Spatiotemporally resolved subcellular phosphoproteomics

Significance As one of the most important post-translational modifications, phosphorylation is both highly abundant and dynamically regulated in cells. Characterizing subcellular phosphoproteome with high temporal resolution should shed light on their contributions to diverse cellular processes. By integrating an activatable proximity labeling enzyme with an orthogonal phosphorylation enrichment scheme, we have developed the SubMAPP strategy for mapping the phosphorylation dynamics of subcellular proteome in living systems. The high sensitivity of SubMAPP enabled the identification of phosphoproteins and phosphorylation sites in the endoplasmic reticulum (ER), revealing ER-to-mitochondria protein translocation under ER stress.