The level of phosphorylation for each site was quantified at 5, 15, 30 and 60 s of TCR/CD28 stimulation, relative to the corresponding level in unstimulated cells. These experiments targeted a period of signaling that has thus far been largely uncharacterized using MS-based proteomics or traditional biochemical assays, which have mostly been used at later timepoints. Our measurements map in unprecedented detail the earliest intracellular events and reveal that even within the first minute of TCR/CD28 co-stimulation, dramatic and diverse biochemical changes occur within the cell, preparing the ground for later events. To analyze these data, we took a knowledge-based/modelguided approach, which is summarized in Fig. S1A in File S2. Regulated changes in phosphorylation occurred as early as 5 s after stimulation, with the number of regulated sites increasing to 138 after 60 s of stimulation. Time courses of phosphorylation fall into four distinct clusters, which reveal that the abundance of some phosphopeptides increase, others decrease, and some changes occur earlier than others. These results clearly demonstrate that even within the first 60 s of TCR stimulation there are diverse patterns of phosphorylation dynamics. Regulated sites map to proteins with various cellular functions, including pivotal signaling factors such as receptors, adapter proteins, phospholipases, phosphatases and kinases from multiple distinct kinase families. In the group of sites showing rapid dynamics we find wellestablished TCR signaling proteins such as LCK, LAT, PLCG1 among many others. These results attest to rapid, multi-functional signaling Salvianolic-acid-B downstream of the TCR, consistent with the known diversity of pathways that emanate from the receptor. Indeed, subsequent enrichment analysis revealed that among the proteins with detected phosphorylation changes, the most frequent pathway association was with the TCR pathway. At the same time, other pathways, such as those influencing metabolism and protein synthesis, were also detected. These results suggest that TCR signaling may influence these general cellular functions Mepiroxol quickly, consistent with evidence that T cells make committed decisions within 60 s of antigen contact. This study of pTyr site dynamics has revealed processes that have been systematically overlooked in the past because of the speed with which they occur. We have monitored the phosphosite dynamics of early TCR signaling with finer temporal resolution than in previous proteomic studies of TCR signaling and with greater breadth than earlier studies of early TCR signaling events employing relatively low-throughput assays, and we developed a mechanistic model for TCR signaling that reproduces measured time courses of phosphorylation for a greater number of specific sites than previously developed models for immunoreceptor signaling. We detected over 100 pTyr sites that undergo greater than twofold changes in abundance during the first minute of TCR signaling. Even on these short timescales, time courses show distinct patterns: the abundances of some pTyr 
sites increase, others decrease, and some changes occur sooner than others. The proteins containing these sites map to diverse cellular functions and include kinases, phospholipases, actin regulators, and transcription factors, many of which are known players in T-cell activation. The significance of these results is that by 60 s, which in many studies is taken as an early time point for measurement, significant changes have already occurred.