Protein Turnover Dynamics Analysis With Subcellular Spatial Resolution

Area of Science:

• Cell Biology
• Proteomics
• Biochemistry

Background:

• Protein homeostasis, involving synthesis and degradation, is vital for cellular function and implicated in aging.
• Existing methods for studying protein turnover in subcellular compartments are often invasive or limited in scope.
• Understanding protein dynamics within specific cellular locations is essential for disease research.

Purpose of the Study:

• To develop a novel protocol for simultaneous measurement of proteome-wide protein turnover rates and subcellular localization.
• To provide an unbiased approach for assessing compartment-specific protein dynamics.
• To enable detailed analysis of protein spatial and temporal distributions.

Main Methods:

• Simultaneous Proteome Localization and Turnover (SPLAT) analysis.
• Dynamic stable isotope labeling of amino acids in cell culture (dynamic SILAC) for temporal resolution.
• Multi-step differential ultracentrifugation for subcellular localization.
• 2D liquid chromatography fractionation and tandem mass tags (TMT) for enhanced depth and reduced acquisition time.

Main Results:

• SPLAT analysis successfully measures protein turnover rates and subcellular localization simultaneously.
• The protocol provides proteome-wide, compartment-specific turnover rates.
• It reveals both spatial and temporal distributions of proteins, including temporally distinct localizations within protein pools.
• Hyperplexing of dynamic SILAC and TMT LOPIT-DC enhances data acquisition and depth.

Conclusions:

• SPLAT analysis offers a powerful, unbiased tool for studying protein dynamics across subcellular compartments.
• This method significantly advances the ability to compare organellar proteome turnover rates.
• The protocol facilitates a deeper understanding of protein homeostasis and its role in age-associated diseases.