Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails

  • Kevin A. Janssen
  • Mariel Coradin
  • Congcong Lu
  • Simone Sidoli
  • Benjamin A. GarciaEmail author
Focus: Protein Post-translational Modifications: Research Article


The analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has been critical to the advancement of the field of epigenetics. The most sensitive and accurate workflow is similar to the canonical proteomics analysis workflow (bottom-up MS), where histones are digested into short peptides (4-20 aa) and quantitated in extracted ion chromatograms. However, this limits the ability to detect even very common co-occurrences of modifications on histone proteins, preventing biological interpretation of PTM crosstalk. By digesting with GluC rather than trypsin, it is possible to produce long polypeptides corresponding to intact histone N-terminal tails (50-60 aa), where most modifications reside. This middle-down MS approach is used to study distant PTM co-existence. However, the most sensitive middle-down workflow uses weak cation exchange-hydrophilic interaction chromatography (WCX-HILIC), which is less robust than conventional reversed-phase chromatography. Additionally, since the buffer systems for middle-down and bottom-up proteomics differ substantially, it is cumbersome to toggle back and forth between both experimental setups on the same LC system. Here, we present a new workflow using porous graphitic carbon (PGC) as a stationary phase for histone analysis where bottom-up and middle-down sized histone peptides can be analyzed simultaneously using the same reversed-phase buffer setup. By using this protocol for middle-down sized peptides, we identified 406 uniquely modified intact histone tails and achieved a correlation of 0.85 between PGC and WCX-HILIC LC methods. Together, our method facilitates the analysis of single and combinatorial histone PTMs with much simpler applicability for conventional proteomics labs than the state-of-the-art middle-down MS.


Histones Epigenetics Proteomics Middle-down PTMs Chromatography 



We gratefully acknowledge funding from NIH grants GM110174, AI118891, and CA196539.

Supplementary material

13361_2019_2303_MOESM1_ESM.docx (1.2 mb)
ESM 1 (DOCX 1233 kb)


  1. 1.
    Strahl, B.D., Allis, C.D.: The language of covalent histone modifications. Nature. 403(6765), 41–45 (2000)CrossRefGoogle Scholar
  2. 2.
    Jenuwein, T., Allis, C.D.: Translating the histone code. Science. 293(5532), 1074–1080 (2001)CrossRefGoogle Scholar
  3. 3.
    Kouzarides, T.: Chromatin modifications and their function. Cell. 128(4), 693–705 (2007)CrossRefGoogle Scholar
  4. 4.
    Bannister, A.J., Kouzarides, T.: Regulation of chromatin by histone modifications. Cell Res. 21(3), 381–395 (2011)CrossRefGoogle Scholar
  5. 5.
    Rossetto, D., Avvakumov, N., Côté, J.: Histone phosphorylation. Epigenetics. 7(10), 1098–1108 (2012)CrossRefGoogle Scholar
  6. 6.
    Torres-Padilla, M.-E., Parfitt, D.-E., Kouzarides, T., Zernicka-Goetz, M.: Histone arginine methylation regulates pluripotency in the early mouse embryo. Nature. 445, 214 (2007)CrossRefGoogle Scholar
  7. 7.
    Casas-Delucchi, C.S., Brero, A., Rahn, H.-P., Solovei, I., Wutz, A., Cremer, T., Leonhardt, H., Cardoso, M.C.: Histone acetylation controls the inactive X chromosome replication dynamics. Nat. Commun. 2, 222–222 (2011)CrossRefGoogle Scholar
  8. 8.
    Huang, H., Sabari, B.R., Garcia, B.A., Alllis, C.D., Zhao, Y.: SnapShot: histone modifications. Cell. 159(2), 458–458 e1 (2014)CrossRefGoogle Scholar
  9. 9.
    Fischle, W., Wang, Y., Allis, C.D.: Histone and chromatin cross-talk. Curr. Opin. Cell Biol. 15(2), 172–183 (2003)CrossRefGoogle Scholar
  10. 10.
    Lee, J.S., Smith, E., Shilatifard, A.: The language of histone crosstalk. Cell. 142(5), 682–685 (2010)CrossRefGoogle Scholar
  11. 11.
    Hunter, T.: The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol. Cell. 28(5), 730–738 (2007)CrossRefGoogle Scholar
  12. 12.
    Atlasi, Y., Stunnenberg, H.G.: The interplay of epigenetic marks during stem cell differentiation and development. Nat. Rev. Genet. 18, 643 (2017)CrossRefGoogle Scholar
  13. 13.
    Bonaldi, T., Imhof, A., Regula, J.T.: A combination of different mass spectroscopic techniques for the analysis of dynamic changes of histone modifications. Proteomics. 4(5), 1382–1396 (2004)CrossRefGoogle Scholar
  14. 14.
    Sidoli, S., Bhanu, N.V., Karch, K.R., Wang, X., Garcia, B.A.: Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: from histone extraction to data analysis. J. Vis. Exp. (111), e54112 (2016)Google Scholar
  15. 15.
    Janssen, K.A., Sidoli, S., Garcia, B.A.: Recent achievements in characterizing the histone code and approaches to integrating epigenomics and systems biology. Methods Enzymol. 586, 359–378 (2017)CrossRefGoogle Scholar
  16. 16.
    Sidoli, S., Garcia, B.A.: Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies. Methods Mol. Biol. 1528, 121–148 (2017)CrossRefGoogle Scholar
  17. 17.
    Liao, R., Wu, H., Deng, H., Yu, Y., Hu, M., Zhai, H., Yang, P., Zhou, S., Yi, W.: Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS. Anal. Chem. 85(4), 2253–2259 (2013)CrossRefGoogle Scholar
  18. 18.
    Maile, T.M., Izrael-Tomasevic, A., Cheung, T., Guler, G.D., Tindell, C., Masselot, A., Liang, J., Zhao, F., Trojer, P., Classon, M., Arnott, D.: Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method. Mol. Cell. Proteomics. 14(4), 1148–1158 (2015)CrossRefGoogle Scholar
  19. 19.
    Garcia, B.A., Mollah, S., Ueberheide, B.M., Busby, S.A., Muratore, T.L., Shabanowitz, J., Hunt, D.F.: Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat. Protoc. 2(4), 933–938 (2007)CrossRefGoogle Scholar
  20. 20.
    Sidoli, S., Lin, S., Xiong, L., Bhanu, N.V., Karch, K.R., Johansen, E., Hunter, C., Mollah, S., Garcia, B.A.: Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) analysis for characterization and quantification of histone post-translational modifications. Mol. Cell. Proteomics. 14(9), 2420–2428 (2015)CrossRefGoogle Scholar
  21. 21.
    Sidoli, S., Simithy, J., Karch, K.R., Kulej, K., Garcia, B.A.: Low resolution data-independent acquisition in an LTQ-Orbitrap allows for simplified and fully untargeted analysis of histone modifications. Anal. Chem. 87(22), 11448–11454 (2015)CrossRefGoogle Scholar
  22. 22.
    Sidoli, S., Fujiwara, R., Garcia, B.A.: Multiplexed data independent acquisition (MSX-DIA) applied by high resolution mass spectrometry improves quantification quality for the analysis of histone peptides. Proteomics. 16(15–16), 2095–2105 (2016)CrossRefGoogle Scholar
  23. 23.
    Krautkramer, K.A., Reiter, L., Denu, J.M., Dowell, J.A.: Quantification of SAHA-dependent changes in histone modifications using data-independent acquisition mass spectrometry. J. Proteome Res. 14(8), 3252–3262 (2015)CrossRefGoogle Scholar
  24. 24.
    Shi, L., Shi, J., Shi, X., Li, W., Wen, H.: Histone H3.3 G34 mutations alter histone H3K36 and H3K27 methylation in cis. J. Mol. Biol. 430(11), 1562–1565 (2018)CrossRefGoogle Scholar
  25. 25.
    Mao, H., Han, G., Xu, L., Zhu, D., Lin, H., Cao, X., Yu, Y., Chen, C.D.: Cis-existence of H3K27me3 and H3K36me2 in mouse embryonic stem cells revealed by specific ions of isobaric modification chromatogram. Stem Cell Res Ther. 6(1), 132–132 (2015)CrossRefGoogle Scholar
  26. 26.
    Marchione, D.M., Garcia, B.A., Wojcik, J.: Proteomic approaches for cancer epigenetics research. Expert Rev Proteomics. 16(1), 33–47 (2019)CrossRefGoogle Scholar
  27. 27.
    Voigt, P., LeRoy, G., Drury, W.J., Zee, B.M., Son, J., Beck, D.B., Young, N.L., Garcia, B.A., Reinberg, D.: Asymmetrically modified nucleosomes. Cell. 151(1), 181–193 (2012)CrossRefGoogle Scholar
  28. 28.
    Garcia, B.A.: What does the future hold for top down mass spectrometry? J. Am. Soc. Mass Spectrom. 21(2), 193–202 (2010)CrossRefGoogle Scholar
  29. 29.
    Young, N.L., DiMaggio, P.A., Plazas-Mayorca, M.D., Baliban, R.C., Floudas, C.A., Garcia, B.A.: High throughput characterization of combinatorial histone codes. Mol. Cell. Proteomics. 8(10), 2266–2284 (2009)CrossRefGoogle Scholar
  30. 30.
    Shvartsburg, A.A., Zheng, Y., Smith, R.D., Kelleher, N.L.: Ion mobility separation of variant histone tails extending to the “middle-down” range. Anal. Chem. 84(10), 4271–4276 (2012)CrossRefGoogle Scholar
  31. 31.
    Tran, J.C., Zamdborg, L., Ahlf, D.R., Lee, J.E., Catherman, A.D., Durbin, K.R., Tipton, J.D., Vellaichamy, A., Kellie, J.F., Li, M., Wu, C., Sweet, S.M.M., Early, B.P., Siuti, N., LeDuc, R.D., Compton, P.D., Thomas, P.M., Kelleher, N.L.: Mapping intact protein isoforms in discovery mode using top-down proteomics. Nature. 480(7376), 254–U141 (2011)CrossRefGoogle Scholar
  32. 32.
    Shaw, J.B., Li, W., Holden, D.D., Zhang, Y., Griep-Raming, J., Fellers, R.T., Early, B.P., Thomas, P.M., Kelleher, N.L., Brodbelt, J.S.: Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. J. Am. Chem. Soc. 135(34), 12646–12651 (2013)Google Scholar
  33. 33.
    Ansong, C., Wu, S., Meng, D., Liu, X., Brewer, H.M., Kaiser, B.L.D., Nakayasu, E.S., Cort, J.R., Pevzner, P., Smith, R.D., Heffron, F., Adkins, J.N., Paša-Tolić, L.: Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella typhimurium in response to infection-like conditions. Proc. Natl. Acad. Sci. U. S. A. 110(35), 10153–10158 (2013)Google Scholar
  34. 34.
    Sidoli, S., Lin, S., Karch, K.R., Garcia, B.A.: Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry. Anal. Chem. 87(6), 3129–3133 (2015)CrossRefGoogle Scholar
  35. 35.
    Sidoli, S., Lu, C., Coradin, M., Wang, X., Karch, K.R., Ruminowicz, C., Garcia, B.A.: Metabolic labeling in middle-down proteomics allows for investigation of the dynamics of the histone code. Epigenetics Chromatin. 10(1), 34 (2017)CrossRefGoogle Scholar
  36. 36.
    Zamdborg, L., LeDuc, R.D., Glowacz, K.J., Kim, Y.B., Viswanathan, V., Spaulding, I.T., Early, B.P., Bluhm, E.J., Babai, S., Kelleher, N.L.: ProSight PTM 2.0: improved protein identification and characterization for top down mass spectrometry. Nucleic Acids Res. 35(Web Server issue), W701–W706 (2007)CrossRefGoogle Scholar
  37. 37.
    Baliban, R.C., DiMaggio, P.A., Plazas-Mayorca, M.D., Young, N.L., Garcia, B.A., Floudas, C.A.: A novel approach for untargeted post-translational modification identification using integer linear optimization and tandem mass spectrometry. Mol. Cell. Proteomics. 9(5), 764–779 (2010)CrossRefGoogle Scholar
  38. 38.
    Pesavento, J.J., Mizzen, C.A., Kelleher, N.L.: Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4. Anal. Chem. 78(13), 4271–4280 (2006)CrossRefGoogle Scholar
  39. 39.
    DiMaggio, P.A., Young, N.L., Baliban, R.C., Garica, B.A., Floudas, C.A.: A mixed integer linear optimization framework for the identification and quantification of targeted post-translational modifications of highly modified proteins using multiplexed electron transfer dissociation tandem mass spectrometry. Mol. Cell. Proteomics. 8(11), 2527–2543 (2009)CrossRefGoogle Scholar
  40. 40.
    Guan, S.H., Burlingame, A.L.: Data processing algorithms for analysis of high resolution MSMS spectra of peptides with complex patterns of posttranslational modifications. Mol. Cell. Proteomics. 9(5), 804–810 (2010)CrossRefGoogle Scholar
  41. 41.
    Sidoli, S., Garcia, B.A.: Middle-down proteomics: a still unexploited resource for chromatin biology. Expert Rev Proteomics. 14(7), 617–626 (2017)CrossRefGoogle Scholar
  42. 42.
    West, C., Elfakir, C., Lafosse, M.: Porous graphitic carbon: a versatile stationary phase for liquid chromatography. J. Chromatogr. A. 1217(19), 3201–3216 (2010)CrossRefGoogle Scholar
  43. 43.
    Karch, K.R., Sidoli, S., Garcia, B.A.: Identification and quantification of histone PTMs using high-resolution mass spectrometry. In: Marmorstein, R. (ed.) Methods in Enzymology, pp. 3–29. Academic Press, Cambridge (2016)Google Scholar
  44. 44.
    Jung, H.R., Sidoli, S., Haldbo, S., Sprenger, R.R., Schwämmle, V., Pasini, D., Helin, K., Jensen, O.N.: Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS. Anal. Chem. 85(17), 8232–8239 (2013)CrossRefGoogle Scholar
  45. 45.
    Yuan, Z.-F., Sidoli, S., Marchione, D.M., Simithy, J., Janssen, K.A., Szurgot, M.R., Garcia, B.A.: EpiProfile 2.0: a computational platform for processing epi-proteomics mass spectrometry data. J. Proteome Res. 17(7), 2533–2541 (2018)CrossRefGoogle Scholar
  46. 46.
    Greer, S.M., Sidoli, S., Coradin, M., Jespersen, M.S., Schwämmle, V., Jensen, O.N., Garcia, B.A., Brodbelt, J.S.: Extensive characterization of heavily modified histone tails by 193 nm ultraviolet photodissociation mass spectrometry via a middle–down strategy. Anal. Chem. 90(17), 10425–10433 (2018)CrossRefGoogle Scholar
  47. 47.
    Sidoli, S., Schwämmle, V., Ruminowicz, C., Hansen, T.A., Wu, X., Helin, K., Jensen, O.N.: Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones. Proteomics. 14(19), 2200–2211 (2014)CrossRefGoogle Scholar
  48. 48.
    Schräder, C.U., Ziemianowicz, D.S., Merx, K., Schriemer, D.C.: Simultaneous proteoform analysis of histones H3 and H4 with a simplified middle-down proteomics method. Anal. Chem. 90(5), 3083–3090 (2018)CrossRefGoogle Scholar
  49. 49.
    Lin, S., Wein, S., Gonzales-Cope, M., Otte, G.L., Yuan, Z.-F., Afjehi-Sadat, L., Maile, T., Berger, S.L., Rush, J., Lill, J.R., Arnott, D., Garcia, B.A.: Stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry. Mol. Cell. Proteomics. 13(9), 2450 (2014)CrossRefGoogle Scholar
  50. 50.
    Liao, R., Zheng, D., Nie, A., Zhou, S., Deng, H., Gao, Y., Yang, P., Yu, Y., Tan, L., Qi, W., Wu, J., Li, E., Yi, W.: Sensitive and precise characterization of combinatorial histone modifications by selective derivatization coupled with RPLC-EThcD-MS/MS. J. Proteome Res. 16(2), 780–787 (2017)CrossRefGoogle Scholar
  51. 51.
    Garabedian, A., Baird, M.A., Porter, J., Fouque, K.J.D., Shliaha, P.V., Jensen, O.N., Williams, T.D., Fernandez-Lima, F., Shvartsburg, A.A.: Linear and differential ion mobility separations of middle-down proteoforms. Anal. Chem. 90(4), 2918–2925 (2018)CrossRefGoogle Scholar
  52. 52.
    Guo, Q., Sidoli, S., Garcia, B.A., Zhao, X.: Assessment of quantification precision of histone post-translational modifications by using an ion trap and down to 50 000 cells as starting material. J. Proteome Res. 17(1), 234–242 (2018)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  • Kevin A. Janssen
    • 1
    • 2
  • Mariel Coradin
    • 1
    • 2
  • Congcong Lu
    • 2
  • Simone Sidoli
    • 2
    • 3
  • Benjamin A. Garcia
    • 1
    • 2
    Email author
  1. 1.Biochemistry and Molecular Biophysics Graduate Group, Department of Biochemistry and Biophysics, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Epigenetics Institute, Department of Biochemistry and Biophysics, Perelman School of MedicineUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Department of BiochemistryAlbert Einstein College of MedicineNew YorkUSA

Personalised recommendations