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Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

作者： Meiby Fernandez-Rojas1, Cassandra N. Fuller1, Lilian Valadares Tose1, Matthew Willetts2, Melvin A. Park2, Natarajan V. Bhanu3, Benjamin A. Garcia3, Francisco Fernandez-Lima1,4 1Department of Chemistry and Biochemistry,Florida International University, 2Bruker Daltonics Inc., 3Department of Biochemistry and Molecular Biophysics,Washington University School of Medicine, 4Biomolecular Sciences Institute,Florida International University
简介：

Overview

This study presents an analytical workflow utilizing liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for the high-confidence analysis of histone modifications. The method significantly enhances the identification of post-translational modifications (PTMs) and their positional isomers.

Key Study Components

Area of Science

  • Neuroscience
  • Biochemistry
  • Epigenetics

Background

  • Histones play a crucial role in gene regulation through various modifications.
  • Identifying these modifications is essential for understanding epigenetic mechanisms.
  • Traditional methods face challenges in separating isomers.
  • Incorporating TIMS offers a new dimension for improved separation.

Purpose of Study

  • To develop a method for efficient histone extraction and analysis.
  • To enhance the identification of PTMs and their isomers.
  • To correlate environmental factors with epigenetic variations.

Methods Used

  • Histone extraction protocol achieving 95% efficiency.
  • LC-TIMS-ToF MS/MS for high-resolution analysis.
  • Passive technology to increase sensitivity in experiments.
  • Screening for PTMs present in histones.

Main Results

  • Successful separation of isomer peptides using TIMS.
  • Improved identification of PTM positions.
  • Enhanced ability to analyze epigenetic variations.
  • Potential correlations between biological factors and the epigenome.

Conclusions

  • The proposed method significantly improves the analysis of histone modifications.
  • Incorporating TIMS simplifies the identification of isomers.
  • This approach opens avenues for further research into epigenetic influences.

Frequently Asked Questions

What is LC-TIMS-ToF MS/MS?
It is an analytical technique combining liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry for detailed molecular analysis.
How does TIMS improve histone analysis?
TIMS adds an extra dimension of separation, enhancing the ability to distinguish between isomeric peptides.
What are post-translational modifications?
PTMs are chemical modifications that occur on proteins after translation, affecting their function and activity.
Why is histone modification important?
Histone modifications play a key role in regulating gene expression and maintaining cellular functions.
What potential applications does this method have?
This method can be used to study epigenetic changes in response to environmental factors and disease states.

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

标签: Histone Modification,    Liquid Chromatography,    Trapped Ion Mobility Spectrometry,    Time-of-flight Mass Spectrometry,    Posttranslational Modifications,    PTM Identification,    Peptide Separation,    Epigenetic Variations,    High-throughput Analysis,    Biological Samples,    Protein Confirmation,    Gene Regulation,    Data Acquisition,    Retention Time,    Fragmentation Analysis,   
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