A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

Abstract

Mass spectrometry–based proteomics1 can reveal protein-protein interactions on a large scale2,3, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway4,5,6, we employ stable isotopic amino acids in cell culture (SILAC)7 to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

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Figure 1: Strategy to study activated EGFR complex.
Figure 2: Quantification of protein ratios from peptide doublets.
Figure 3: Subcellular co-localization of selected proteins by confocal microscopy and FRET.

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Acknowledgements

We thank other members of our laboratory for help and fruitful discussions and comments on the manuscript. Akhilesh Pandey provided the GST-SH2 (Grb2) domain construct. Work in the Center for Experimental BioInformatics (CEBI) is supported by a generous grant by the Danish National Research foundation.

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Correspondence to Matthias Mann.

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The authors declare no competing financial interests.

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Blagoev, B., Kratchmarova, I., Ong, S. et al. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21, 315–318 (2003). https://doi.org/10.1038/nbt790

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