Chromatin enrichment for proteomics


During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a 'snapshot' of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses.

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Figure 1: Outline of the ChEP procedure.
Figure 2: Comparison of protein enrichment after isolation of ChEP-based gelatinous chromatin and the 'classical' chromatin pellet isolated according to Shiio et al.35.
Figure 3: Typical results from ChEP-based chromatin proteomics.


  1. 1

    Kustatscher, G. et al. Proteomics of a fuzzy organelle: interphase chromatin. EMBO J. 33, 648–664 (2014).

    CAS  Article  Google Scholar 

  2. 2

    Solomon, M.J., Larsen, P.L. & Varshavsky, A. Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53, 937–947 (1988).

    CAS  Article  Google Scholar 

  3. 3

    Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).

    CAS  Article  Google Scholar 

  4. 4

    Ishihama, Y. et al. Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards. Nat. Biotechnol. 23, 617–621 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Geiger, T., Cox, J., Ostasiewicz, P., Wiśniewski, J.R. & Mann, M. Super-SILAC mix for quantitative proteomics of human tumor tissue. Nat. Methods 7, 383–385 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Geiger, T. et al. Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics. Nat. Protoc. 6, 147–157 (2011).

    CAS  Article  Google Scholar 

  7. 7

    Wiśniewski, J.R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).

    Article  Google Scholar 

  8. 8

    Kohwi-Shigematsu, T., deBelle, I., Dickinson, L.A., Galande, S. & Kohwi, Y. Identification of base-unpairing region-binding proteins and characterization of their in vivo binding sequences. Methods Cell Biol. 53, 323–354 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Lewis, C.D. & Laemmli, U.K. Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell 29, 171–181 (1982).

    CAS  Article  Google Scholar 

  10. 10

    Ohta, S. et al. The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell 142, 810–821 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Kadonaga, J.T. & Tjian, R. Affinity purification of sequence-specific DNA binding proteins. Proc. Natl. Acad. Sci. USA 83, 5889–5893 (1986).

    CAS  Article  Google Scholar 

  12. 12

    Himeda, C.L. et al. Quantitative proteomic identification of six4 as the Trex-binding factor in the muscle creatine kinase enhancer. Mol. Cell. Biol. 24, 2132–2143 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Mittler, G., Butter, F. & Mann, M. A SILAC-based DNA protein interaction screen that identifies candidate binding proteins to functional DNA elements. Genome Res. 19, 284–293 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Hoshino, A. & Fujii, H. Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J. Biosci. Bioeng. 108, 446–449 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Akiyoshi, B., Nelson, C.R., Ranish, J.A. & Biggins, S. Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit. Genes Dev. 23, 2887–2899 (2009).

    CAS  Article  Google Scholar 

  16. 16

    Fujita, T. & Fujii, H. Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS ONE 6, e26109 (2011).

    CAS  Article  Google Scholar 

  17. 17

    Agelopoulos, M., McKay, D.J. & Mann, R.S. Developmental regulation of chromatin conformation by Hox proteins in Drosophila. Cell Rep. 1, 350–359 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Byrum, S.D., Raman, A., Taverna, S.D. & Tackett, A.J. ChAP-MS: a method for identification of proteins and histone posttranslational modifications at a single genomic locus. Cell Rep. 2, 198–205 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Pourfarzad, F. et al. Locus-specific proteomics by TChP: targeted chromatin purification. Cell Rep. 4, 589–600 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013).

    CAS  Article  Google Scholar 

  21. 21

    Fujita, T. et al. Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Sci. Rep. 3, 3171 (2013).

    Article  Google Scholar 

  22. 22

    Déjardin, J. & Kingston, R.E. Purification of proteins associated with specific genomic loci. Cell 136, 175–186 (2009).

    Article  Google Scholar 

  23. 23

    Kliszczak, A.E., Rainey, M.D., Harhen, B., Boisvert, F.M. & Santocanale, C. DNA mediated chromatin pull-down for the study of chromatin replication. Sci. Rep. 1, 95 (2011).

    Article  Google Scholar 

  24. 24

    Sirbu, B.M., Couch, F.B. & Cortez, D. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat. Protoc. 7, 594–605 (2012).

    CAS  Article  Google Scholar 

  25. 25

    Alabert, C. et al. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat. Cell Biol. 16, 281–293 (2014).

    CAS  Article  Google Scholar 

  26. 26

    Lambert, J.P., Mitchell, L., Rudner, A., Baetz, K. & Figeys, D. A novel proteomics approach for the discovery of chromatin-associated protein networks. Mol. Cell. Proteomics 8, 870–882 (2009).

    CAS  Article  Google Scholar 

  27. 27

    Wang, C.I. et al. Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nat. Struct. Mol. Biol. 20, 202–209 (2013).

    CAS  Article  Google Scholar 

  28. 28

    Soldi, M. & Bonaldi, T. The proteomic investigation of chromatin functional domains reveals novel synergisms among distinct heterochromatin components. Mol. Cell. Proteomics 12, 764–780 (2013).

    CAS  Article  Google Scholar 

  29. 29

    Villar-Garea, A. & Imhof, A. The analysis of histone modifications. Biochim. Biophys. Acta 1764, 1932–1939 (2006).

    CAS  Article  Google Scholar 

  30. 30

    Vermeulen, M. et al. Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4. Cell 131, 58–69 (2007).

    CAS  Article  Google Scholar 

  31. 31

    Vermeulen, M. et al. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell 142, 967–980 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Nikolov, M. et al. Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns. Mol. Cell. Proteomics 10, M110.005371 (2011).

    Article  Google Scholar 

  34. 34

    Spruijt, C.G. et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 152, 1146–1159 (2013).

    CAS  Article  Google Scholar 

  35. 35

    Shiio, Y. et al. Quantitative proteomic analysis of chromatin-associated factors. J. Am. Soc. Mass Spectrom. 14, 696–703 (2003).

    CAS  Article  Google Scholar 

  36. 36

    Kubota, T., Hiraga, S., Yamada, K., Lamond, A.I. & Donaldson, A.D. Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint. Mol. Cell. Proteomics 10, M110.005561 (2011).

    Article  Google Scholar 

  37. 37

    Kim, D.R., Gidvani, R.D., Ingalls, B.P., Duncker, B.P. & McConkey, B.J. Differential chromatin proteomics of the MMS-induced DNA damage response in yeast. Proteome Sci. 9, 62 (2011).

    CAS  Article  Google Scholar 

  38. 38

    Monte, E. et al. Systems proteomics of cardiac chromatin identifies nucleolin as a regulator of growth and cellular plasticity in cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 305, H1624–H1638 (2013).

    CAS  Article  Google Scholar 

  39. 39

    Khoudoli, G.A. et al. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition. Curr. Biol. 18, 838–843 (2008).

    CAS  Article  Google Scholar 

  40. 40

    Chou, D.M. et al. A chromatin localization screen reveals poly (ADP ribose)-regulated recruitment of the repressive polycomb and NuRD complexes to sites of DNA damage. Proc. Natl. Acad. Sci. USA 107, 18475–18480 (2010).

    CAS  Article  Google Scholar 

  41. 41

    Franklin, S. et al. Quantitative analysis of the chromatin proteome in disease reveals remodeling principles and identifies high-mobility group protein B2 as a regulator of hypertrophic growth. Mol. Cell Proteomics 11, M111.014258 (2012).

    Article  Google Scholar 

  42. 42

    Kaguni, L.S. & Lehman, I.R. Eukaryotic DNA polymerase-primase: structure, mechanism and function. Biochim. Biophys. Acta 950, 87–101 (1988).

    CAS  Article  Google Scholar 

  43. 43

    Fujita, N. et al. MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer. Cell 113, 207–219 (2003).

    CAS  Article  Google Scholar 

  44. 44

    Nowak, D.E., Tian, B. & Brasier, A.R. Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation. Biotechniques 39, 715–725 (2005).

    CAS  Article  Google Scholar 

  45. 45

    Zeng, P.Y., Vakoc, C.R., Chen, Z.C., Blobel, G.A. & Berger, S.L. In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. Biotechniques 41, 694 (2006).

    CAS  Article  Google Scholar 

  46. 46

    Choi, N.M. & Boss, J.M. Multiple histone methyl and acetyltransferase complex components bind the HLA-DRA gene. PLoS ONE 7, e37554 (2012).

    CAS  Article  Google Scholar 

  47. 47

    Ong, S.E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660 (2006).

    CAS  Article  Google Scholar 

  48. 48

    Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  Article  Google Scholar 

  49. 49

    Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    CAS  Article  Google Scholar 

  50. 50

    Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    CAS  Article  Google Scholar 

  51. 51

    Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  Article  Google Scholar 

  52. 52

    Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).

    CAS  Article  Google Scholar 

  53. 53

    Spitzer, M., Wildenhain, J., Rappsilber, J. & Tyers, M. BoxPlotR: a web tool for generation of box plots. Nat. Methods 11, 121–122 (2014).

    CAS  Article  Google Scholar 

  54. 54

    Timinszky, G. et al. A macrodomain-containing histone rearranges chromatin upon sensing PARP1 activation. Nat. Struct. Mol. Biol. 16, 923–929 (2009).

    CAS  Article  Google Scholar 

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We thank F. de Lima Alves and L. Peil for their assistance with mass spectrometric analyses and N. Hegarat and H. Hochegger for testing this protocol on chicken DT40 cells. The Wellcome Trust generously supported this work through a Senior Research Fellowship to J.R. (084229), two Wellcome Trust Centre Core Grants (077707 and 092076) and an instrument grant (091020). G.K. was supported by a Federation of European Biochemical Societies (FEBS) long-term fellowship.

Author information




G.K. and J.R. conceived the chromatin enrichment procedure. G.K., K.L.H.W. and C.F. performed the experiments. G.K. and J.R. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Juri Rappsilber.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The effect of RNase treatment on ChEP-isolated chromatin fractions.

(a) Boxplot showing the impact of RNase digestion on various functional categories of proteins. Proteins with functions related to RNA processing (purple) are depleted by RNase treatment, presumably because they are cross-linked to chromatin indirectly via RNA. Proteins without expected chromatin function (i.e. contaminants, blue) are also somewhat reduced by RNase treatment, but proteins with canonical chromatin functions are not (red). (b) Boxplot showing that RNase treatment reduces the co-purification of ribosomes, which are typical contaminants of chromatin fractions. Note that the family of serine / arginine (SR)-rich splicing factors is not affected by RNase treatment, as these proteins generally act co-transcriptionally and are thus likely to be cross-linked directly to DNA or other chromatin factors.

Supplementary information

Supplementary Figure 1

The effect of RNase treatment on ChEP-isolated chromatin fractions. (PDF 152 kb)

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Kustatscher, G., Wills, K., Furlan, C. et al. Chromatin enrichment for proteomics. Nat Protoc 9, 2090–2099 (2014).

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