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Pervasive Charge Solvation Permeates Native-like Protein Ions and Dramatically Influences Top-down Sequencing Data
Daniel A Polasky1, Sugyan M Dixit1, Michael F Keating1
1Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
Journal of the American Chemical Society
|March 24, 2020
Summary
Labeling proteins with trimethyl pyrylium (TMP) modifies fragmentation pathways for improved top-down sequencing. This method enhances proteomic analysis by controlling charge and improving sequence coverage for intact protein analysis.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biochemistry
Background:
- Post-translational modifications generate diverse proteoforms, complicating disease-related proteomic analysis.
- Top-down sequencing of intact proteins is advantageous but faces challenges in achieving complete fragmentation.
Purpose of the Study:
- To develop novel strategies for enhanced fragmentation of intact proteins for comprehensive top-down sequencing.
- To investigate the impact of chemical labeling on protein ion fragmentation pathways.
Main Methods:
- Proteins were labeled with trimethyl pyrylium (TMP) at lysine residues.
- Acidic side chains were capped using carbodiimide chemistry.
- Fragmentation pathways were analyzed using mass spectrometry, supported by molecular dynamics simulations.
Main Results:
- TMP labeling shifts fragmentation from charge-directed to charge-remote pathways by reducing mobile protons.
- Capping acidic residues restores charge-directed fragmentation, leading to more even sequence coverage.
- Fragmentation can be directed almost exclusively to TMP-labeled lysine residues.
- Intact protein ions exhibit a high capacity for charge solvation, involving structural changes like helix formation.
Conclusions:
- Chemical modification of intact proteins offers a powerful approach to control fragmentation for top-down sequencing.
- Understanding charge solvation in gas-phase protein ions is crucial for advancing comprehensive protein analysis.
- These findings provide insights into the physical forces influencing top-down sequencing efficiency.
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