Abstract
Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today’s commonly used high throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications), and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annotated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html), a fully annotated 2-D map is created online. It is a multi-dimensional representation of a proteome, that contains interpreted PMF data in addition to protein identification results. This “MS-imaging” method represents a major step towards the development of a clinical molecular scanner.
Reproduced with permission of (Binz, Muller et al., 1999). Copyright (1999) American Chemical Society
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
Preview
Unable to display preview. Download preview PDF.
7. References
Appel, R., Bairoch, A., & Hochstrasser, D. (1994). Trends Biochem. Sci., 19, 258–260.
Appel, R., Palagi, P., Walther, D., Vargas, J., Sanchez, J., Ravier, F., et al. (1997). Melanie II—a third-generation software package for analysis of two-dimensional electrophoresis images: I. Features and user interface. Electrophoresis, 18(15), 2724–2734.
Appel, R., Vargas, J., Palagi, P., Walther, D., & Hochstrasser, D. (1997). Melanie II—a third-generation software package for analysis of two-dimensional electrophoresis images: II. Algorithms. Electrophoresis, 18(15), 2735–2748.
Bienvenut, W., Sanchez, J., Karmime, A., Rouge, V., Rose, K., Binz, P., et al. (1999). Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot. Anal Chem, 71(21), 4800–4807.
Binz, P., Muller, M., Walther, D., Bienvenut, W., Gras, R., Hoogland, C., et al. (1999). A molecular scanner to automate proteomic research and to display proteome images. Anal Chem, 71(21), 4981–4988.
Binz, P., Wilkins, M., Gasteiger, E., Bairoch, A., Appel, R., & Hochstrasser, D. (1999). In R. Kellner, F. Lottspeich & H. Meyer (Eds.), Microcharacterisation of proteins (2nd ed., pp. 277–300). Berlin: Wiley-VCH.
Blackledge, J., & Alexander, A. (1995). Polyethylene membrane as a sample support for direct MALDI MS of high mass proteins. Anal. Chem., 67, 843–848.
Caprioli, R., Farmer, T., & Gile, J. (1997). Molecular imaging of biological samples localization of peptides and proteins using MALDI-TOF-MS. Anal. Chem., 69, 4751–4760.
Eckerskorn, C., Strupat, K., Schleuder, D., Hochstrasser, D., Sanchez, J., Lottspeich, F., et al. (1997). Analysis of proteins by direct scanning IR-MALDI-MS after 2-D PAGE separation and electroblotting. Anal. Chem., 69, 2888–2892.
Fabris, D., Vestling, M., Cordero, M., Doroshenko, V., Cotter, R., & C, F. (1995). Rapid Commun Mass Spectrom., 9(11), 1051–1055.
Hochstrasser, D. (1998). Proteome in perspective. Clin. Chem. Lab. Med., 36(11), 825–836.
Hochtrasser, D., Appel, R., Vargas, R., Perrier, R., Vurlod, J., Ravier, F., et al. (1991). MD-Computing, 8, 85–91.
Hoogland, C., Sanchez, J., Tonella, L., Binz, P., Bairoch, A., Hochstrasser, D., et al. (2000). 28(1), 286–288.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(259), 680–685.
McComb, M., Oleschuk, R., Chow, A., Ens, W., Standing, K., Perreault, H., et al. (1998). Characterization of hemoglobin variants by MALDI-TOF MS using a polyurethane membrane as the sample support. Anal Chem, 70(24), 300.
McComb, M. E., Oleschuk, R. D., Manley, D. M., Donald, L., A. Chow, O’Neil, J. D. J., et al. (1997). Use of a non-porous polyurethane membrane as a sample support for MALDI-TOF MS of peptides and proteins. Rapid Commun. Mass Spectrom., 11, 1716–1722.
Ogorzalek Loo, R., Mitchell, C., Stevenson, T., Martin, S., Hines, W., Juhasz, P., et al. (1997). Sensitivity and mass accuracy for proteins analyzed directly from polyacrylamide gels: implications for proteome mapping. Electrophoresis, 18(3–4), 382–390.
Sanchez, J., & Hochstrasser, D. (1998). In A. Link (Ed.), Method in molecular biology: 2-d proteome analysis protocol (Vol. 112, pp. 227–233). Totowa, NJ: Humana press.
Scheler, C., Lamer, S., Pan, Z., Li, X., Salnikov, J., & Jungblut, P. (1998). Peptide mass fingerprint sequence coverage from differently stained proteins on 2-DE patterns by MALDI-MS. Electrophoresis, 19, 918–927.
Schreiner, M., Strupat, K., Lottspeich, F., & Eckerskorn, C. (1996). UV-MALDI-MS of electroblotted proteins. Electrophoresis, 17, 954–961.
Traini, M., Gooley, A. A., Ou, K., Wilkins, M. R., Tonella, L., Sanchez, J.-C., et al. (1998). Towards an automated approach for protein identification in proteome projects. Electrophoresis, 19, 1941–1949.
Vestling, M., & Fenselau, C. (1994). PVDF: an interface for gel electrophoresis and MALDI-MS. Biochem. Soc. Trans., 22(2), 547–551.
Wilkins, M., & al., e. (1999). High throughput mass spectrometry discovery of protein post translational modification. J. Mol. Biol., 289, 645–657.
Wilkins, M., Sanchez, J., Gooley, A., Appel, R., Humphery-Smith, J., Hochstrasser, D., et al. (1995). Progress with proteome projects: Why all proteins expressed by genome should be identified and how to do it. Biotechnology & genetic Engineering Reviews, 13, 19–50.
Wilkins, M., Sanchez, J., Williams, K., & Hochstrasser, D. (1996). Current challenges and futures applications for protein maps and post-translational vector maps in proteome project. Electrophoresis, 17, 830–838.
Williams, K., & Hochstrasser, D. (1997). In Proteome Research: New Frontiers in functional genomics (pp. 1–12). Berlin: Springer-Verlag.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Springer
About this chapter
Cite this chapter
Binz, P. et al. (2005). Signal Traitment and Virtual Images Production (1/2). In: Bienvenut, W.V. (eds) Acceleration and Improvement of Protein Identification by Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/1-4020-3319-2_4
Download citation
DOI: https://doi.org/10.1007/1-4020-3319-2_4
Publisher Name: Springer, Dordrecht
Print ISBN: 978-1-4020-3318-6
Online ISBN: 978-1-4020-3319-3
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)