Mol. & Cell. Proteomics 2016

In March 2016, Matthew Makowski and Michiel Vermeulen and colleagues (Radboud University, Nijmegen, The Netherlands) published a paper in Molecular and Cellular Proteomics:


Makowski MM, Willems E, Jansen PW, Vermeulen M. Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification. Mol Cell Proteomics. 2016 Mar;15(3):854-65. doi: 10.1074/mcp.M115.053082.



In recent years, cross-linking mass spectrometry (XL-MS) has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, XL-MS 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 (PRPP) 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.