Simultaneous Proteome Localization and Turnover Analysis, MS3 Data

The functionality of proteins is dependent on their spatial and temporal distributions, neither of which is directly measured by static protein abundance. Here we report a mass spectrometry-based proteomics workflow and data analysis pipeline, named Simultaneous Proteome Localization and Turnover (SPLAT), to concurrently examine the turnover dynamics and subcellular distributions of whole cell proteomes under perturbation. SPLAT builds on prior work in protein turnover measurements and subcellular localization profiling, by combining dynamic stable isotope labeling, differential ultracentrifugation, and kinetic modeling to concurrently measure changes in protein turnover and subcellular localization under perturbation in one experiment.
Briefly, dynamic SILAC labeled cell lysates were fractionated with ultracentrifugation, digested using a modified FASP protocol, and multiplexed with TMT-10 plex tags. The pooled sample was then fractionated with RPLC and injected into an Orbitrap Fusion Tribrid mass spectrometer coupled to an LC with electrospray ionization source operated in data dependent acquisition mode.
Detailed methods can be found in the submission files.