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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Lescuyer, P., Hochstrasser, D. & Rabolloud, T. How shall we use proteomics toolbox for biomarker discovery? J. Prot. Res. 6, 3371–3375 (2007).
Frank, R. & Hargreaves, R. Clinical biomarkers in drug discovery and development. Nat. Rev. Drug Discov. 2, 566–580 (2003).
Barnea, E., Sorkin, R., Ziv, T., Beer, I. & Admon, A. Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins. Proteomics 5, 3367–3375 (2005).
Herbert, B.R., Righetti, P.G., Citterio, A. & Boschetti, E. Sample preparation and pre-fractionation techniques for electrophoresis-based proteomics. in Proteome Research (eds. Wilkins M.R., Appel R.D., Williams K.L. & Hochstrasser D.F.) 15–40 (Springer, Berlin-Heidelberg, 2007).
Hirabayashi, J. Lectin-based structural glycomics: glycoproteomics and glycan profiling. Glycoconj. J. 21, 35–40 (2004).
Rush, J. et al. Immuno-affinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 23, 94–101 (2005).
Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P. & Jorgensen, T.J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium oxide microcolumns. Mol. Cell. Proteomics 4, 873–886 (2005).
Pieper, R. et al. The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3, 1345–1364 (2003).
Echan, L.A., Tang, H.Y., Ali-Khan, N., Lee, K. & Speicher, D.W. Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma. Proteomics 5, 3292–3303 (2005).
Bhattacharya, D., Mukhopadhyay, D. & Chakrabarti, A. Hemoglobin depletion from red blood cell cytosol reveals new proteins in 2-D gel-based proteomics study. Proteomics Clin. Appl. 1, 561–564 (2007).
Shen, Y. et al. Characterization of the human blood plasma proteome. Proteomics 5, 4034–4045 (2005).
Ulhen, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell. Proteomics 4, 1920–1932 (2005).
Thulasiraman, V. et al. Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands. Electrophoresis 26, 3561–3571 (2005).
Boschetti, E., Lomas, L., Citterio, A. & Righetti, P.G. Romancing the 'hidden proteome', Anno Domini two zero zero seven. J. Chromatogr. A 1153, 277–290 (2007).
Hober, S. & Uhlen, M. Human protein atlas and the use of microarray technologies. Curr. Opin. Biotechnol. 19, 30–35 (2008).
Castagna, A. et al. Exploring the hidden human urinary proteome via ligand library beads. J. Proteome Res. 4, 1917–1930 (2005).
Sennels, L. et al. Proteomic analysis of human blood serum using peptide library beads. J. Proteome Res. 6, 4055–4062 (2007).
Guerrier, L. et al. Exploring the platelets proteome via combinatorial hexapeptide ligand libraries. J. Proteome Res. 6, 4290–4303 (2007).
Guerrier, L. et al. Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins. J. Chromatogr. 1176, 192–205 (2007).
Roux-Dalvai, F. et al. Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced spectrometry. Mol. Cell. Proteomics. (in the press) (2008).
D'Ambrosio, C. et al. Exploring the chicken egg white proteome with combinatorial ligand libraries. J. Proteome Res. (in the press) (2008).
Lam, K.S. et al. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354, 82–84 (1991).
Buettner, J.A., Dadd, C.A., Baumbach, G.A., Masecar, B.L. & Hammond, D.J. Chemically derived peptide libraries: a new resin and methodology for lead identification. Int. J. Peptide Protein Res. 47, 70–83 (1996).
Huang, P.Y. et al. Affinity purification of von Willebrand factor using ligands derived from peptide libraries. Bioorg. Med. Chem. 4, 699–708 (1996).
Bastek, P.D., Land, J.M., Baumbach, G.A., Hammond, D.H. & Carbonell, R.G. Discovery of alpha-1-proteinase inhibitor binding peptide from the screening of a solid phase combinatorial library. Sep. Sci. Technol. 35, 1681–1706 (2000).
Kaufman, D.B et al. Affinity purification of fibrinogen using a ligand from a peptide library. Biotechnol. Bioeng. 77, 278–289 (2002).
Lehman, A., Gholami, S., Hahn, M. & Lam, K.S. Image subtraction approach to screening one-bead-one-compound combinatorial libraries with complex protein mixtures. J. Comb. Chem. 8, 562–570 (2006).
Lathrop, J.T., Fijalkowska, I. & Hammond, D. The bead blot: a method for identifying ligand–protein and protein–protein interactions using combinatorial libraries of peptide ligands. Anal. Biochem. 361, 65–76 (2007).
Boschetti, E., Monsarrat, B. & Righetti, P.G. The invisible proteome: how to capture the low abundance proteins via combinatorial ligand libraries. Curr. Proteomics 4, 198–208 (2007).
Righetti, P.G., Boschetti, E., Lomas, L. & Citterio, A. Protein equalizer technology: the quest for a 'democratic proteome'. Proteomics 6, 3980–3992 (2006).
Harrison, P.R. Selective precipitation of ribonucleic acid from a mixture of total cellular nucleic acids extracted from cultured mammalian cells. Biochem. J. 121, 27–31 (1961).
Marmur, J. A Procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3, 208–218 (1961).
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).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2008.59
This article is cited by
-
Insight of low-abundance proteins in rice leaves under Cd stress using combinatorial peptide ligand library technology
Analytical and Bioanalytical Chemistry (2020)
-
Plasma proteomic study of acute mountain sickness susceptible and resistant individuals
Scientific Reports (2018)
-
The impact of growth hormone on proteomic profiles: a review of mouse and adult human studies
Clinical Proteomics (2017)
-
Optimized sample treatment protocol by solid-phase peptide libraries to enrich for protein traces
Amino Acids (2013)
-
Serum protein profiles as potential biomarkers for infectious disease status in pigs
BMC Veterinary Research (2012)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.