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Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization

Nature Protocols volume 1, pages 1790–1798 (2006)Cite this article

Abstract

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 α and ε (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.

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Figure 1: Outline of a standard approach to protein identification.
Figure 2: Scheme showing the chemistry involved in N-terminal purification.
Figure 3: Gantt chart for a typical N-terminal peptide-purification experiment.
Figure 4: Isolation of N-terminal peptides from chicken skeletal muscle soluble fraction.
Figure 5: Isolation of N-terminal peptides from E. coli cell lysate.

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Acknowledgements

This work was supported by the Engineering and Physical Sciences Research Council.

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Authors and Affiliations

  1. Proteomics and Functional Genomics Group, Faculty of Veterinary Science, University of Liverpool, Crown Street, Liverpool, L69 7ZJ, UK

    Lucy McDonald & Robert J Beynon

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  1. Lucy McDonald
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Correspondence to Robert J Beynon.

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McDonald, L., Beynon, R. Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization. Nat Protoc 1, 1790–1798 (2006). https://doi.org/10.1038/nprot.2006.317

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