Time‐Resolved Study of In Situ Generated Protein Subcomplexes by Tandem‐Trapped Ion Mobility Spectrometry

We present an advanced analytical strategy that exploits the unique capabilities of tandem-trapped ion mobility spectrometry (Tandem-TIMS) to combine high-resolution ion mobility separation with targeted collisional activation and controlled gas-phase trapping. This approach enables the in situ generation of subcomplexes from native-like protein complexes of a selected charge state and allows direct evaluation of their kinetic stability and structural integrity in the gas phase. Using this workflow, we investigated the gas-phase stability of in situ generated streptavidin subunits. Streptavidin tetramers 15+ were mobility-selected and subjected to CID at 160 V between TIMS-1 and TIMS-2, producing monomers, dimers, and trimers. The resulting subcomplexes were then stored in TIMS-2 for up to 10.3 s, during which their collision cross-section distributions remained unchanged, indicating high kinetic stability in the gas phase. Overall, this study highlights the versatility of Tandem-TIMS as an analytical platform for advanced, mobility-resolved measurements that provide new structural and kinetic insights into biological systems.