Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational...

Area of Science:

• Cell Biology
• Biochemistry
• Proteomics

Background:

• Intermediate filaments (IFs) are crucial cytoskeletal components providing mechanical resilience.
• Dysfunctional IFs are linked to human diseases, and their regulation by post-translational modifications (PTMs) is vital for cellular stress responses.
• The diversity of IF proteins and tissue-specific expression pose challenges in studying PTMs.

Purpose of the Study:

• To develop and optimize biochemical methods for isolating IF proteins and their PTMs from various mouse tissues.
• To enable organism-wide monitoring of IF PTM signatures for a better understanding of their role as stress responders.
• To investigate age-related changes in IF expression levels and PTMs in different mouse tissues.

Main Methods:

• Biochemical isolation of total, detergent-soluble, and detergent-resistant IF protein fractions from nine mouse tissues.
• Optimized protocol using lysing matrix and automated homogenization for rapid IF protein isolation.
• Downstream analysis including mass spectrometry-based PTM profiling.

Main Results:

• Successful isolation and profiling of IF proteins and their PTMs from diverse mouse tissues.
• Demonstration of an automated, high-throughput method for IF analysis.
• New data showing parallel changes in IF protein expression levels and PTMs in mouse brain and liver during aging.

Conclusions:

• The developed methods facilitate comprehensive analysis of IFs and their PTMs across multiple tissues and experimental conditions.
• These techniques are valuable for studying IFs in disease models and aging research.
• Age-related changes in IFs suggest a role in the aging process and cellular stress response.