Integral Synthesis and Clearance Analysis via DIA (ISDia) Reveals Coordinated Protein Dynamics Regulation during Endoplasmic Reticulum Stress

Endoplasmic Reticulum (ER) stress disrupts protein homeostasis and impacts protein dynamics, driving cellular responses critical for survival, development and disease. However, no current proteome-wide technology enables simultaneous identification of proteins undergoing altered synthesis and clearance and distinguish their relative contribution during ER stress. To fill this gap, we developed Integral Synthesis and clearance analysis via DIA (ISDia), a robust mass spectrometry-based platform that integrates pulsed-SILAC labeling with data-independent acquisition (DIA) to quantify heavy and light peptide changes and determine the drivers of protein dynamics with high proteome coverage under non-steady-state conditions. Using ISDia, we uncover diverse regulatory mechanisms by which protein synthesis and clearance are modulated to control protein abundances during ER stress, revealing PERK dependent and independent regulatory mechanisms across subcellular compartments, complexes and isoforms. These findings highlight the potential of ISDia as a powerful and widely applicable platform for elucidating protein dynamic regulatory mechanisms.