Targeted Quantitation of Phosphotyrosine-Containing Proteins in T-Cell Receptor Signaling Using a SureQuant-Based...

Area of Science:

• Immunology
• Proteomics
• Cell Signaling

Background:

• T-cell receptor (TCR) signaling is vital for immune responses.
• Tyrosine phosphorylation is a key regulatory mechanism in TCR signaling.
• Low abundance of phosphotyrosine peptides challenges conventional detection methods.

Purpose of the Study:

• To develop and validate a targeted proteomics method for quantifying phosphotyrosine peptides in TCR signaling.
• To assess the sensitivity and reproducibility of the SureQuant approach for low-abundance peptides.
• To investigate dynamic changes in phosphotyrosine signaling during T-cell activation.

Main Methods:

• Development of a SureQuant-based targeted mass spectrometry assay.
• Utilizing triggered data acquisition with heavy isotope-labeled peptides.
• Stimulation of primary T-cells with anti-CD3/CD28 antibodies to monitor signaling.

Main Results:

• Successful quantification of changes in key phosphotyrosine peptides in primary T-cells.
• Demonstrated high sensitivity and reproducibility of the SureQuant method for low-abundance phosphotyrosine peptides.
• Provided a systematic view of TCR signaling dynamics with distinct phosphorylation patterns.

Conclusions:

• The SureQuant approach accurately quantifies low-abundance phosphotyrosine peptides in TCR signaling.
• This method offers a valuable tool for precise analysis of signaling pathways and post-translational modifications.
• The framework can be extended to study other signaling networks and immune cell functions.