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Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification.


ABSTRACT: In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures.

SUBMITTER: Makowski MM 

PROVIDER: S-EPMC4813705 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification.

Makowski Matthew M MM   Willems Esther E   Jansen Pascal W T C PW   Vermeulen Michiel M  

Molecular & cellular proteomics : MCP 20151111 3


In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for stru  ...[more]

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