Profiling Proteins and Phosphorylation Sites During T Cell Activation Using an Integrated Thermal Shift Assay

Area of Science:

• Immunology
• Proteomics
• Cellular Biology

Background:

• T cell activation is crucial for adaptive immunity, involving naive T cells differentiating into effector cells.
• Understanding the functional significance of protein and phosphorylation changes during T cell activation is challenging.
• Existing proteomic methods provide abundance data but often lack insights into functional regulation.

Purpose of the Study:

• To develop a novel assay combining protein abundance and thermal stability measurements.
• To investigate the functional significance of protein and phosphorylation dynamics during T cell activation.
• To apply this method to identify key regulatory proteins and phosphorylation sites.

Main Methods:

• Development of the Phosphorylation Integrated Thermal Shift Assay (PITSA).
• PITSA integrates protein/phosphorylation site abundance and thermal stability measurements in a single experiment.
• Application of PITSA to study T cell activation using tandem mass tags (TMT) quantitative proteomics.

Main Results:

• Quantified abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites.
• Identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins.
• Demonstrated PITSA's ability to reveal functionally relevant changes in protein regulation.

Conclusions:

• PITSA is a powerful tool for simultaneously assessing protein abundance and thermal stability.
• The method provides novel insights into the functional regulation of proteins and phosphorylation sites during T cell activation.
• PITSA can be broadly applied to generate hypotheses about protein function in various biological systems.