Comparing Solution-Phase and Gas-Phase Protein Stability Using Ion Mobility and Differential Mobility Mass Spectrometry

A critical quality attribute for many proteins concerning biopharmaceutical products is the proteins stability. Current stability assays are lengthy but some quick stability assays to determine the proteins melting temperature include thermal ramps of a protein using a fluorescent dye to monitor unfolding. Protein unfolding has also been studies with mass spectrometry through collision induced unfolding (CIU) whereby the ion temperature in the trap collision cell is increased and unfolding changes are tracked in the ion mobility cell. However, whether a protein retains its structural properties in the gas phase has been a topic of wide discussion. Here, we present that CIU and solution thermal melts provide the same information on protein stability using a homo-tetrameric model protein, pyruvate kinase, and four of its point mutants. Efforts to employ a second ion mobility technique differential mobility spectrometry, to determine gas phase stability will also be discussed.