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Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification
- Source :
- Molecular & Cellular Proteomics, 15, 3, pp. 854-865, Molecular & Cellular Proteomics, 15, 854-865
- Publication Year :
- 2016
-
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.
- Subjects :
- 0301 basic medicine
Models, Molecular
Resolution (mass spectrometry)
Immunoprecipitation
Chromosomal Proteins, Non-Histone
lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4]
Peptide
Cell Cycle Proteins
Mass spectrometry
Topology
Biochemistry
Mass Spectrometry
Special Issue: Chromatin Biology and Epigenetics
Analytical Chemistry
03 medical and health sciences
Protein structure
Affinity chromatography
Ribose-Phosphate Pyrophosphokinase
Humans
Protein Structure, Quaternary
GeneralLiterature_REFERENCE(e.g.,dictionaries,encyclopedias,glossaries)
Molecular Biology
Tandem affinity purification
chemistry.chemical_classification
Minichromosome Maintenance Proteins
Chemistry
Proteomics and Chromatin Biology
Chromatin
030104 developmental biology
Cross-Linking Reagents
Multiprotein Complexes
Chromatography, Liquid
HeLa Cells
Subjects
Details
- ISSN :
- 15359476
- Database :
- OpenAIRE
- Journal :
- Molecular & Cellular Proteomics, 15, 3, pp. 854-865, Molecular & Cellular Proteomics, 15, 854-865
- Accession number :
- edsair.doi.dedup.....db2d3b4f57a9ee44bb70d8fa043498fa