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REVIEWS
THE ABCS (AND XYZS) OF PEPTIDE SEQUENCING
Hanno Steen* and Matthias Mann
Abstract | Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the components of small protein complexes and large organelles, to determine post-translational modifications and in sophisticated functional screens. The key but little understood technology in mass-spectrometry-based proteomics is peptide sequencing, which we describe and review here in an easily accessible format.
To sequence a protein ten years ago, a substantial amount had to be purified and a technique known as Edman degradation had to be used. This method, which was developed by Peer Edman, relies on the identification of amino acids that have been chemically cleaved in a stepwise fashion from the amino terminus of the protein and requires much expertise. Often no sufficiently long or unambiguous peptide sequence could be assigned and the method failed completely if the protein was acetylated at its amino terminus or was otherwise blocked to the Edman reaction, which requires a free amino terminus. During the 1990s, mass spectrometry (MS), in which biomolecules are ionized and their mass is measured by following their specific trajectories in a vacuum system, displaced Edman degradation, because it is much more sensitive and can fragment the peptides in seconds instead of hours or days1.
Furthermore, MS does not require proteins or peptides to be purified to homogeneity and has no problem identifying blocked or otherwise modified proteins. In the last few years, further breathtaking technological advances have established MS not only as the definitive tool to study the primary structure of proteins, but also as a central technology for the field of proteomics (for recent proteomics reviews, see REFS 26).
Protein MS facilities have proliferated and many biologists now have access to a service to which they can submit a sample and are handed back a list of proteins that have been identified by MS. This arrangement frequently works quite well for the identification of single spots or bands, but it is our experience that biologists generally do not have the necessary background to critically interpret
the results of more challenging MS experiments in particular, the many kinds of advanced proteomic screens that are now possible. Often, the results...