biostudies-literatureLiu XNIGMS NIH HHS31861-31870https://www.ebi.ac.uk/biostudies/studies/S-EPMC7749342biostudies-literatureUnknown117(50)Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.Proceedings of the National Academy of Sciences of the United States of AmericaDriving integrative structural modeling with serial capture affinity purification.PMC7749342R01 GM112639R35 GM118068Liu XBanks CASAbmayr SMWorkman JLWashburn MPWen ZZhang YUnruh JRFlorens LSlaughter BDHao YLange JJfalseDriving integrative structural modeling with serial capture affinity purification.Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.2020-01-01T00:00:00Z2020 Dec2022-02-10T09:55:21.837Z2021-02-20T15:56:10ZS-EPMC77493423325757810.1073/pnas.2007931117