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Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Published on: February 27, 2020
An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS
Maria D Person1, Terrence J Monks, Serrine S Lau
1Center for Molecular and Cellular Toxicology, Division of Pharmacology & Toxicology, College of Pharmacy, The University of Texas at Austin, 78712, USA.
This study introduces an unbiased proteomics strategy to identify novel posttranslational modifications and chemical adducts. The method uses mass spectrometry (MS) and tandem mass spectrometry (MS/MS) for accurate characterization of protein modifications.
Area of Science:
- Proteomics
- Chemical Biology
- Mass Spectrometry
Background:
- Identifying novel posttranslational modifications (PTMs) and chemical adducts in proteins is challenging.
- Existing methods often require prior assumptions about modification type or location.
- Understanding chemically induced PTMs is crucial for toxicology and drug development.
Purpose of the Study:
- To develop and demonstrate an unbiased strategy for identifying unknown posttranslational modifications and chemical adducts in proteins.
- To characterize chemically induced modifications using mass spectrometry-based approaches.
- To investigate the formation of novel adducts and the influence of protein structure on these reactions.
Main Methods:
- Comparative matrix-assisted laser/desorption ionization (MALDI) MS of protein digests.
- Targeted HPLC-electrospray ionization (ESI)-MS/MS for peptide fragmentation.
- Manual de novo sequencing for determining amino acid sequence and modification type.
- Analysis of histone H4 and cytochrome c for chemically induced modifications.
Main Results:
- Successfully identified methylation and acetylation sites on histone H4.
- Characterized a novel cyclized diquinone adduct on cytochrome c formed by benzoquinone.
- Demonstrated the method's efficacy in detecting unpredicted chemical adducts and understanding their formation.
- Highlighted the influence of protein structure on chemical adduction.
Conclusions:
- The developed unbiased strategy effectively identifies novel PTMs and chemical adducts without prior assumptions.
- This approach is particularly valuable for studying modifications induced by exogenous chemicals.
- Characterization of adducts like the cyclized diquinone species provides insights into chemical-protein interactions and their structural determinants.
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