Coupling Fluorescence‐Activated Cell Sorting and Targeted LC‐MS/MS for Epi‐Proteomic Analysis of Normal Leukocytes

Abstract

Fluorescence‐activated cell sorting (FACS) is widely used to isolate subtypes of various immune cells from human blood. Most recently, these subpopulations have been subjected to next generation sequencing to elucidate changes in gene expression associated with differentiation. Histone modifications are essential for regulating chromatin and maintaining gene expression, and few studies have investigated global alterations in histone modifications required for differentiation. Here, we describe an approach for integrating FACS and targeted mass spectrometry to define a global “epiproteomic” signature for white blood cells from normal individuals. Using our multiple‐reaction monitoring LC‐MS/MS method, which can measure 90+ histone modifications on all core histones, we assessed changes in histone modifications from different collection methods and cell types. First, collection buffer conditions were optimized to allow for sorting directly into histone extraction buffer, thereby reducing the likelihood of changes in histone modifications that could occur during long sorts. Next, we sorted neutrophils, monocytes, B cells, and T cells and identified differences in histones modifications across these broad cell types. Finally, FACS was used to sort naïve, TH1, and TH2 T cells from human peripheral blood for histone analysis, revealing little differences in histone marks in these closely‐related subtypes. This method allows for interrogation of global changes in histone modifications in distinct cell types, defining an “epi‐proteomic” signature that can be used to more fully understand differentiation in hematopoiesis and disease development.Support or Funding InformationThis work was carried out with financial support from the National Resource for Translational and Developmental Proteomics (P41GM108569).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.