Proteomics: A Shotgun Approach without Two‐dimensional Gels

Claire Delahunty, The Scripps Research Institute, La Jolla, California, USA
John R Yates, The Scripps Research Institute, La Jolla, California, USA

Published online: January 2006

DOI: 10.1038/npg.els.0006197

Proteome analysis has been most commonly accomplished by the combination of two-dimensional electrophoresis and mass spectrometry, but this method suffers some drawbacks that limit its usefulness for large-scale analysis. New ‘shotgun’ techniques allow for the detection of proteins from complex mixtures and are rapidly replacing gel-based methods for certain applications.

Keywords: proteomics; mass spectrometry; tandem mass spectrometry; two-dimensional gels; shotgun proteomics; multidimensional protein identification technique; microcapillary liquid chromatography; matrix assisted laser desorption/ionization

Figure 1. General strategy for proteome characterization. Proteins are purified using 1DE or 2DE (see text) before being digested with an endoproteinase. The collection of peptides is introduced to the mass spectrometer. MS data are used to search available databases and protein identification is made.
Figure 2. MALDI- TOF mass spectrometry. Sample is codeposited with matrix on a stainless steel plate and advanced to the mass spectrometer by ionization with a laser. The TOF analyzer separates ions based on the time it takes them to travel the length of the flight tube.
Figure 3. Tandem mass spectrometry. Two mass analyzers are separated by an ion activation device. A mixture of peptides is ionized and then separated in the first segment by their mass to charge (m/z) ratios. Selected ions are then advanced to the ion activation device where they are fragmented.
Figure 4. MuDPIT technique. Complex peptide mixture is loaded onto a biphasic microcapillary column packed with strong cation exchange (SCX) and reverse-phase (RP) materials. Peptides are first displaced from the SCX to the RP by a salt gradient and then eluted directly into the mass spectrometer. After re-equilibration, the process is repeated with increasing salt steps until all peptides are eluted.
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 References
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 Further Reading
    book Blackstock W and Mann M (eds.) (2000) Trends in Proteomics. London: Elsevier.
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Related Sub-Topics:

Techniques in Cell Biology | Protein Structure | Mass Spectrometry | Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism
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Article Title: Proteomics: A Shotgun Approach without Two‐dimensional Gels
 
How to Cite close
Delahunty, Claire, and Yates, John R(Jan 2006) Proteomics: A Shotgun Approach without Two‐dimensional Gels. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0006197]