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Mol Cell Proteomics. 2016 Mar;15(3):854-65. doi: 10.1074/mcp.M115.053082. Epub 2015 Nov 11.

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

Author information

1
From the ‡Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands;
2
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.

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.

PMID:
26560067
PMCID:
PMC4813705
[Available on 2017-03-01]
DOI:
10.1074/mcp.M115.053082
[Indexed for MEDLINE]
Free PMC Article

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