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Protocol for mapping murine transcription factor interactomes and composite motifs combining affinity purification mass spectrometry and ChIP-seq

Year: 2025

Type: article

Abstract: Mass spectrometry (MS)-based approaches have significantly advanced our ability to study protein interaction networks in an unbiased manner. Here, we present a protocol that uses affinity purification (AP)-MS to identify interaction partners of a biotinylated transcription factor of interest, isolated from primary murine T cells. The resulting interactome data are integrated with motif analyses from chromatin immunoprecipitation sequencing (ChIP-seq) experiments. This combined approach facilitates the concurrent identification of protein interactors and composite DNA motifs, with each dataset corroborating the findings of the other. For complete details on the use and execution of this protocol, please refer to Gabele et al . 1 • A protocol to identify transcription factor complexes • Steps for affinity purification mass spectrometry interactome analysis • Integration of interactomics and ChIP-seq for the identification of composite motifs • Guidance on combinatorial transcription factor motif analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Mass spectrometry (MS)-based approaches have significantly advanced our ability to study protein interaction networks in an unbiased manner. Here, we present a protocol that uses affinity purification (AP)-MS to identify interaction partners of a biotinylated transcription factor of interest, isolated from primary murine T cells. The resulting interactome data are integrated with motif analyses from chromatin immunoprecipitation sequencing (ChIP-seq) experiments. This combined approach facilitates the concurrent identification of protein interactors and composite DNA motifs, with each dataset corroborating the findings of the other.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104184