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

Meiby Fernandez-Rojas; Cassandra N. Fuller; Lilian Valadares Tose; Matthew Willetts; Melvin A. Park; Natarajan V. Bhanu; Benjamin A. Garcia; Francisco Fernandez-Lima

doi:10.3791/65589

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

JoVE Journal
Chemistry

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JoVE Journal Chemistry

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

999 Views

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05:52 min

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January 12, 2024

DOI:

10.3791/65589-v

DOI:

10.3791/65589-v

•

05:52 min

January 12, 2024

•

16 Views

Meiby Fernandez-Rojas¹, Cassandra N. Fuller¹, Lilian Valadares Tose¹, Matthew Willetts², Melvin A. Park², Natarajan V. Bhanu³, Benjamin A. Garcia³, Francisco Fernandez-Lima^1,4

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

Transcript

A method is proposed using histone extraction protocol with 95%efficiency. Together with LC-TIMS-ToF MS/MS, in a short time, it is possible to obtain a screening for those PTMs present in the histone. And above all, the possibility of separating isomers.

The separation of isomeric peptides though possible using tandem mass spectrometry alone is often difficult. By incorporating TIMS, we introduce an extra dimension of separation, simplifying many positional isomer identifications. In addition, passive technology increases the sensitivity of these experiments, further improving our ability to analyze epigenetic variations.

With the increased ability to identify PTM positions, there comes the ability to further determine the correlation between various environmental or biological factors, and their effects on the epigenome. Methods that improve and facilitate analysis of histones can be applied to several research areas, including medical and environmental chemistry. The dynamics of protein confirmation in native or damaged states will be established from the implementation of TIMS MS/MS with HDX.

Through LC-TIMS-ToF MS/MS and Nano LC-TIMS-ToF MS/MS, the differences in PTM existing in various biological sample will be charted.

Summary

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).