Coupling Fluorescence-Activated Cell Sorting and Targeted Analysis of Histone Modification Profiles in Primary Human Leukocytes

  • Jeannie M. Camarillo
  • Suchitra Swaminathan
  • Nebiyu A. Abshiru
  • Jacek W. Sikora
  • Paul M. ThomasEmail author
  • Neil L. KelleherEmail author
Focus: Protein Post-translational Modifications: Research Article


Histone posttranslational modifications (PTMs) are essential for regulating chromatin and maintaining gene expression throughout cell differentiation. Despite the deep level of understanding of immunophenotypic differentiation pathways in hematopoietic cells, few studies have investigated global levels of histone PTMs required for differentiation and maintenance of these distinct cell types. Here, we describe an approach to couple fluorescence-activated cell sorting (FACS) with targeted mass spectrometry to define global “epi-proteomic” signatures for primary leukocytes. FACS was used to sort closely and distantly related leukocytes from normal human peripheral blood for quantitation of histone PTMs with a multiple reaction monitoring LC-MS/MS method measuring histone PTMs on histones H3 and H4. We validate cell sorting directly into H2SO4 for immediate histone extraction to decrease time and number of steps after FACS to analyze histone PTMs. Relative histone PTM levels vary in T cells across healthy donors, and the majority of PTMs remain stable up to 2 days following initial blood draw. Large differences in the levels of histone PTMs are observed across the mature lymphoid and myeloid lineages, as well as between different types within the same lineage, though no differences are observed in closely related T cell subtypes. The results show a streamlined approach for quantifying global changes in histone PTMs in cell types separated by FACS that is poised for clinical deployment.


Histones Posttranslation modifications Fluorescence-activated cell sorting Proteomics Targeted LC-MS/MS 



This work was carried out with a financial support from The Paul G. Allen Family Foundation (Award #11715), NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, and the National Resource for Translational and Developmental Proteomics supported by P41 GM108569.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Statement of Human Rights

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

13361_2019_2255_MOESM1_ESM.xlsx (20 kb)
ESM 1 (XLSX 20 kb)


  1. 1.
    Seumois, G., Chavez, L., Gerasimova, A., Lienhard, M., Omran, N., Kalinke, L., Vedanayagam, M., Ganesan, A.P.V., Chawla, A., Djukanović, R., Ansel, K.M., Peters, B., Rao, A., Vijayanand, P.: Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility. Nat. Immunol. 15, 777 (2014)CrossRefGoogle Scholar
  2. 2.
    Poropatich, K., Fontanarosa, J., Swaminathan, S., Dittmann, D., Chen, S., Samant, S., Zhang, B.: Comprehensive T-cell immunophenotyping and next-generation sequencing of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinomas. J. Pathol. 243, 354–365 (2017)CrossRefGoogle Scholar
  3. 3.
    Mikkelsen, T.S., Ku, M., Jaffe, D.B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T.K., Koche, R.P., Lee, W., Mendenhall, E., O'Donovan, A., Presser, A., Russ, C., Xie, X., Meissner, A., Wernig, M., Jaenisch, R., Nusbaum, C., Lander, E.S., Bernstein, B.E.: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 448, 553–560 (2007)CrossRefGoogle Scholar
  4. 4.
    Terskikh, A.V., Miyamoto, T., Chang, C., Diatchenko, L., Weissman, I.L.: Gene expression analysis of purified hematopoietic stem cells and committed progenitors. Blood. 102, 94–101 (2003)CrossRefGoogle Scholar
  5. 5.
    Hystad, M.E., Myklebust, J.H., Bo, T.H., Sivertsen, E.A., Rian, E., Forfang, L., Munthe, E., Rosenwald, A., Chiorazzi, M., Jonassen, I., Staudt, L.M., Smeland, E.B.: Characterization of early stages of human B cell development by gene expression profiling. J. Immunol. 179, 3662–3671 (2007)CrossRefGoogle Scholar
  6. 6.
    Durek, P., Nordström, K., Gasparoni, G., Salhab, A., Kressler, C., de Almeida, M., Bassler, K., Ulas, T., Schmidt, F., Xiong, J., Glažar, P., Klironomos, F., Sinha, A., Kinkley, S., Yang, X., Arrigoni, L., Amirabad, A.D., Ardakani, F.B., Feuerbach, L., Gorka, O., Ebert, P., Müller, F., Li, N., Frischbutter, S., Schlickeiser, S., Cendon, C., Fröhler, S., Felder, B., Gasparoni, N., Imbusch, C.D., Hutter, B., Zipprich, G., Tauchmann, Y., Reinke, S., Wassilew, G., Hoffmann, U., Richter, A.S., Sieverling, L., Chang, H.-D., Syrbe, U., Kalus, U., Eils, J., Brors, B., Manke, T., Ruland, J., Lengauer, T., Rajewsky, N., Chen, W., Dong, J., Sawitzki, B., Chung, H.-R., Rosenstiel, P., Schulz, M.H., Schultze, J.L., Radbruch, A., Walter, J., Hamann, A., Polansky, J.K.: Epigenomic profiling of human CD4+ T cells supports a linear differentiation model and highlights molecular regulators of memory development. Immunity. 45, 1148–1161 (2016)CrossRefGoogle Scholar
  7. 7.
    Avni, O., Lee, D., Macian, F., Szabo, S.J., Glimcher, L.H., Rao, A.: T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat. Immunol. 3, 643–651 (2002)CrossRefGoogle Scholar
  8. 8.
    Fields, P.E., Kim, S.T., Flavell, R.A.: Cutting edge: changes in histone acetylation at the IL-4 and IFN-gamma loci accompany Th1/Th2 differentiation. J. Immunol. 169, 647–650 (2002)CrossRefGoogle Scholar
  9. 9.
    Ono, C., Yu, Z., Kasahara, Y., Kikuchi, Y., Ishii, N., Tomita, H.: Fluorescently activated cell sorting followed by microarray profiling of helper T cell subtypes from human peripheral blood. PLoS One. 9, e111405 (2014)CrossRefGoogle Scholar
  10. 10.
    Wei, G., Wei, L., Zhu, J., Zang, C., Hu-Li, J., Yao, Z., Cui, K., Kanno, Y., Roh, T.-Y., Watford, W.T., Schones, D.E., Peng, W., Sun, H.-w., Paul, W.E., O'Shea, J.J., Zhao, K.: Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells. Immunity. 30, 155–167 (2009)CrossRefGoogle Scholar
  11. 11.
    Stein, E.M., Garcia-Manero, G., Rizzieri, D.A., Tibes, R., Berdeja, J.G., Savona, M.R., Jongen-Lavrenic, M., Altman, J.K., Thomson, B., Blakemore, S.J., Daigle, S.R., Waters, N.J., Suttle, A.B., Clawson, A., Pollock, R., Krivtsov, A., Armstrong, S.A., DiMartino, J., Hedrick, E., Lowenberg, B., Tallman, M.S.: The DOT1L inhibitor pinometostat reduces H3K79 methylation and has modest clinical activity in adult acute leukemia. Blood. 131, 2661–2669 (2018)CrossRefGoogle Scholar
  12. 12.
    Kurmasheva, R.T., Sammons, M., Favours, E., Wu, J., Kurmashev, D., Cosmopoulos, K., Keilhack, H., Klaus, C.R., Houghton, P.J., Smith, M.A.: Initial testing (stage 1) of tazemetostat (EPZ-6438), a novel EZH2 inhibitor, by the Pediatric Preclinical Testing Program. Pediatr. Blood Cancer. 64, e26218 (2017)Google Scholar
  13. 13.
    Italiano, A., Soria, J.C., Toulmonde, M., Michot, J.M., Lucchesi, C., Varga, A., Coindre, J.M., Blakemore, S.J., Clawson, A., Suttle, B., McDonald, A.A., Woodruff, M., Ribich, S., Hedrick, E., Keilhack, H., Thomson, B., Owa, T., Copeland, R.A., Ho, P.T.C., Ribrag, V.: Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open-label, phase 1 study. Lancet Oncol. 19, 649–659 (2018)CrossRefGoogle Scholar
  14. 14.
    Peach, S.E., Rudomin, E.L., Udeshi, N.D., Carr, S.A., Jaffe, J.D.: Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules. Mol. Cell. Proteomics. 11, 128–137 (2012)CrossRefGoogle Scholar
  15. 15.
    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, 933–938 (2007)CrossRefGoogle Scholar
  16. 16.
    Zheng, Y., Sweet, S.M., Popovic, R., Martinez-Garcia, E., Tipton, J.D., Thomas, P.M., Licht, J.D., Kelleher, N.L.: Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3. Proc. Natl. Acad. Sci. U. S. A. 109, 13549–13554 (2012)CrossRefGoogle Scholar
  17. 17.
    Zheng, Y., Thomas, P.M., Kelleher, N.L.: Measurement of acetylation turnover at distinct lysines in human histones identifies long-lived acetylation sites. Nat. Commun. 4, 2203 (2013)CrossRefGoogle Scholar
  18. 18.
    MacLean, B., Tomazela, D.M., Shulman, N., Chambers, M., Finney, G.L., Frewen, B., Kern, R., Tabb, D.L., Liebler, D.C., MacCoss, M.J.: Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 26, 966–968 (2010)CrossRefGoogle Scholar
  19. 19.
    Bunkenborg, J., Garcia, G.E., Paz, M.I., Andersen, J.S., Molina, H.: The minotaur proteome: avoiding cross-species identifications deriving from bovine serum in cell culture models. Proteomics. 10, 3040–3044 (2010)CrossRefGoogle Scholar
  20. 20.
    Zee, B.M., Levin, R.S., DiMaggio, P.A., Garcia, B.A.: Global turnover of histone post-translational modifications and variants in human cells. Epigenetics Chromatin. 3, 22 (2010)CrossRefGoogle Scholar
  21. 21.
    Pesavento, J.J., Bullock, C.R., LeDuc, R.D., Mizzen, C.A., Kelleher, N.L.: Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry. J. Biol. Chem. 283, 14927–14937 (2008)CrossRefGoogle Scholar
  22. 22.
    Whitney, A.R., Diehn, M., Popper, S.J., Alizadeh, A.A., Boldrick, J.C., Relman, D.A., Brown, P.O.: Individuality and variation in gene expression patterns in human blood. Proc. Natl. Acad. Sci. U. S. A. 100, 1896–1901 (2003)CrossRefGoogle Scholar
  23. 23.
    Beguelin, W., Popovic, R., Teater, M., Jiang, Y., Bunting, K.L., Rosen, M., Shen, H., Yang, S.N., Wang, L., Ezponda, T., Martinez-Garcia, E., Zhang, H., Zheng, Y., Verma, S.K., McCabe, M.T., Ott, H.M., Van Aller, G.S., Kruger, R.G., Liu, Y., McHugh, C.F., Scott, D.W., Chung, Y.R., Kelleher, N., Shaknovich, R., Creasy, C.L., Gascoyne, R.D., Wong, K.K., Cerchietti, L., Levine, R.L., Abdel-Wahab, O., Licht, J.D., Elemento, O., Melnick, A.M.: EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell. 23, 677–692 (2013)CrossRefGoogle Scholar
  24. 24.
    Martinez-Garcia, E., Popovic, R., Min, D.-J., Sweet, S.M.M., Thomas, P.M., Zamdborg, L., Heffner, A., Will, C., Lamy, L., Staudt, L.M., Levens, D.L., Kelleher, N.L., Licht, J.D.: The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells. Blood. 117, 211–220 (2011)CrossRefGoogle Scholar
  25. 25.
    Oyer, J.A., Huang, X., Zheng, Y., Shim, J., Ezponda, T., Carpenter, Z., Allegretta, M., Okot-Kotber, C.I., Patel, J.P., Melnick, A., Levine, R.L., Ferrando, A., Mackerell Jr., A.D., Kelleher, N.L., Licht, J.D., Popovic, R.: Point mutation E1099K in MMSET/NSD2 enhances its methyltranferase activity and leads to altered global chromatin methylation in lymphoid malignancies. Leukemia. 28, 198–201 (2014)CrossRefGoogle Scholar
  26. 26.
    Souroullas, G.P., Jeck, W.R., Parker, J.S., Simon, J.M., Liu, J.Y., Paulk, J., Xiong, J., Clark, K.S., Fedoriw, Y., Qi, J., Burd, C.E., Bradner, J.E., Sharpless, N.E.: An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632–640 (2016)CrossRefGoogle Scholar
  27. 27.
    Elsaesser, S.J., Goldberg, A.D., Allis, C.D.: New functions for an old variant: no substitute for histone H3.3. Curr. Opin. Genet. Dev. 20, 110–117 (2010)CrossRefGoogle Scholar
  28. 28.
    Sweet, S.M., Li, M., Thomas, P.M., Durbin, K.R., Kelleher, N.L.: Kinetics of re-establishing H3K79 methylation marks in global human chromatin. J. Biol. Chem. 285, 32778–32786 (2010)CrossRefGoogle Scholar
  29. 29.
    Ansel, K.M., Lee, D.U., Rao, A.: An epigenetic view of helper T cell differentiation. Nat. Immunol. 4, 616–623 (2003)CrossRefGoogle Scholar
  30. 30.
    Creyghton, M.P., Cheng, A.W., Welstead, G.G., Kooistra, T., Carey, B.W., Steine, E.J., Hanna, J., Lodato, M.A., Frampton, G.M., Sharp, P.A., Boyer, L.A., Young, R.A., Jaenisch, R.: Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. U. S. A. 107, 21931–21936 (2010)CrossRefGoogle Scholar
  31. 31.
    Li, B., Carey, M., Workman, J.L.: The role of chromatin during transcription. Cell. 128, 707–719 (2007)CrossRefGoogle Scholar
  32. 32.
    Schuettengruber, B., Chourrout, D., Vervoort, M., Leblanc, B., Cavalli, G.: Genome regulation by polycomb and trithorax proteins. Cell. 128, 735–745 (2007)CrossRefGoogle Scholar
  33. 33.
    Schmidl, C., Renner, K., Peter, K., Eder, R., Lassmann, T., Balwierz, P.J., Itoh, M., Nagao-Sato, S., Kawaji, H., Carninci, P., Suzuki, H., Hayashizaki, Y., Andreesen, R., Hume, D.A., Hoffmann, P., Forrest, A.R.R., Kreutz, M.P., Edinger, M., Rehli, M.: Transcription and enhancer profiling in human monocyte subsets. Blood. 123, e90–e99 (2014)CrossRefGoogle Scholar
  34. 34.
    Barski, A., Cuddapah, S., Cui, K., Roh, T.-Y., Schones, D.E., Wang, Z., Wei, G., Chepelev, I., Zhao, K.: High-resolution profiling of histone methylations in the human genome. Cell. 129, 823–837 (2007)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of Chemistry, Molecular Biosciences and the National Resource for Translational and Developmental ProteomicsNorthwestern UniversityEvanstonUSA
  2. 2.Department of Medicine, Division of RheumatologyNorthwestern University Feinberg School of MedicineChicagoUSA

Personalised recommendations