Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis-Top-Down Mass Spectrometry

Kevin Jooß, Luis F. Schachner, Rachel Watson, Zachary B. Gillespie, Sarah A. Howard, Marcus A. Cheek, Matthew J. Meiners, Amin Sobh, Jonathan D. Licht, Michael-Christopher Keogh, Neil L. Kelleher

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

We report a novel platform [native capillary zone electrophoresis-top-down mass spectrometry (nCZE-TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ∼200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE-TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ∼3 orders of magnitude. The nCZE-TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE-TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.
Original languageEnglish
Pages (from-to)5151-5160
JournalAnalytical chemistry
Volume93
Issue number12
DOIs
Publication statusPublished - 30 Mar 2021
Externally publishedYes

Funding

This work was supported by the National Institute of General Medical Sciences P41GM108569 for the National Resource for Translational and Developmental Proteomics at Northwestern University and NIH Grants R01AM115739 (Kelleher lab) and R01CA195732 (Licht Lab), RF1AG063903 from the National Institute on Aging (Kelleher lab), P30DA018310 from the National Institute on Drug Abuse (Kelleher lab), a Leukemia and Lymphoma Society Specialized Center of Excellence Grant (Licht Lab), NIH Grants R44GM116584 and R44CA212733 (EpiCypher), and an AACR Myeloma Fellowship (A.S.). Bottom-up (histone) analysis was performed by the Northwestern Proteomics Core Facility, supported by NCI CCSG P30CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center, instrumentation award (S10OD025194) from NIH Office of Director, and the National Resource for Translational and Developmental Proteomics supported by P41GM108569. L.F.S. is a Gilliam Fellow of the Howard Hughes Medical Institute. Research in this publication is also supported by Thermo Fisher Scientific and a fellowship associated with the Chemistry of Life Processes Predoctoral Training Grant T32GM105538 at Northwestern University. We thank SCIEX for their support including Dr. Fang Wang for the valuable discussions and insightful suggestions throughout this research project.

FundersFunder number
National Resource for Translational and Developmental ProteomicsT32GM105538
National Institutes of HealthR01AM115739, R01CA195732, RF1AG063903
National Institute on Drug AbuseR44CA212733, R44GM116584
American Association for Cancer Research
National Institute on AgingP30DA018310
National Cancer InstituteS10OD025194, P30CA060553
National Institute of General Medical SciencesP41GM108569
Northwestern University

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