TcellSubC

TcellSubC maps the subcellular proteome of primary human CD4+ T cells using optimized fractionation and high-resolution isoelectric focusing coupled with liquid chromatography-tandem mass spectrometry (HiRIEF-LC-MS/MS) to quantify protein localization and translocation across cytosolic, nuclear, and membrane fractions under steady-state conditions and following T cell receptor (TCR) stimulation.

Key Features:

• HiRIEF-LC-MS/MS workflow: Uses high-resolution isoelectric focusing coupled with liquid chromatography-tandem mass spectrometry optimized for limited amounts of primary cells.
• Optimized fractionation: Separates proteins into cytosolic, nuclear, and membrane fractions for subcellular assignment.
• Quantitative coverage: Quantifies the distribution of 6,572 proteins across subcellular compartments.
• Temporal profiling: Captures proteome states at steady-state and after TCR stimulation at 15 minutes and 1 hour.
• Translocation detection: Identifies a subset of 237 potentially translocating proteins between compartments.
• Microscopic validation: Confirms localization of selected proteins by microscopy.

Scientific Applications:

• T cell signaling and trafficking: Enables analysis of protein localization changes and translocation events in response to TCR stimulation.
• Cell biology of CD4+ T cells: Provides a subcellular proteome atlas for investigating compartment-specific protein functions in primary human CD4+ T cells.
• Biomarker and target discovery: Supports identification of proteins with dynamic localization that may serve as biomarkers or therapeutic targets in T cell–related research.

Methodology:

Optimized fractionation and high-resolution isoelectric focusing coupled with liquid chromatography-tandem mass spectrometry (HiRIEF-LC-MS/MS) were used to generate and quantify subcellular proteomes at steady-state and at 15 minutes and 1 hour after TCR stimulation, with selected localizations validated by microscopy.