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Mass spectrometry; Co-immunoprecipitation; Interactome; Proteome; Computational tools
Abstract:
Identifying the partners of a given protein (the interactome) may
provide leads about the protein's function and the molecular mechanisms
in which it is involved. One of the alternative strategies used to
characterize 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.
