Protein phosphorylation-dephosphorylation events play a primary role in 
regulation of almost all aspects of cell function including signal transduction, 
cell cycle, or apoptosis. Thus far, T cell phosphoproteomics have focused on 
analysis of phosphotyrosine residues, and little is known about the role of 
serine/threonine phosphorylation in early activation of the T cell receptor 
(TCR). Therefore, we performed a quantitative mass spectrometry-based analysis 
of the global phosphoproteome of human primary T cells in response to 5 min of 
TCR activation with anti-CD3 antibody. Combining immunoprecipitation with an 
antiphosphotyrosine antibody, titanium dioxide phosphopeptide enrichment, 
isobaric tag for the relative and absolute quantitation methodology, and strong 
cation exchange separation, we were able to identify 2814 phosphopeptides. These 
unique sites were employed to investigate the site-specific phosphorylation 
dynamics. Five hundred and seventeen phosphorylation sites showed TCR-responsive 
changes. We found that upon 5 min of stimulation of the TCR, specific serine and 
threonine kinase motifs are overrepresented in the set of responsive 
phosphorylation sites. These phosphorylation events targeted proteins with many 
different activities and are present in different subcellular locations. Many of 
these proteins are involved in intracellular signaling cascades related mainly 
to cytoskeletal reorganization and regulation of small GTPase-mediated signal 
transduction, probably involved in the formation of the immune synapse.