title: 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.
authors.affiliation: From the ‡Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands;
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Makowski
authors.affiliation: From the ‡Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands;
authors.forename: Esther
authors.initials: E
authors.lastname: Willems
authors.affiliation: From the ‡Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands;
authors.forename: Pascal W T C
authors.initials: PW
authors.lastname: Jansen
authors.affiliation: From the ‡Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands; §Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG Utrecht, the Netherlands M.Vermeulen@ncmls.ru.nl.
authors.forename: Michiel
authors.initials: M
authors.lastname: Vermeulen
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry / Q000302:isolation & purification; D002843:Chromatin / Q000378:metabolism*; D002853:Chromatography, Liquid; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000302:isolation & purification; D003432:Cross-Linking Reagents; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000302:isolation & purification; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification*; D020836:Protein Structure, Quaternary; D012268:Ribose-Phosphate Pyrophosphokinase / Q000737:chemistry / Q000302:isolation & purification; D000097722:Cohesins
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D064110:Minichromosome Maintenance Proteins; D046912:Multiprotein Complexes; D012268:Ribose-Phosphate Pyrophosphokinase; D000097722:Cohesins