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Immunoprecipitation-Mass Spectrometry (IP-MS) of Protein-Protein Interactions of Nuclear-Localized Plant Proteins

Methods Mol Biol. 2023;2698:163-181. doi: 10.1007/978-1-0716-3354-0_11.


Transcription factors that act within a gene regulatory network (GRN) often interact with other proteins such as chromatin remodeling factors, histone modifiers, and other co-regulators. Characterizing these interactions is crucial for understanding the function and mechanism of action of a transcription factor. Here, a method for the identification of protein-protein interactions of nuclear-localized, transcription-associated factors is described. The method is based on the immunoprecipitation (IP) of a fluorophore-tagged target, followed by mass spectrometry (MS), peptide identification, and quantification of interacting proteins. By applying label-free quantification to IPs and their input protein extracts, statistically controlled protein enrichment ratios uncover high-confidence interaction partners of the target. A complete step-by-step procedure, including sample preparation, MS settings, data analysis, and visualization is provided.

PMID:37682475 | DOI:10.1007/978-1-0716-3354-0_11

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