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Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species in response to a cold environment. The activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms., (© 2018 Hsu et al.)

The state of protein phosphorylation can be a key determinant of cellular physiology such as early-stage cancer, but the development of phosphoproteins in biofluids for disease diagnosis remains elusive. Here we demonstrate a strategy to isolate and identify phosphoproteins in extracellular vesicles (EVs) from human plasma as potential markers to differentiate disease from healthy states. We identified close to 10,000 unique phosphopeptides in EVs isolated from small volumes of plasma samples. Using label-free quantitative phosphoproteomics, we identified 144 phosphoproteins in plasma EVs that are significantly higher in patients diagnosed with breast cancer compared with healthy controls. Several biomarkers were validated in individual patients using paralleled reaction monitoring for targeted quantitation. This study demonstrates that the development of phosphoproteins in plasma EV as disease biomarkers is highly feasible and may transform cancer screening and monitoring.