Expanding Differential Ion Mobility Separations into the MegaDalton Range

• Published in: Analytical chemistry (Washington) 2024-04, Vol.96 (14), p.5392-5398
• Main Authors: Wörner, Tobias P., Thurman, Hayden A., Makarov, Alexander A., Shvartsburg, Alexandre A.
• Format: Article
• Language: English
• Subjects: Beta decay; Biomolecules; Dissociation; Electron capture; High definition; Ion Mobility Spectrometry; Ionic mobility; Ionization; Ions; Macromolecules; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Mobility; Monoclonal antibodies; Proteins; Proteins - chemistry; Survival; Viruses
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.3c05012

Along with mass spectrometry (MS), ion mobility separations (IMS) are advancing to ever larger biomolecules. The emergence of electrospray ionization (ESI) and native MS enabled the IMS/MS analyses of proteins up to ∼100 kDa in the 1990s and whole protein complexes and viruses up to ∼10 MDa since the 2000s. Differential IMS (FAIMS) is substantially orthogonal to linear IMS based on absolute mobility K and offers exceptional resolution, unique selectivity, and steady filtering readily compatible with slower analytical methods such as electron capture or transfer dissociation (ECD/ETD). However, the associated MS stages had limited FAIMS to ions with m/z < 8000 and masses under ∼300 kDa. Here, we integrate high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer that can handle m/z up to 80,000 and MDa-size ions in the native ESI regime. In the initial evaluation, the oligomers of monoclonal antibody adalimumab (148 kDa) are size-selected up to at least the nonamers (1.34 MDa) with m/z values up to ∼17,000. This demonstrates the survival and efficient separation of noncovalent MDa assemblies in the FAIMS process, opening the door to novel analyses of the heaviest macromolecules.