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Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library

Abstract

Deciphering the protein composition of complex extracts or discovering biologically relevant polypeptides is frequently hindered by large dynamic concentration ranges. The presence of high-abundance proteins suppresses the signal of low-abundance ones, and the most rare proteins are frequently below the sensitivity of available analytical methods. This protocol addresses this problem by using highly diversified hexapeptide ligand libraries for capturing proteins. A protein extract is mixed with the library and because the library has equal amounts of each ligand, theoretically the maximum amount of each protein bound is the same. Under overloading conditions this has the effect of diluting those proteins present in excess of the ligand concentration and concentrating those of relatively lower abundance. Unbound components are washed out and captured species are finally desorbed. The entire sample treatment process takes about half a day and yields a protein solution that could be used as such for mass spectrometry investigations.

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Figure 1: Schematic representation of the process for reduction of the dynamic concentration range of proteins constituting a mixture.
Figure 2: 2D map analyses of proteins from a platelet extract.
Figure 3: Analysis of a red blood cell lysate before and after treatment with combinatorial hexapeptide ligand library.

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Acknowledgements

We thank the following scientists for their contribution to the experimental data mentioned in this document: F Roux-Dalvai, A. Gonzales de Peredo, B. Monsarrat, S. Claverol, M. Jandrot-Perrus, C. Simo' and F. Fortis. P.G.R. is supported by Fondazione Cariplo (Milano).

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Correspondence to Egisto Boschetti.

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Guerrier, L., Righetti, P. & Boschetti, E. Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library. Nat Protoc 3, 883–890 (2008). https://doi.org/10.1038/nprot.2008.59

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