ABSTRACT 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.
Pedrete et al. (Thu,) studied this question.