic, New York, pp 241–261Google Scholar
  26. 26.
    Aletras A, Barlos K, Gatos D, Koutsogianni S, Mamos P (1995) Epitheor Klin Farmacol Farmakokinet Int Ed 9:129Google Scholar
  27. 27.
    Frolov A, Hoffmann R (2008) Ann N Y Acad Sci 1126:253–256CrossRefGoogle Scholar
  28. 28.
    Garcia MC (2005) J Chromatogr B 825:111–123CrossRefGoogle Scholar
  29. 29.
    Apffel A, Fischer S, Goldberg G, Goodley PC, Kuhlmann FE (1995) J Chromatogr A 712:177–190CrossRefGoogle Scholar
  30. 30.
    Chong BE, Yan F, Lubman DM, Miller FR (2001) Rapid Commun Mass Spectrom 15:291–296CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  1. 1.Institute of Bioanalytical Chemistry, Center for Biotechnology and BiomedicineFaculty of Chemistry and Mineralogy, Leipzig UniversityLeipzigGermany

Personalised recommendations