Dissecting Heterogeneous Populations of Protein-Complex Samples Using Direct Mass Technology

Charge detection mass spectrometry (CDMS) is an emerging technique for the qualitative analysis of heterogeneous biological samples by individually measuring the charge and m/z of ions to determine their mass. The commercialization of CDMS with Orbitrap-based CDMS has allowed for broader use of the technique within the native mass spectrometry (nMS) field. However, CDMS is still primarily used qualitatively, with only a few studies applying it for quantitation in the MDa range. Here, we demonstrate how Orbitrap-based CDMS/Direct Mass Technology (DMT) can be used for quantitation of heterogeneous transthyretin (TTR) tetramer samples <100 kDa. First, we demonstrate DMT can resolve wild-type (WT) and C-terminally tagged (CT) TTR homotetramers and hybrid tetramers (i.e., tetramers containing both types of subunits), enabling kinetic analysis of hybrid TTR formation. The CT-TTR homotetramer dissociates quicker, providing evidence that the C-terminal modification (-ASGENLFYQ) decreases the stability of the tetramer. We hypothesize this is due to altering important noncovalent interactions between subunits. Next, DMT is used to resolve and quantify thyroxine binding events to WT-TTR and CT-TTR, which can be utilized to determine binding affinity. Interestingly, CT-TTR has a higher affinity for thyroxine than WT-TTR, which indicates that the C-terminus is involved in TTR-ligand binding. In both examples, overlapping m/z signals that cannot be distinguished by conventional nMS are separated in CDMS through the additional charge measurement, enabling an accurate determination of mass and abundance. Overall, these results establish CDMS as a quantitative approach for heterogeneous protein samples and highlight its potential for broader applications in complex biological systems.