Abstract
Currently, cell-based screenings yield a multitude of small molecule modulators of diverse biological processes. The most demanding step in the course of elucidation of the mode of action of biologically active compounds is the identification of the target proteins. Although there is no generic approach available, affinity-based chemical proteomics is the most widely applied methodology. Particularly, quantitative chemical proteomics has proven very powerful in the identification of the putative targets of small molecules. Here we describe the procedure for identification of target proteins for small molecules employing affinity chromatography and the stable isotope labeling in cell culture (SILAC) for quantitative proteomics.
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References
Ziegler S et al (2013) Target identification for small bioactive molecules: finding the needle in the haystack. Angew Chem Int Ed Engl 52:2744–2792
Yan W, Chen SS (2005) Mass spectrometry-based quantitative proteomic profiling. Brief Funct Genomic Proteomic 4:27–38
Ong SE et al (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386
Ong S-E et al (2012) Identifiying cellular targets of small-molecule probes and drugs with biochemical enrichment and SILAC. Methods Mol Biol 803:129–140
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372
Neuhoff V et al (1988) Improved staining of proteins in polyacrylamide gels including isoelectric-focusing gels with clear background at nanogram sensitivity using Coomassie brilliant blue G-250 and R-250. Electrophoresis 9:255–262
Rappsilber J et al (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
Perkins DN et al (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567
Eng JK et al (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5:976–989
Cox J, Mann M (2012) 1D and 2D annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data. BMC Bioinformatics 13(Suppl 16):S12
Nodwell MB, Sieber SA (2012) ABPP methodology: introduction and overview. Top Curr Chem 324:1–41
Blagoev B, Mann M (2006) Quantitative proteomics to study mitogen-activated protein kinases. Methods 40:243–250
Bendall SC et al (2008) Prevention of amino acid conversion in SILAC experiments with embryonic stem cells. Mol Cell Proteomics 7:1587–1597
Van Hoof D et al (2007) An experimental correction for arginine-to-proline conversion artifacts in SILAC-based quantitative proteomics. Nat Methods 4:677–678
Park SK et al (2009) A computational approach to correct arginine-to-proline conversion in quantitative proteomics. Nat Methods 6:184–185
Glatter T et al (2012) Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion. J Proteome Res 11:5145–5156
Acknowledgement
This work was supported by the Max-Planck-Gesellschaft and the European Union (funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement no. 268309).
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Vendrell-Navarro, G., Brockmeyer, A., Waldmann, H., Janning, P., Ziegler, S. (2015). Identification of the Targets of Biologically Active Small Molecules Using Quantitative Proteomics. In: Hempel, J., Williams, C., Hong, C. (eds) Chemical Biology. Methods in Molecular Biology, vol 1263. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2269-7_21
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DOI: https://doi.org/10.1007/978-1-4939-2269-7_21
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