Characterization of a Protein Interactome by Co-Immunoprecipitation and Shotgun Mass Spectrometry

TURCK, C. W.; MACCARRONE, G.; MARTINS-DE-SOUZA, D.; BONFIGLIO, J.J.; SILBERSTEIN, S.

Multiplex Biomarker Techniques

Springer

Lugar: New York; Año: 2017; p. 223 - 234

Identifying the partners of a given protein (the interactome) may provide leads about the protein?s func- tion and the molecular mechanisms in which it is involved. One of the alternative strategies used to char- acterize protein interactomes consists of co-immunoprecipitation (co-IP) followed by shotgun mass spectrometry. This enables the isolation and identification of a protein target in its native state and its interactome from cells or tissue lysates under physiological conditions. In this chapter, we describe a co-IP protocol for interactome studies that uses an antibody against a protein of interest bound to protein A/G plus agarose beads to isolate a protein complex. The interacting proteins may be further fractionated by SDS-PAGE, followed by in-gel tryptic digestion and nano liquid chromatography high-resolution tandem mass spectrometry (nLC ESI-MS/MS) for identification purposes. The computational tools, strategy for protein identification, and use of interactome databases also will be described.