The Malaysian Journal of Analytical Sciences Vol 15 No 2 (2011): 273 – 287

 

 

 

PEPTIDE SEPARATION BY CAPILLARY ELECTROPHORESIS WITH ULTRAVIOLET DETECTION: SOME SIMPLE APPROACHES TO ENHANCE DETECTION SENSITIVITY AND RESOLUTION

 

(Pemisahan Peptida Oleh Elektroforesis Rerambut Dengan Pengesan Ultra-Lembayung: Pendekatan Mudah Untuk Meningkatkan Isyarat Pengesanan Dan Resolusi)

 

L. Noumie Surugau

 

Industrial Chemistry Program,

School of Science and Technology,

Universiti Malaysia Sabah,

Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia.

 

*Corresponding author: lnoumie@ums.edu.my

 

 

Abstract

Capillary electrophoresis (CE) is one of the leading separation technologies for analysis of water-soluble analytes. CE has many advantages over the more established methods such as liquid chromatography and gel electrophoresis particularly in rapid analysis, require very little sample, use less or no toxic organic solvent, high peak efficiency and ease of automation. Despite the many attractive advantages of CE, CE users continue to seek improvements particularly on detection sensitivity, resolution and selectivity.  This paper presented several simple approaches to improve detection sensitivity using simple sample pre-concentration called field-enhanced sample injection (FESI) and chromatographic-based ZipTip C18 pre-concentrator.  Also, some improvements in the resolution of complex peptides mixture when using two strategies namely, capillary coating and manipulation of the hydrophobicity of peptides using perfluorinated acids as background electrolyte (BGE), which have anionic conjugate base forms with hydrophobic character. As test compounds, standard peptide mixture and proteins digests were used for these studies. The results showed that FESI has significantly enhanced the detection signal of peptide standards and bovine serum albumin (BSA) tryptic digests. As for the use of ZipTip C18 pre-concentrator, selective enhancement in detection signal was particularly notable on the late migrating peptides. Coating the capillary proved to have little changes on the CE of peptides when used in conjunction with acidic BGE. Electropherograms of BSA tryptic peptides in pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) showed interesting profile, with notable resolution improvement for peptides with close similarity in electrophoretic mobilities.

 

Keywords: Capillary electrophoresis, peptide, protein digests, pre-concentration, detection sensitivity, resolution.

 

References

1.       Washburn, M.P., Wolters, D., Yates III, J.R. 2001. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19: 242-248.

2.       Wittke, S., Fliser, D., Haubitz, M., Bartel, S., Krebs, R., Hausadel, F., Hillmann, M., Golovko, I., Koester, P., Haller, H., Kaiser, T., Mischak, H.,Weissinger, E.M. 2003. Determination of peptides and proteins in human urine with capillary electrophoresis–mass spectrometry, a suitable tool for the establishment of new diagnostic markers. J. Chromatogr. A 1013(1-2): 173-181.

3.       Sassi, A.P., Andell III, F., Bitter, H.-M., Brown, M.P.S., Chapman, R.G., Espiritu, J. Greenquist, A.C., Guyon, I., Horchi-Alegre, M., Stults, K.L., Wainright, A., Heller, J.C., Stults, J.T. 2005. An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serum. Electrophoresis 26(7-8): 1500-1512.

4.       Wittke, S., Mischak, H., Walden, M., Kolch, W., Rädler, T., Wiedemann, K. 2005. Discovery of biomarkers in human urine and cerebrospinal fluid by capillary electrophoresis coupled to mass spectrometry: Towards new diagnostic and therapeutic approaches. Electrophoresis 26(7-8): 1476-1487.

5.       Kaiser, T., Wittke, S., Just, I., Krebs, R., Bartel, S., Fliser, D., Mischak, H., Weissinger, E.M. 2004. Capillary electrophoresis coupled to mass spectrometer for automated and robust polypeptide determination in body fluids for clinical use. Electrophoresis 25(13): 2044-2055.

6.       Hau, J., Devaud, S., Blank, I. 2005. Detection of Amadori compounds by capillary electrophoresis coupled to tandem mass spectrometry. Electrophoresis 26(13): 2077-2083.

7.       Ullsten, S., Zuberovic, A., Wetterhall, M., Hardenborg, E., Markides, K.E., Berquist, J. 2004. A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.  Electrophoresis 25(13): 2090-2099.

8.       Moring, S.E., Reel, R.T., van Soest, R.E.J., Lauer, H.H. 1993. Optical improvements of a Z-shaped cell for high-sensitivity UV absorbance detection in capillary electrophoresis. Anal. Chem.  65(23): 3454-3459.

9.       Chervet, J.P., Van Soest, R.E.J., Ursem, M. 1991. Z-shaped flow cell for UV detection in capillary electrophoresis. J. Chromatogr. A 543: 439-449.

10.    Heiger, D. N. 1992. High Performance Capillary Electrophoresis. Hewlett-Packard, USA. pp. 100–101.

11.    Wang, T., Aiken, J.H., Huie, C.W., Hartwick, R.A. 1991. Nanoliter-scale multireflection cell for absorption detection in capillary electrophoresis.  Anal. Chem. 63(14): 1372-1376.

12.    Cifuentes, A., Rodriguez, M.A., Garcia-Montelongo, J. F. 1996. Rectangular capillary electrophoresis: study of some dispersive effects. J. Chromatogr. A 737(2): 243-253.

13.    Stutz, H. 2005. Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix-assisted laser desorption/ionization and electrospray-mass spectrometry detection. Electrophoresis 26(7-8): 1254-1290.

14.    Simpson, D. C., Smith, R.D. 2005. Combining capillary electrophoresis with mass spectrometry for applications in proteomics. Electrophoresis 26(7-8): 1291-1305.

15.    Timperman, A.T., Oldenburg, K.E., Sweedler, J.V. 1995. Native fluorescence detection and spectral differentiation of peptides containing tryptophan and tyrosine in capillary electrophoresis. Anal. Chem. 67(19): 3421-3426.

16.    Tong, W., Yeung, E.S. 1996. Determination of insulin in single pancreatic cells by capillary electrophoresis and laser-induced native fluorescence. J. Chromatogr. B: Biomed. Sci. Appl. 685(1): 35-40.

17.    Voegel, P.D., Baldwin, R.P. 1997. Electrochemical detection in capillary electrophoresis. Electrophoresis 18(12): 2267-2278.

18. Staller, T.D., Sepaniak, M.J. 1997. Chemiluminescence detection in capillary electrophoresis. Electrophoresis 189(12-13): 2291-2296.

19.    Grossman, P.D., Wilson, K.J., Petrie, G., Lauer, H.J. 1988. Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis. Anal. Biochem. 173(2): 265-270.

20.    Popa, T.V., Mant, C.T., Hodges, R.S. 2004. Capillary electrophoresis of cationic random coil peptide standards: Effect of anionic ion-pairing reagents and comparison with reversed-phase chromatography. Electrophoresis 25(9): 1219-1229.

21.    Mant, C.T., Hodges, R.S. 1991. High-Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation, CRC Press, Boca Raton, FL.

22.     Ensing, K., de Boer, T., Schreuder, N., de Zeeuw, R. 1999. Separation and identification of neuropeptide Y, two of its fragments and their degradation products using capillary electrophoresis–mass spectrometry. J. Chromatogr. B: Biomed. Sci. Appl. 727(1-2): 53-61.

23.    Zhang, C.-X., Thormann, W. 1996. Head-Column Field-Amplified Sample Stacking in Binary System Capillary Electrophoresis: A Robust Approach Providing over 1000-Fold Sensitivity Enhancement. Anal. Chem. 68(15): 2523-2532.

24.    Monton, M. R. N., Terabe, S. 2004. Field-enhanced sample injection for high-sensitivity analysis of peptides and proteins in capillary electrophoresis–mass spectrometry. J. Chromatogr. 1032(1-2): 203-211.

25.    Ledger, R., Tucker, I.G., Walker, G.F. 2002. Quantitative capillary electrophoresis assay for the proteolytic stability of luteinizing hormone-releasing hormones. J. Chromatogr. B 769(2): 235-242.

26.    http://www.expasy.org/tools/peptidecutter/.

27.    Popa, T.V., Mant, C.T., Hodges, R.S. 2006. Ion-interaction–capillary zone electrophoresis of cationic proteomic peptide standards. J. Chromatogr. A 1111(2): 192-199.

 

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