Characterizing Proteins from 2-DE Gels by Internal Sequence Analysis of Peptide Fragments
For years, proteolytic digest and peptide purification were essential tools for the biochemist wishing to determine the primary structure of a protein. The strategies changed with the availability of a polypeptide gas-phase sequencer and the advent of DNA recombinant technology. However, blocked N-termini and changing trends in molecular cloning techniques, such as PCR, brought back “internal” sequencing as well, a term coined by Aebersold et al. in their seminal paper on the in situ micropreparative digestion of electroblotted proteins (1). Since then, many practical improvements of the original digest recipe and alternative approaches have been proposed (2). Considerable effort has also been expended to interface in situ digestion with micro-liquid chromotography (LC), peptide sequencers, and mass spectrometers. The ability to generate a set of specific peptides (e.g., tryptic), together with recent advances in biopolymer mass spectrometry provide a novel approach to protein identification. Accurate masses of several protein fragments compose a “peptide mass fingerprint,” theoretically sufficient for unambiguous searching of sequence repositories (3). It is therefore expected that enzymatic proteolysis will become even more widely used in the future.
KeywordsAmmonium Bicarbonate Peptide Mass Fingerprint Chemical Sequencing Practical Improvement Enzymatic Proteolysis
- 1.Aebersold, R. H., Leavitt, J., Saavedra, R.A., Hood, L.E., and Kent, S. B. (1987) Internal amino acid sequence analysis of proteins separated by one or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc. Natl. Acad. Sci. USA 84, 6970–6974.PubMedCrossRefGoogle Scholar
- 9.Tempst, P., Erdjument-Bromage, H., Casteels, P., Geromanos, S., Lui, M., Powell, M., et al. (1996) MALDI-TOF mass spectrometry in the protein biochemistry lab: from characterization of cell cycle regulators to the quest for novel antibiotics, in Mass Spectrometry in Biological Sciences (Burlingame, A. L. and Carr, S. A., eds.), Humana Press, Totowa, NJ, pp. 105–133.Google Scholar
- 10.Erdjument-Bromage, H., Geromanos, S., Chodera, A., and Tempst, P. (1993) Successful peptide sequencing with femtomole level PTH-analysis: a commentary, in Techniques in Protein Chemistry IV (Angeletti H. R., ed.), Academic, San Diego, CA, pp. 419–426.Google Scholar