Expanding Differential Ion Mobility Separations into the MegaDalton Range.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Ion mobility separations (IMS) and mass spectrometry (MS) have advanced for analyzing larger biomolecules.
• Electrospray ionization (ESI) and native MS enabled analysis of proteins up to 100 kDa and complexes/viruses up to 10 MDa.
• Differential FAIMS offers orthogonal separation but was limited to ions under 300 kDa.

Purpose of the Study:

• To integrate high-definition FAIMS with a high-capacity Orbitrap mass spectrometer (Q-Exactive Orbitrap UHMR).
• To extend the mass range of FAIMS analysis for very large biomolecular assemblies.
• To demonstrate the capability for analyzing megadalton (MDa) ions in the native ESI regime.

Main Methods:

• Integration of high-definition FAIMS with the Q-Exactive Orbitrap UHMR mass spectrometer.
• Utilized native ESI for ionization of large biomolecules.
• Size-selected oligomers of monoclonal antibody adalimumab (148 kDa).

Main Results:

• Successfully analyzed nonamers (1.34 MDa) of adalimumab with mass-to-charge (m/z) up to ~17,000.
• Demonstrated the survival and efficient separation of noncovalent MDa assemblies within the FAIMS process.
• The UHMR mass spectrometer handled ions with m/z up to 80,000 and MDa-size ions.

Conclusions:

• High-definition FAIMS coupled with the Q-Exactive Orbitrap UHMR enables analysis of the heaviest macromolecules.
• This technological advancement opens new avenues for studying large, noncovalent biomolecular assemblies.
• Expands the scope of IMS/MS for characterizing complex biological systems.