Temporal Tracking of Insulin Action on the Cell Surface of Proteins at a Resolution of Ten Seconds

The ligand–receptor signaling occurring on the
cell surface
governs cell growth, proliferation, and survival via rapidly triggering
a cascade of events. Here, we for the first time report an in situ perturbation-free and rapid surface proteomic profiling
at a temporal resolution of ten seconds. By this innovation, about
1022 cell surface-associated proteins were reproducibly identified
and quantified. It is noteworthy that, upon a model ligand insulin
stimulus, a few rapid-responding proteins at 10 s to 2 min were identified,
e.g., CNNM3. Moreover, temporal response patterns were established
for the members of GLUT4 storage vesicles (GSVs; responsible for glucose
transportation) and confirmed with five known GSV proteins. This pattern
was then exploited to uncover seven new regulatory proteins (LDLR,
HFE, ECE1, MRC2, CORO1C, CPD, and BST2). Collectively, we showed a
powerful surface proteomic tool to decipher rapid signaling of cell-surface
proteins and to uncover new subunits involved in rapidly trafficking
vesicles.

细胞表面发生的配体-受体信号传导通过迅速触发的级联反应来调控细胞生长、增殖与存活。本研究首次报告了在十秒时间分辨率下，进行原位无干扰且快速表面蛋白质组学分析的成果。通过此创新技术，成功且可重复地鉴定并量化了约1022种细胞表面相关蛋白。值得注意的是，在胰岛素这一模型配体的刺激下，10秒至2分钟内识别出了一些快速响应蛋白，例如CNNM3。此外，建立了GLUT4储存囊泡（GSV，负责葡萄糖运输）的成员的时间响应模式，并通过五种已知的GSV蛋白进行了验证。随后，利用这一模式发掘了七种新的调控蛋白（LDLR、HFE、ECE1、MRC2、CORO1C、CPD和BST2）。总之，本研究展示了一种强大的表面蛋白质组学工具，用以解读细胞表面蛋白的快速信号传导，并揭示参与快速转运囊泡的新亚基。