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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

Abstract

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.

Keywords

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

Notes

Acknowledgements

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)

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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

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