Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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


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.

This is a preview of subscription content, log in to check access.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6


  1. 1.

    Strahl, B.D., Allis, C.D.: The language of covalent histone modifications. Nature. 403(6765), 41–45 (2000)

  2. 2.

    Jenuwein, T., Allis, C.D.: Translating the histone code. Science. 293(5532), 1074–1080 (2001)

  3. 3.

    Kouzarides, T.: Chromatin modifications and their function. Cell. 128(4), 693–705 (2007)

  4. 4.

    Bannister, A.J., Kouzarides, T.: Regulation of chromatin by histone modifications. Cell Res. 21(3), 381–395 (2011)

  5. 5.

    Rossetto, D., Avvakumov, N., Côté, J.: Histone phosphorylation. Epigenetics. 7(10), 1098–1108 (2012)

  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)

  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)

  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)

  9. 9.

    Fischle, W., Wang, Y., Allis, C.D.: Histone and chromatin cross-talk. Curr. Opin. Cell Biol. 15(2), 172–183 (2003)

  10. 10.

    Lee, J.S., Smith, E., Shilatifard, A.: The language of histone crosstalk. Cell. 142(5), 682–685 (2010)

  11. 11.

    Hunter, T.: The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol. Cell. 28(5), 730–738 (2007)

  12. 12.

    Atlasi, Y., Stunnenberg, H.G.: The interplay of epigenetic marks during stem cell differentiation and development. Nat. Rev. Genet. 18, 643 (2017)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  26. 26.

    Marchione, D.M., Garcia, B.A., Wojcik, J.: Proteomic approaches for cancer epigenetics research. Expert Rev Proteomics. 16(1), 33–47 (2019)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

  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)

Download references


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

Author information

Correspondence to Benjamin A. Garcia.

Electronic Supplementary Material


(DOCX 1233 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Janssen, K.A., Coradin, M., Lu, C. et al. Quantitation of Single and Combinatorial Histone Modifications by Integrated Chromatography of Bottom-up Peptides and Middle-down Polypeptide Tails. J. Am. Soc. Mass Spectrom. 30, 2449–2459 (2019). https://doi.org/10.1007/s13361-019-02303-6

Download citation


  • Histones
  • Epigenetics
  • Proteomics
  • Middle-down
  • PTMs
  • Chromatography