Phosphorylation dysregulation has been implicated in various diseases including cancer. The phosphorylation change of proteins in secretome may be a novel source for the discovery of biomarkers and drug targets. In this study, the phosphoproteins in cancer secretome (phosphosecretome) were globally analyzed for the first time by phosphoproteomics. One hundred forty-two phosphorylation sites on 62 unique phosphopeptides representing 49 nonredundant proteins were identified, several of which are known as secreted proteins involved in carcinogenesis, invasion, and metastasis. Most of them were first found as secreted proteins with no previously known function. Protein sublocation analysis showed that 33 proteins were found to be sectreted as phosphoproteins, in which 27 (81.81%) were secreted by a nonclassic, ER/Golgi-independent pathway, suggestting that the phosphorylation modification of these proteins might play an important role in their nonconventional secretion processes. Their protein kinases and regulatory phosphosites involved in the secretion regulation of these phosphoproteins, such as stanniocalcin 2, annexin A2, and HSP90 alphạ, were first identified. The phosphosecretome data enriched the secretome database and phosphoproteome database, and will help us to discover cancer biomarkers and drug targets, illustrating the mystery of the nonclassic protein secretion pathway.
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