Abstract
Proteins separated by one-dimensional or two-dimensional gel electrophoresis can be digested in-gel with trypsin and the recovered peptides can be sequenced de novo using triple quadrupole or hybrid quadrupole time-of-flight instruments equipped with a nanoelectrospray ion source. The peptide sequences determined provide useful information for identification of proteins by homology searching for cloning of the cognate genes by PCR based approaches.
Similar content being viewed by others
References
Pandey, A. and Mann, M. (2000) Proteomics to study genes and genomes. Nature 405, 837–846.
Chalmers, M. J. and Gaskell, S. J. (2000) Advances in mass spectrometry for proteome analysis. Curr. Opin. Biotechnol. 11, 384–390.
Lahm, H. W. and Langen, H. (2000) Mass spectrometry: a tool for the identification of proteins separated by gels. Electrophoresis 21, 2105–2114.
Jensen, O. N., Podtelejnikov, A. V., and Mann, M. (1997) Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching. Anal. Chem. 69, 4741–4750.
Berndt, P., Hobohm, U., and Langen, H. (1999) Reliable automatic protein identification from matrixassisted laser desorption/ionization mass spectrometric peptide fingerprints. Electrophoresis 20, 3521–3526.
Wilm, M., Shevchenko, A., Houthaeve, T., et al. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nanoelectrospray mass spectrometry. Nature 379, 466–469.
Zachariae, W., Shevchenko, A., Andrews, P.D., et al. (1998) Mass spectrometric analysis of the anaphasepromoting complex from yeast: identification of a subunit related to cullins. Science 279, 1216–1219.
Neubauer, G., King, A., Rappsilber, J., et al. (1998) Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genetic 20, 46–50.
Mann, M. and Wilm, M. (1994) Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 66, 4390–4399.
Shevchenko, A., Sunyaev, S., Loboda, A., Shevchenko, A., Bork, P., Ens, W., Standing, K.G., (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI quadropole time-of-flight mass spectometry and BLAST homology searching. Anal. Chem. 73, 1917–1926
Loboda, A. V., Krutchinsky, A. N., Bromirski, M., Ens, W., and Standing, K. G. (2000) A tandem quadrupole/time-of-flight mass spectrometer with a matrix-assisted laser desorption/ionization source: design and performance. Rapid Commun. Mass. Spectrom. 14, 1047–1057.
Shevchenko, A., Loboda, A., Shevchenko, A., Ens, W., and Standing, K. G. (2000) MALDI Quadrupole Time-of-Flight mass spectrometry: a powerful tool for proteomic research. Anal. Chem. 72, 2132–2141.
Roepstorff, P. and Fohlman, J. (1984) Proposed nomenclature for sequence ions. Biomed. Mass Spectrom. 11, 601.
Shevchenko, A., Wilm, M., and Mann, M. (1997) Peptide sequencing by mass spectrometry for homology searches and cloning of genes. J. Protein Chem. 16, 481–490.
Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., et al. (1996) FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817–827.
McNagny, K. M., Petterson, I., Rossi, F., et al. (1997) Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoetic progenitors. J. Cell Biol. 138, 1395–1407.
Morris, H. R., Paxton, T., Dell, A., et al. (1996) High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 10, 889–896.
Shevchenko, A., Chernushevich, I., Ens, W., et al. (1997) Rapid ‘De Novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024.
Wilm, M., Neubauer, G., L., T., Shevchenko, A., and Bachi, A. (1999) De novo sequencing of proteins with mass spectrometry using the differential scanning technique, in Proteome and protein analysis (Kamp, R. M., Kyriakidis, D., and Choli-Papadopoulou, T., eds.), Springer, Berlin, Heidelberg, New York, pp. 65–79.
Shevchenko, A., Jensen, O. N., Wilm, M., and Mann, M. (1996) Sample preparation techniques for femtomole sequencing of proteins from polyacrylamide gels, in Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland OR, p. 331.
Wilm, M., Neubauer, G., and Mann, M. (1996) Parent ion scans of unseparated peptide mixtures. Anal. Chem. 68, 527–533.
Wilm, M. and Mann, M. (1996) Analytical properties of the nano electrospray ion source. Anal. Chem. 66, 1–8.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.
Fernandez-Patron, C., Calero, M., Collazo, P. R., et al. (1995) Protein reverse staining: high-efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224, pp. 203–211.
Rabilloud, T. (1990) Mechanism of protein silver staining in polyacrylamide gels: A 10-year synthesis. Electrophoresis 11, 785–794.
Thiede, B., Lamer, S., Mattow, J., et al. (2000) Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion. Rapid. Commun. Mass Spectrom. 14, 496–502.
Andersen, J. S., Küster, B., Podtelejnikov, A., Mortz, E., and Mann, M. (1999) Common peptide contaminants observed by nanoelectrospray MS in low level sequencing of gel-separated proteins, in Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas TX, pp. 405,406.
Fenyo, D. (2000) Identifying the proteome: software tools. Curr. Opin. Biotechnol. 11, 391–395.
Chernushevich, I. V. (2000) Duty cycle improvement for a QqTOF mass spectrometer and its use for precursor ion scans. Eur. J. Mass Spectrom., in press.
Shevchenko, A., Chernushevich, I., and Mann, M. (1998) High sensitivity analysis of gel separated proteins by a quadrupole-TOF tandem mass spectrometer, in Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, p. 237.
Lingner, J., Hughes, T. R., Shevchenko, A., Mann, M., Lundblad, V., and Cech, T. R. (1997) Reverse transcriptase motifs in the catalytic subunits of telomerase. Science 276, 561–567.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Shevchenko, A., Chernushevich, I., Shevchenko, A. et al. “De novo” sequencing of peptides recovered from in-gel digested proteins by nanoelectrospray tandem mass spectrometry. Mol Biotechnol 20, 107–118 (2002). https://doi.org/10.1385/MB:20:1:107
Issue Date:
DOI: https://doi.org/10.1385/MB:20:1:107