Isolation of N-terminal peptides
McDonald, L. & Beynon, R.J. (2006) Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization. Nature Protocols 1, 1790 - 1798 [PUBMED] [PDF]
We describe a protocol for selective extraction of the amino (N)-terminal-most peptide of a protein or a mixture of proteins after proteolysis. The first stage of the protocol blocks the free amino groups alpha and epsilon (the latter being lysyl residues) on the intact proteins by acetylation. In the second stage, proteolysis of the acetylated proteins yields a mixture of N-terminally acetylated (true N-terminal) and non-acetylated (internal and carboxy-terminal) peptides. Affinity capture of peptides bearing free amino groups using an immobilized amine-reactive reagent removes internal peptides from the mixture. The unbound fraction is highly enriched in N-terminal peptides, which can be analyzed without further treatment. This method is compatible with a range of proteolytic enzymes and fragmentation methods, and should take 2 d to complete. The N-terminal peptides can then be analyzed by mass spectrometry. This low cost, rapid method is readily adopted using off the shelf reagents.
Faculty of 1000 Member Comments Ruedi Aebersold, Swiss Federal Institute of Technology Zurich, Switzerland "Tech Advance The paper describes a method for the selective isolation of N-terminal peptides from the digests of complex protein samples. The method is significant for proteomics because the reduction of sample complexity achieves results in more efficient, more sensitive and less redundant proteome analyses and at the same time provides direct experimental information about the N terminus of native proteins."