Abstract
Separation in capillary electrophoresis (CE) is based on the movement of charged compounds inside a background electrolyte under an applied potential. Because the mechanism of separation of CE differs from that of conventional high-performance liquid chromatography (HPLC), where separation is based on the analyte’s hydrophobic properties, CE is often used as a complementary technique to HPLC. In addition, because CE is performed in narrow capillaries at atmospheric pressure, it is used as an alternative to HPLC, capable of handling small sample volumes while providing shorter analysis times with higher efficiency. For the analysis of amino acid, protein, and peptide mixtures in small volume samples such as in single cells, CE has rapidly evolved as a preferred separation technique. The combination of a high-efficiency separation technique, such as CE, with mass spectrometry (MS) detection provides a powerful system for the analysis of complex biological mixtures. In this chapter, a theoretical and practical approach to achieving high-performance CE-MS is discussed and the utility of CE-MS for the analysis of amino acids, peptides, and proteins is demonstrated.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Swanek, F. D., Ferris, S. S., and Ewing, A. G. (1996) Capillary electrophoresis for the analysis of single cells: electrochemical, mass spectrometric, and radiochemical detection, in Handbook of Capillary Electrophoresis (Landers, J. P., ed.), CRC, Boca Raton, FL, pp. 495–521.
Kennedy, R. T., Oates, M. D., Cooper, B. R., Nickerson, B., and Jorgenson, J. W. (1989) Microcolumn separations and the analysis of single cells. Science 246, 57–63.
Kennedy, R. T., St. Claire, R. L., White, J. G., and Jorgenson, J. W. (1987) Chemical analysis of single neurons by open tubular liquid chromatography. Mikrochim. Acta. 2, 37–45.
Kennedy, R. T. and Jorgenson, J. W. (1989) Quantitative analysis of individual neurons by open tubular liquid chromatography with voltammetric detection. Anal. Chem. 61, 436–441.
Oates, M. D., Cooper, B. R., and Jorgenson, J. W. (1990) Quantitative amino acid analysis of individual snail neurons by open tubular liquid chromatography. Anal. Chem. 62, 1573–1577.
Cooper, B. R., Jankowski, J. A., Leszyczyszyn, D. J., Wrightman, R. M., and Jorgenson, J. W. (1992) Quantitative determination of catecholamines in individual bovine adrenomedullary cells by reversed-phase microcolumn liquid chromatography with electrochemical detection. Anal. Chem. 64, 691–694.
Ewing, A. G., Wallingford, R. A., and Olefirowicz, T. M. (1989) Capillary electrophoresis. Anal. Chem. 61, 292A–303A.
Olefirowicz, T. M. and Ewing, A. G. (1990) Capillary electrophoresis in 2 and 5 microns diameter capillaries: application to cytoplasmic analysis. Anal. Chem. 62, 1872–1876.
Olefirowicz, T. M. and Ewing, A. G. (1991) Capillary electrophoresis for sampling single nerve cells. Chemia. 45, 106–108.
Hogan, B. L. and Yeung, E. S. (1992) Determination of intracellular species at the level of a single erythrocyte via capillary electrophoresis with direct and indirect fluorescence detection. Anal. Chem. 64, 2841–2845.
Olefirowicz, T. M. and Ewing, A. G. (1990) Dopamine concentration in the cytoplasmic compartment of single neurons determined by capillary electrophoresis. J. Neurosci. Meth. 34, 11–15.
Jorgenson, J. W. and Lukacs, K. D. (1983) Capillary zone electrophoresis. Science 222, 266–272.
Gordon, M. J., Huang, X., Pentoney, Jr., S. L., and Zare, R. N. (1988) Capillary electrophoresis. Science 242, 224–248.
Gilman, S. D. and Ewing, A. G. (1995) Recent advances in the application of capillary electrophoresis to neuroscience. J. Cap. Elec. 2, 1–13.
Yeung, E. S. (1994) Chemical analysis of single human erythrocytes. Acc. Chem. Res. 27, 409–414.
Jankowski, J. A., Tracht, S., and Sweedler, J. V. (1995) Assaying single cells with capillary electrophoresis. Trends Anal. Chem. 14, 170–176.
Cannon, Jr., D. M., Winograd, N., and Ewing, A. G. (2000) Quantitative chemical analysis of single cells. Ann. Rev. Biophys. Biomol. Struct. 29, 239–263.
Zhang, X., Stuart, J. N., and Sweedler, J. V. (2002) Capillary electrophoresis with wavelength-resolved laser-induced fluorescence detection. Anal. Bioanal. Chem. 373, 332–343.
Preisler J., Hu, P., Rejtar, T., and Karger, B. L. (2000) Capillary electrophoresis—matrix assisted laser desorption/ionization time-of-flight mass spectrometry using a vacuum deposition interface. Anal. Chem. 72, 4785–4795.
Kennedy, R. T., German, I., Thompson, J. E., and Witowski, S. R. (1999) Fast analytical-scale separations by capillary electrophoresis and liquid chromatography. Chem. Rev. 99, 3081–3131.
Pertsen, J. R. and Mohammad, A. D. eds., (2001) Clinical and Forensic Applications of Capillary Electrophoresis, Humana, Totowa, NJ.
Von Brocke, A., Nicholson, G., and Bayer, E. (2001) Recent advances in capillary electrophoresis/electrospray-mass spectrometry. Electrophoresis 22, 1251–1266.
Moini, M. (2002) Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures. Anal. Bioanal. Chem. 373, 466–480.
Bocek, P., Deml, M., Gebauer, P., and Dolnik, V. (1988) Analytical Isotachophoresis (Radola, B. J., ed.), VCH, New York.
Dobos, D. (1975) Electrochemical Data: A Handbook for Electrochemists in Industry and Universities. Elsevier Scientific, New York.
Macka, M., Andersson, P., and Haddad, P. R. (1998) Changes in electrolyte pH due to electrolysis during capillary zone electrophoresis. Anal. Chem. 70, 743–749.
Timperman, A., Tracht, S. E., and Sweedler, J. V. (1996) Dynamic on-column pH monitoring in capillary electrophoresis: application to volume-limited outlet vials. Anal. Chem. 68, 2693–2698.
Bello, M. S. (1996) Electrolytic modification of a buffer during a capillary electrophoresis run. J. Chromatogr. A744, 81–91.
Corstjens, H., Billiet, H. A., Frank, J., and Luyben, K. C. (1996) Variation of the pH of the background electrolyte due to electrode reactions in capillary electrophoresis: theoretical approach and in situ measurement. Electrophoresis 17, 137–143.
Kebarle, P. and Tang, L. (1993) From ions in solution to ions in the gas phase-the mechanism of electrospray mass spectrometry. Anal. Chem. 65, 972A–86A.
Van Berkel, G. J. (1997) The electrolytic nature of electrospray, in Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications (Cole, R. B., ed.), Wiley, New York, pp. 65–105.
Smith, A. D. and Moini, M. (2001) Control of electrochemical reactions at the capillary electrophoresis outlet/electrospray emitter electrode under CE-ESI MS through the application of redox buffers. Anal. Chem. 73, 240–246.
Bruinis, A. P. (1997) ESI source design and dynamic range considerations, in Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications (Cole, R.B., ed.), Wiley, New York, pp. 107–136.
Longfei, J. and Moini, M. (2000) Development of multi-ESI-sprayer, multi-atmospheric-pressure-inlet mass spectrometry and its application to accurate mass measurement using time-of-flight mass spectrometry. Anal. Chem. 72, 20–24, and 885.
Bateman, K. P. (1999) Electrochemical properties of capillary electrophoresis-nanoelectrospray mass spectrometry. J. Am. Soc. Mass Spectrom. 10, 309–317.
Chang, H. T. and Yeung, E. S. (1993) Oncolumn digestion of protein for peptide mapping by capillary zone electrophoresis with laser-induced native fluorescence detection. Anal. Chem. 65, 2947–2951.
Moini, M. (2001) Design and performance of a universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique. Anal. Chem. 73, 3497–3501.
Moini, M., Cao, P., and Bard, A. J. (1999) Hydroquinone as a buffer additive for suppression of bubbles formed by electrochemical oxidation of the CE buffer at the outlet electrode in capillary electrophoresis/electrospray ionization-mass spectrometry. Anal. Chem. 71, 1658–1661.
Rindgen, D, Turesky, R. J., and Vouros, P. (1995) The application of CE/ESI/MS/ MS for detection of IQ adducts of DNA, in Proce. 43 th ASMS Conf. Mass Spectrometry and Allied Topics, p. 590.
Soga, T. and Heiger, D. N. (2000) Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Anal. Chem. 72, 1236–1241.
Schultz, C. L. and Moini, M. (2003) The analysis of underivatized amino acids and their D/L enantiomers using sheathless CE-MS. Anal. Chem. 75, 1508–1513.
Moseley, M. A., Jorgenson, W. J., Shabanowitz, J., Hunt, D. F., and Tomer, K. B. (1992) Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides. J. Am. Soc. Mass Spectrom. 3, 289–300.
Teerlink, T. (1994) Derivatization of posttranslationally modified amino acids. J. Chromatogr. B659, 185–207.
Schegg, K. M., Denslow, N. D., Anderson, T. T., Bao, Y., Cohen, S. A., Mahrenholz, A. M., and Mann, K. (1997) Quantitation and identification of proteins by amino acid analysis: ABRF-96AAA collaborative trial, in Techniques in Protein Chemistry VIII (Marshak, D. R., ed.), Academic, San Diego, CA, pp. 207–216.
Prata, C., Bonnafous, P., Fraysse, N, Treilhou, M., Poinsot, V., and Couderc, F. (2001) Recent advances in amino acid analysis by capillary electrophoresis. Electrophoresis 22, 4129–4138.
Vecchione, G., Margaglione, G., Grandone, E., et al. (1999) Determining sulfur-containing amino acids by capillary electrophoresis. A fast novel method for total homocyste(e)ine human plasma. Electrophoresis 20, 569–574.
Nyhan, W. L. (1984) Abnormalities in Amino Acid Metabolism in Clinical Medicine. Appleton Century Crofts, East Norwalk, CT.
Soga, T. and Heiger, D. N. (2000) Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Anal. Chem. 72, 1236–1241.
Marchelli, R., Dossena, A., and Palla, G. (1996) The potential of enantioselective analysis as a quality control tool. Trends in Food Sci. Tech. 7, 113–119.
Zumwalt, R. W., Kuo, K. C., and Gehrke, C. W., eds. (1987) Amino Acid Analysis by Gas Chromatography (Vol I–III). CRC, Boca Raton, FL.
Deyl, Z., Hyanek. J., and Horikova, M. (1986) Profiling of amino acids in body fluids and tissues by means of liquid chromatography. J. Chromatogr. 379, 177–250.
Duncan, M. W. and Poljak, A. (1998) Amino Acid Analysis of Peptides and Proteins on the Femtomole Scale by Gas Chromatography/Mass Spectrometry. Anal. Chem. 70, 890–896.
Kwon, J.-Y and Moini, M. (2001) Analysis of underivatized amino acid mixtures using high performance liquid chromatography/dual oscillating nebulizer atmospheric pressure microwave induced plasma ionization-mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 117–122.
He, T., Quinn, D., Fu, E., and Wang, Y. K. (1999) Analysis of diagnostic metabolites by capillary electrophoresis-mass spectrometry. J. Chromatogr. B727, 43–52.
Verleysen, K. and Sandra, P. (1998) Separation of chiral compounds by capillary electrophoresis. Electrophoresis 19, 2798–2833.
Kuhn, R., Stoecklin, F., and Erni, F. (1992) Chiral separations by host-guest complexation with cyclodexterin and crown ether in capillary zone electrophoresis. Chromatographia 33, 32–36.
Verleysen, K., Bosch, T. V. and Sandra, P. (1999) Comparison of highly sulfated α-, β-, and γ-cyclodextrins and 18-crown-6-tetracarboxylic acid for the enantiomeric separation of some amino acids and derivatives by capillary electrophoresis. Electrophoresis 20, 2650–2655.
D’Aniello, A., Lee, J. M., Petrucelli, L., and Maddalena Di Fiore, M. (1998) Regional decreases of free D-aspartate levels in Alzheimer’s disease. Neurosci. Lett. 250, 131–134.
Schell, M. J., Cooper, O. B., and Snyder, S. H. (1997) D-Aspartate localizations imply neuronal and neuroendocrine roles. Proc. Natl. Acad. Sci. 94, 2013–2018.
Schell, M. J., Brady Jr., R. O., Molliver, M. E., and Snyder, S. H. (1997) D-serine as a neuromodulator: regional and developmental localizations in rat brain glia resemble NMDA receptors. J. Neurosci. 17, 1604–1615.
Lubec, G. and Lubec, B (1993) Racemization rates of amino acids for dating ancient samples. Amino Acids 4, 1–3.
Kyba, E. P., Timko, J. M., Kaplan, L. J., de Jong, F., Gokel, G. W., and Cram, D. J. (1978) Host-guest complexation. 11. Survey of chiral recognition of amine and amino ester salts by dilocular bisdinaphthyl hosts. J. Am. Chem. Soc. 100, 4555–4568.
Sousa, L. S., Sogah, G. D. Y., Hoffman, D. H., and Cram, D. J. (1978) Host-guest complexation. 12. Total optical resolution of amine and amino ester salts by chromatography. J. Am. Chem. Soc. 100, 4569–4576.
Behr, J. P., Girodeau, J. M., Heyward, R. C., Lehn, J. M., and Sauvage, J. P. (1980) Molecular receptors. Functionalized and chiral macrocyclic polyethers derived from tartaric acid. Helv. Chim. Acta. 63, 2096–2111.
Behr, J. P., Lehn, J. M., and Vierling, P. (1982) Molecular receptors. Structural effects and substrate recognition in binding of organic and biogenic ammonium ions by chiral polyfunctional macrocyclic polyethers bearing amino acid and other side-chains. Helv. Chim. Acta. 65, 1853–1867.
Banks, J. F. and Gulcicek, E. E. (1997) Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry. Anal. Chem. 69, 3973–3978.
Wu, J., He, L., Li, M. X., Parus, S., and Lubman, D. M. (1997) Online capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of-flight mass detector. J. Am. Soc. Mass Spectrom. 8, 1237–1246.
Roberts, G. D., Johnson, W. P., Burman, S., Anumula K. R., and Carr, S. A. (1995) An integrated strategy for structural characterization of the protein and carbohydrate components of monoclonal antibodies: application to anti-respiratory syncytial virus MAb. Anal. Chem. 67, 3613–3625.
Yates, J. R. 3rd, Speicher, S., Griffin, P. R., and Hunkapiller, T. (1993) Peptide mass maps: a highly informative approach to protein identification. Anal. Biochem. 214, 397–408.
Kitagishi, K. (1997) Comparison with other analytical methods, in Handbook of Capillary Electrophoresis Applications (Shintani, H. and Polonsky J., eds.), Chapman & Hall, New York, pp. 137–146.
Wahl, J. H., Udseth, H. R., and Smith, R. D. (1996) Capillary electrophoresis—mass spectrometry in peptide mapping, in New Methods in Peptide Mapping for the Characterization of Proteins (Hancock, W. S., ed.), CRC, Boca Raton, FL, pp. 143–179.
Banks, J. F. and Dresch, T. (1996) Detection of fast capillary electrophoresis peptide and protein separations using electrospray ionization with a time-of-flight mass spectrometer. Anal. Chem. 68, 1480–1485.
Jiang, L. and Moini, M. (1995) Design and performance of a high resolution electrospray ion source for a magnetic sector mass spectrometer with a heated capillary inlet. J. Am. Soc. Mass Spectrom. 6, 1256–1261.
Moini, M. (1994) Ultramark 1621 as a calibration/reference compound for mass spectrometry. II. Positive-and negative-ion electrospray ionization. Rapid Commun. Mass Spectrom. 8, 711–714.
Laude, D. A., Stevenson, E., and Robinson, J. M. (1997) Electrospray ionization/fourier transform ion cyclotron resonance mass spectrometry, in Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications (Cole, R.B., ed.), Wiley, New York, NY, pp. 291–319.
Chernushevich, I. V., Ens, W., and Standing, K. G. (1997) Electrospray ionization time-of-flight mass spectrometry, in Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation and Applications (Cole, R.B., ed.), Wiley, New York, NY, pp. 203–234.
Lazar, I. M., Xin, B., et al. (1997) Design of a time-of-flight mass spectrometer as a detector for capillary electrophoresis. Anal. Chem. 69, 3205–3211.
Fang, L., Zhang, R., Williams, E. R., and Zare, R. N. (1994) Online time-of-flight mass spectrometric analysis of peptides separated by capillary electrophoresis. Anal. Chem. 66, 3696–3701.
Muddiman, D. C., Rockwood, A. L., Gao, Q., Severs, J. C., Udseth, H. R., and Smith, R. D. (1995) Application of sequential paired covariance to capillary electrophoresis electrospray ionization time-of-flight mass spectrometry: Unraveling the signal from the noise in the electropherogram. Anal. Chem. 67, 4371–4375.
Wu, J. T., Qian, M. M. G., Li, M. M. X., Liu, L., and Lubman, D. M. (1996) Use of an ion trap storage/reflectron time-of-flight mass spectrometer as a rapid and sensitive detector for capillary electrophoresis in protein digest analysis. Anal. Chem. 68, 3388–3396.
Yates, J. R., III, Zhou, J., Griffin, P. R., and Hood, L. E. (1990) Computer aided interpretation of low energy MS/MS mass spectra of peptides, in Techniques in Protein Chemistry II (Villafranca, J. J., ed.), Academic, New York, pp. 477–486.
Cao, P. and Moini, M. (1998) Capillary electrophoresis/electrospray ionization high mass accuracy time-of-flight mass spectrometry for protein identification using peptide mapping. Rapid. Commun. Mass Spectrom. 12, 864–870.
Mann, M. and Wilm, M. (1994) Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399.
Chong, B.E., Wall, D.B., Lubman, D.M., and Flynn, S.J. (1997) Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid. Commun. Mass Spectrom. 11, 1900–1908.
Arnold, R. J. and Reily, J. P. (1999) Observation of Escherichia coli ribosomal proteins and their posttranslational modifications by mass spectrometry. Anal. Biochem. 269, 105–112.
Reid, G. E., Shang, H., Hogan, J. M., Lee, G. U., and McLuckey, S. A. (2002) Gas-phase concentration, purification, and identification of whole proteins from complex mixtures. J. Am. Chem. Soc. 124, 7353–7362.
Jensen, P. K., Pasa-Tolic, L., Anderson, G. A., et al. (1999) Probing proteomes using capillary isoelectric focusing-electrospray ionization fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 71, 2076–2084.
Johnson, D. H., Naylor, S., Rhode, E., and Tomlinson, A. J. (1998) Comparison of protein mixtures in aqueous humor by membrane preconcentration-capillary electrophoresis-mass spectrometry. Electrophoresis 19, 2361–2370.
Cao, P. and Moini, M. (1998) Analysis of peptides, proteins, protein digests, and whole human blood by capillary electrophoresis/electrospray ionization-mass spectrometry using an in-capillary electrode sheathless interface. J. Am. Soc. Mass Spectrom. 9, 1081–1088.
Naylor, S. and Tomlinson, A. J. (2001) Capillary electrophoresis-mass spectrometry of biologically active peptides and proteins, in Clinical and Forensic Applications of Capillary Electrophoresis (Mohammad, A. A. and Peterson, J. R., eds.), Humana, Totowa, NJ, pp. 317–353.
Cutalo, J. N., Deterding, L. J., Khaledi, M., and Tomer, K. B. (2002) Separation and characterization of human high-density apolipoproteins using a nonaqueous modifier in capillary electrophoresis-mass spectrometry. Electrophoresis 23, 2296–2305.
Moini, M., Demars, S. M., and Huang, H. (2002) Analysis of carbonic anhydrase in human red Blood cells using capillary electrophoresis/ electrospray ionization-mass spectrometry. Anal. Chem. 74, 3772–3776.
Valaskovic, G. A., Kelleher, N. L., and McLafferty, F. W. (1996) Attomole protein characterization by capillary electrophoresis-mass spectrometry. Science 273, 1199–1202.
Andersson, B., Nyman, P. O., and Strid, L. (1972) Amino acid sequence of human erythrocyte carbonic anhydrase B. Biochem. Biophys. Res. Commun. 48, 670–677.
Terzakis, J. A. and Santegada, E. (2000) Measurement of erythrocytes on diagnostic slides by scanning electron microscopy. Anal. Quantitat. Cytol. Histol. 22, 244–246.
Henderson, L. E., Henriksson, D., and Nyman, P. O. (1973) Amino acid sequence of human erythrocyte carbonic anhydrase C. Biochem. Biophys. Res. Commun. 52, 1388–1394. For a more recent CAII amino acid sequence, see the NCBI database at http://www.ncbi.nlm.nih.gov.
Hutterer, K. M. and Jorgenson, J. W. (1999) Ultrahigh-voltage capillary zone electrophoresis. Anal. Chem. 71, 1293–1297.
Smith, A. D. and Moini, M. (2000) Advances in capillary electrophoresis/electrospray ionization mass spectrometry achieved through the in-capillary electrode technique, in Proc. 48 th ASMS Conf. Mass Spectrom. Alli. Top., pp. 1119–1120.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Moini, M. (2004). Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry of Amino Acids, Peptides, and Proteins. In: Strege, M.A., Lagu, A.L. (eds) Capillary Electrophoresis of Proteins and Peptides. Methods in Molecular Biology™, vol 276. Humana Press. https://doi.org/10.1385/1-59259-798-X:253
Download citation
DOI: https://doi.org/10.1385/1-59259-798-X:253
Publisher Name: Humana Press
Print ISBN: 978-1-58829-017-5
Online ISBN: 978-1-59259-798-7
eBook Packages: Springer Protocols