Protein Identification by In-Gel Digestion and Mass Spectrometric Analysis

  • Michele Learmonth
  • Alastair Aitken
Part of the Springer Protocols Handbooks book series (SPH)


This chapter describes the analysis of proteins that have been separated by one-(or two-) dimensional gel electrophoresis. In-gel digestion of the protein bands or spots from 1-D gels is detailed. Normally the proteins are digested with trypsin, which cleaves after lysine and arginine. Therefore, due to the specificity of this protease, this results in basic charges at both the amino-terminal and carboxy-terminal ends of most peptides. Not all peptides will have basic groups at both ends, since the N-terminus of an intact protein is commonly blocked by an acetyl or a number of other modifications and the C-terminus may well not be a lysine or arginine. Doubly charged peptide species will, however, predominate. These are of high energy, and fragment more easily in tandem (including ion-trap) mass spectrometry (1) . This results in substantial sequence information, leading to more powerful database analysis. There are programs available, such as Sequest (2), that allow the databases to be searched directly with the peptide fragment data (tandem MS-MS data). This results in a large saving of time and effort, since the data need not be interpreted into tentative peptide sequences, which can be extremely tedious. Glu-C may also be used in place of trypsin, although fewer doubly charged species will result and analysis may be limited to the mass fingerprint data. This is perhaps more suitable to mass analysis on the highly sensitive matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers, where few sequence or mass fragment data are obtained in any case.


Trypsin Solution Aqueous Acetonitrile Iodoacetic Acid Aqueous Formic Acid Basic Charge 
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  1. 1.
    Jensen, O. N., Wilm, M., Shevchenko, A., and Mann, M. (1999) Peptide sequencing of 2-DE gel-isolated proteins by nanoelectrospray tandem mass spectrometry. Methods Mol. Biol. 112, 571–588.PubMedGoogle Scholar
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    Yates, J. R., 3rd (1998) Database searching using mass spectrometry data. Electrophoresis 19, 893–900.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Michele Learmonth
    • 1
  • Alastair Aitken
    • 1
  1. 1.School of Biomedical and Clinical Laboratory Sciences, University of EdinburghUK

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