Abstract
Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole-cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of noncovalent complexes on the intact complex level.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pasa-Tolic, L., Lipton, M. S., Masselon, C. D., et al. (2002) Gene expression profiling using advanced mass spectrometric approaches. J. Mass. Spec. 37, 1185–1198.
Goodlet D. R. and Yi, E. C. (2002) Proteomics without polyacrylamide: qualitative and quantitative uses of tandem mass spectrometry in proteome analysis. Funct. Integr. Genomics 2, 138–153.
Pasa-Tolic, L., Jensen, P. K., Anderson, G. A., et al. (1999) High throughput proteome-wide precision measurements of protein expression using mass spectrometry J. Amer. Chem. Soc. 121, 7949–7950.
Tang, Q., Harrata, A. K., and Lee, C. S. (1996) High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis Anal. Chem. 68, 2482–2487.
Martinovic, S., Pasa Tolic, Lj., Masselon, C., Jensen, P. K., Stone, C. L., and Smith, R. D. (2000) Characterization of the human alcohol dehydrogenase isoenzymes by CIEF-ESI-FTMS Electrophoresis 21, 2368–2375.
Tang, Q., Harrata, A. K., and Lee, C. S. (1997) Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69, 3177–3182.
Yang, L., Lee, C. S., Hofstadler, S. A., Pasa-Tolic, L., and Smith, R. D. (1998) Capillary isoelectric focusing-electrospray ionization fourier transform ion cyclotron resonance mass spectrometry for protein characterization Anal. Chem. 70, 3235–3241.
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.
Martinovic, S., Veenstra, T. D., Anderson, G. A., Pasa-Tolic, Lj., and Smith, R. D. (2002) Selective incorporation of isotopically labeled amino acids for identification of intact proteins on proteome-wide level. J. Mass Spectrom. 37, 99–107
Jensen, P. K., Pasa-Tolic, L., Peden, K. K., et al. (2000) Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures. Electrophoresis 21, 1372–1380.
Lyubarskaya, Y. V., Carr, S. A., Dunnington, D., et al. (1998) Screening for highaffinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry Anal. Chem. 1, 4761–5770.
Martinovic, S, Berger, S., Pasa Tolic, Lj., and Smith, R.D. (2000) Separation and detection of intact non-covalent protein complexes from the mixtures by on-line capillary isoelectric focusing-mass spectrometry. Anal. Chem. 72, 5356–5360.
Liu, C. L., Hofstadler, S. A., Bresson, J. A., et al. (1998) Rapid on line dual microdialysis with ESI-MS for direct analysis of complex biological samples and microorganism lysates. Anal. Chem. 70, 1797–1801.
Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Kilar, F. and Hjerten, S. (1989) Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries. Electrophoresis 10, 23–29.
Winger, B. E., Hofstadler, S. A., Bruce, J. E., Udseth, H. R., and Smith, R. D. (1993) High-resolution accurate mass measurements of biomolecules using a new electrospray ionization ion cyclotron resonance mass spectrometer. J. Am. Soc. Mass Spectrom. 4, 566–577.
The ICR-2LS suite of data analysis software is publicly available for noncommercial use. Requests and inquiries should be directed to: Gordon.Anderson@pnl.gov.
Horn, D. M., Zubarev, R. A., and McLafferty, F. W. (2000) Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules. J. Am. Soc. Mass Spectrom. 11, 320–332.
Shen, Y. and Smith, R. D. (2000) High-resolution capillary isoelectric focusing of proteins using highly hydrophilic-substituted cellulose-coated capillaries. J. Microcolumn Separations 12, 135–141.
Pasa-Tolic, L., Bruce, J. E., Lei, Q. P., Anderson, G. A., and Smith, R. D. (1998) In-trap cleanup of proteins from electrospray ionization using soft sustained off-resonance irradiation with fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 70, 405–408.
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
Martinović, S., Paša-Tolić, L., Smith, R.D. (2004). Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes. 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:291
Download citation
DOI: https://doi.org/10.1385/1-59259-798-X:291
Publisher Name: Humana Press
Print ISBN: 978-1-58829-017-5
Online ISBN: 978-1-59259-798-7
eBook Packages: Springer Protocols