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

Area of Science:

• Mass Spectrometry
• Biophysical Chemistry
• Proteomics

Background:

• Charge detection mass spectrometry (CDMS) is an emerging technique for qualitative analysis of heterogeneous biological samples.
• Orbitrap-based CDMS has increased the technique's use in native mass spectrometry (nMS).
• Current applications of CDMS are primarily qualitative, with limited quantitative studies in the MDa range.

Purpose of the Study:

• To demonstrate the quantitative capabilities of Orbitrap-based CDMS/Direct Mass Technology (DMT) for heterogeneous protein samples under 100 kDa.
• To analyze the stability and ligand-binding properties of transthyretin (TTR) variants using DMT.
• To establish CDMS as a viable quantitative method for complex biological systems.

Main Methods:

• Utilized Orbitrap-based CDMS/Direct Mass Technology (DMT) for high-resolution mass analysis.
• Resolved and quantified wild-type (WT) and C-terminally tagged (CT) TTR homotetramers and hybrid tetramers.
• Analyzed kinetic formation of hybrid TTR and quantified thyroxine binding to TTR variants.

Main Results:

• DMT successfully resolved WT and CT TTR homotetramers and hybrid tetramers, enabling kinetic analysis.
• CT-TTR homotetramers showed decreased stability compared to WT-TTR, suggesting C-terminal modification impacts tetramer stability.
• DMT quantified thyroxine binding, revealing CT-TTR has a higher affinity than WT-TTR, indicating C-terminal involvement in ligand binding.
• CDMS separated overlapping m/z signals, enabling accurate mass and abundance determination for heterogeneous samples.

Conclusions:

• Orbitrap-based CDMS/DMT is established as a quantitative approach for heterogeneous protein samples.
• The C-terminal modification of TTR influences tetramer stability and thyroxine binding affinity.
• CDMS shows significant potential for broader applications in analyzing complex biological systems.