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Related Experiment Video

Updated: Jan 3, 2026

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High-Throughput Quantitative Top-Down Proteomics: Histone H4.

Matthew V Holt1, Tao Wang1, Nicolas L Young2,3

  • 1Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine, Houston, TX, USA.

Journal of the American Society for Mass Spectrometry
|November 20, 2019
PubMed
Summary
This summary is machine-generated.

This study presents a quantitative top-down proteomics method for analyzing protein isoforms (proteoforms) with high throughput and sensitivity. The new method accurately quantifies hundreds of histone H4 proteoforms, revealing biological changes in cells.

Keywords:
Dynamics of histone modificationsEpigenetic inhibitorHistone post-translational modificationsHistone proteoformsTop-down proteomics

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Area of Science:

  • Proteomics
  • Molecular Biology
  • Biochemistry

Background:

  • Proteins exist as diverse proteoforms, generated by post-translational modifications (PTMs).
  • Characterizing proteoforms with multiple PTMs is challenging for current proteomics methods.
  • Existing top-down proteomics techniques often lack the quantitative capacity and throughput needed for comprehensive proteoform biology.

Purpose of the Study:

  • To develop and validate a quantitative, reproducible, sensitive, and high-throughput top-down proteomics method.
  • To enable detailed analysis of complex proteoform landscapes, specifically for histone H4.
  • To demonstrate the method's capability in measuring biologically relevant changes in proteoform abundance.

Main Methods:

  • Optimized protein purification from cells or tissues.
  • On-line C3 chromatography for physical separation of proteoforms.
  • Mass spectrometry (MS1 isolation) with Electron Transfer Dissociation (ETD) fragmentation for sequencing.

Main Results:

  • Achieved high throughput (12 replicates/day) and reproducibility (Pearson correlations of 0.89).
  • Quantified hundreds of proteoforms (200-300) across six orders of magnitude with a low limit of quantification (0.001%).
  • Successfully measured known changes in histone H4 proteoforms upon sodium butyrate treatment in SUM159 cells.

Conclusions:

  • Top-down proteomics can be made quantitative, reproducible, sensitive, and high-throughput.
  • The developed method provides a powerful tool for studying proteoform biology and delineating biological changes at the proteoform level.
  • This approach facilitates parametric statistical comparisons of proteoform data, advancing our understanding of cellular regulation.