Tandem-Trapped Ion Mobility Spectrometry as a Platform for Conformationally Resolved Analysis of Protein Complexes and Biologics

Protein structural heterogeneity is central to function, yet remains difficult to resolve with methods that report ensemble averages. Native ion mobility/mass spectrometry (IM/MS) is widely used to analyze intact proteins but does not directly characterize specific subpopulations of the protein structural ensemble. Here, we demonstrate that tandem-trapped-ion mobility spectrometry/tandem-mass spectrometry (Tandem-TIMS) combines accurate measurements of masses and collision cross sections of protein complexes of up to ∼232 kDa with quantitative characterization of their structural ensembles. In line with prior results reported from small protein model systems, we validate that mobility-selected subpopulations of protein complexes are kinetically stable on the measurement time scale. Collisional activation reveals that mobility-selected subpopulations do not interconvert into each other, even if their collision cross sections differ by only about 1%. These results support the view that the mobility-selected subpopulations reflect different protein structures present in solution. Extending to biotherapeutics, we demonstrate the ability of Tandem-TIMS to quantify and characterize low-abundance, structurally distinct species of an antibody construct comprising two single-chain variable fragment (scFv) domains. Together, our results position Tandem-TIMS as a platform for characterizing the structural ensemble of protein complexes and biologics, thereby opening new avenues for the rational design and quality assessment of complex antibody scFv formats relevant to current biotherapeutic development.