Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms

Area of Science:

• Proteomics
• Epigenetics
• Biochemistry

Background:

• Post-translational modifications (PTMs) on proteins, especially histones, regulate gene expression and chromatin structure.
• The combinatorial patterns of histone PTMs, known as the "histone code," are critical for cellular function and have significant health implications.
• Identifying and distinguishing between various histone proteoforms and their isomers, which differ in PTM positions, is a major challenge in proteomics.

Purpose of the Study:

• To develop and apply advanced analytical methods for resolving complex histone proteoforms and their isomers.
• To advance the capability of ion mobility spectrometry (IMS) methods for analyzing histone isomers in biological samples.
• To decipher the intricate "histone code" by accurately identifying PTM connectivity in histone proteins.

Main Methods:

• Middle-down analysis of histones from mouse embryonic stem cells.
• Online liquid chromatography for fractionation of proteoforms with distinct PTM sets.
• Differential or Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) for resolving isomeric PTMs.
• Orbitrap mass spectrometry coupled with electron transfer dissociation (ETD) for species identification.

Main Results:

• Demonstrated the capability of IMS methods to resolve histone isomers from model histone tails.
• Successfully applied middle-down analysis to resolve PTM isomers in histones from biological samples (mouse embryonic stem cells).
• Achieved high-resolution separation and identification of complex histone proteoforms, preserving PTM connectivity.

Conclusions:

• The developed analytical workflow enables the resolution of complex histone proteoforms and isomers, overcoming limitations of previous separation techniques.
• This advancement provides a powerful tool for dissecting the "histone code" in biological systems.
• The findings have significant implications for understanding gene regulation, chromatin dynamics, and diseases associated with aberrant histone modifications.