Separation and Collision Cross Section Measurements of Protein Complexes Afforded by a Modular Drift Tube Coupled to an Orbitrap Mass Spectrometer

New developments in analytical technologies and biophysical methods have advanced the characterization of increasingly complex biomolecular assemblies using native mass spectrometry (MS). Ion mobility methods, in particular, have enabled a new dimension of structural information and analysis of proteins, allowing separation of conformations and providing size and shape insights based on collision cross sections (CCSs). Based on the concepts of absorption-mode Fourier transform (aFT) multiplexing ion mobility spectrometry (IMS), here, a modular drift tube design proves capable of separating native-like proteins up to 148 kDa with resolution up to 45. Coupled with high-resolution Orbitrap MS, binding of small ligands and cofactors can be resolved in the mass domain and correlated to changes in structural heterogeneity observed in the ion-neutral CCS distributions. We also demonstrate the ability to rapidly determine accurate CCSs for proteins with 1-min aFT-IMS-MS sweeps without the need for calibrants or correction factors.

Automated summary

New developments in analytical technologies and biophysical methods have advanced the characterization of increasingly complex biomolecular assemblies. Ion mobility methods, especially, provide a new dimension of structural information and protein analysis based on collision cross sections (CCSs). This article presents a modular drift tube design capable of separating native-like proteins with high resolution, and combines it with high-resolution Orbitrap MS for complex analysis. Additionally, the ability to rapidly determine accurate CCSs for proteins without the need for calibrants or correction factors is demonstrated.