Abstract
Recent developments in technology and instrumentation have made mass spectrometry the method of choice for the identification of gel-separated proteins using rapidly growing sequence databases (1). Proteins with a full-length sequence present in a database can be identified with high certainty and high throughput using the accurate masses obtained by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry peptide mapping (2). Simple protein mixtures can also be deciphered by MALDI peptide mapping (3) and the entire identification process, starting from in-gel digestion (4) and finishing with acquisition of mass spectra and database search, can be automated (5). Only 1–3% of a total digest are consumed for MALDI analysis even if the protein of interest is present on a gel in a subpicomole amount. If no conclusive identification is achieved by MALDI peptide mapping, the remaining protein digest can be analyzed by nanoelectrospray tandem mass spectrometry (Nano ESI-MS/MS) (6). Nano ESI-MS/MS produces data that allow highly specific database searches so that proteins that are only partially present in a database, or relevant clones in an EST database, can be identified (7). It is important to point out that there is no need to determine the complete sequence of peptides in order to search a database-a short sequence stretch consisting of three to four amino acid residues provides enough search specificity when combined with the mass of the intact peptide and the masses of corresponding fragment ions in a peptide sequence tag (8) (see Subheading 3.4.).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Shevchenko A., Jensen O. N., Podtelejnikov A. V., Sagliocco F., Wilm M., Vorm O., et al. (1996) Linking genome and proteome by mass spectrometry: large scale identification of yeast proteins from two dimensional gels. Proc. Natl. Acad. Sci. USA 98, 14,440–14,445.
Jensen O. N., Podtelejnikov P., and Mann M. (1996) Delayed extraction improves specificity in database searches by MALDI peptide maps. Rapid Commun. Mass Spectrom. 10, 1371–1378.
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.
Houthaeve T., Gausepohl H., Mann M., and Ashman K. (1995) Automation of micro-preparation and enzymatic cleavage of gel electrophoretically separated proteins. FEBS Lett. 376, 91–94.
Jensen O. N., Mortensen P., Vorm O., and Mann M. (1997) Automatic acquisition of MALDI spectra using fuzzy logic control. Anal. Chem. 69, 1706–1714.
Wilm M., Shevchenko A., Houthaeve T., Breit S., Schweigerer L., Fotsis T., et al. (1996) Femtomole sequencing of proteins from polyacrylamide gels by nanoelectrospray mass spectrometry. Nature 379, 466–469.
Lamond A. and Mann M. (1997) Cell biology and the genome projects—a concerted strategy for characterizing multiprotein complexes by using mass spectrometry. Trends Cell Biol. 7, 139–142.
Mann M. and Wilm M. (1994) Error tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 86, 4390–4399.
Shevchenko A., Keller P., Scheiffele P., Mann M., and Simons K. (1997) Identification of components of trans-Golgi network-derived transport vesicles and detergent-insoluble complexes by nanoelectrospray tandem mass spectrometry. Electrophoresis 18, 2591–2600.
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., Rourke K. O., Shevchenko A., Ni J., 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., Flamme I., Shevchenko A., Mann M., 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., Langhorn J., Berg M., Bordoli R. S., 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, Standing K. G, Thomson B., Wilm M., et al. (1997) Rapid lsde novors peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 11, 1015–1024.
Shevchenko A., Jensen O. N., Wilm M., and Mann M. (1996) Sample preparation techniques for femtomole sequencing of proteins from polyarylamide 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., Garcia J. R., Madrazo J., Musacchio A., et al. (1995) Protein reverse staining: high efficiency microanalysis of unmodified proteins detected on electrophoresis gels. Anal. Biochem. 224, 203–211.
Wilm M. and Mann M.(1994) Electrospray and Taylor-cone theory, Dole’s beam of macro molecules at last? Int. J. Mass Spectrom. Ion Processes 136, 167–180.
Mann M. (1994) Sequence database searching by mass spectrometric data, in Microcharacterization of Proteins (Kellner R., Lottspeich F., and Meyer H. E., eds.), VCH Weinheim, pp. 223–245.
Shevchenko A., Chernushevich I., and Mann M. (1998) High sensitivity analysis of gel separated proteins by a quadrupole-TOF tandem mass spectrometer, in Proceedings 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
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc.
About this protocol
Cite this protocol
Shevchenko, A., Chernushevich, I., Wilm, M., Mann, M. (2000). De Novo Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry Using Triple Quadrupole and Quadrupole/Time-of-Flight Instruments. In: Chapman, J.R. (eds) Mass Spectrometry of Proteins and Peptides. Methods in Molecular Biology™, vol 146. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-045-4:1
Download citation
DOI: https://doi.org/10.1385/1-59259-045-4:1
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-609-3
Online ISBN: 978-1-59259-045-2
eBook Packages: Springer Protocols