Abstract
T cell activation is a complex biological process of naive cells maturing into effector cells. Proteomic and phospho-proteomic approaches have provided critical insights into this process, yet it is not always clear how changes in individual proteins or phosphorylation sites have functional significance. Here, we developed the Phosphorylation Integrated Thermal Shift Assay (PITSA) that combines the measurement of protein or phosphorylation site abundance and thermal stability into a single tandem mass tags experiment and apply this method to study T cell activation. We quantified the abundance and thermal stability of over 7500 proteins and 5000 phosphorylation sites and identified significant differences in chromatin-related, TCR signaling, DNA repair, and proliferative phosphoproteins. PITSA may be applied to a wide range of biological contexts to generate hypotheses as to which proteins or phosphorylation sites are functionally regulated in a given system as well as the mechanisms by which this regulation may occur.
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•T cells undergo significant proteome/phosphoproteome remodeling during activation.•PITSA simultaneously measures abundance and thermal stability in a single TMT-plex.•PITSA highlights abundance, signaling, and thermal stability changes in DNA repair.•Increased CDK activity correlates with decreased CDK substrate thermal stability.
T cells undergo a significant rearrangement of their proteome and phosphoproteome during activation while accumulating necessary cellular components for rapid cell division. Here, we describe the phosphorylation integrated thermal shift assay, which combines the measurement of protein and phosphorylation site abundance with the measurement of protein and phosphorylation site thermal stability. This assay may help determine which proteome and phosphoproteome changes are functionally relevant in T cell activation by identifying proteins/phosphorylation sites with altered biophysical states, and presumably altered function.