Protocol for analyzing protein interactomes under starvation in fission yeast using in vivo cross-linking

Identifying a protein's interactome can be challenging when studying low-abundance proteins, labile or transient interactions, or cells under starvation conditions. Here, we present a protocol to purify proteins and their interacting partners in the fission yeast Schizosaccharomyces pombe. We use formaldehyde to cross-link proteins in vivo, thereby stabilizing protein complexes prior to immunoprecipitation. We then describe immunoprecipitation procedures and approaches for interpreting mass spectrometry datasets. Finally, this protocol enables the validation of specific protein interactions by western blotting. For complete details on the use and execution of this protocol, please refer to Gálvez-Merchán et al.¹ and Encinar del Dedo et al.².