Improved characterization of large, intact proteins by leveraging ion-ion and ion-photon reactions in the gas-phase

Description:
Currently, state-of-the-art top-down mass spectrometry, defined as the gas-phase interrogation of whole protein ions, can be efficiently applied to the identification of thousands of proteoforms <30 kDa in a single experiment. However, challenges still persist when the mass of the analyzed proteins exceeds the “30 kDa barrier”, primarily due to signal dilution (a combination of charge state and isotopic effects) and excessive spectral congestion (i.e., spectra with overlapping ion signals). Additionally, several proteins of biomedical or biotherapeutic interest, such as immunoglobulins and albumin, present a complex network of disulfide bonds, that typically hinder the sequencing of entire portions of the amino acid chains.
Here we present the results achievable for the characterization of proteins >50 kDa (human albumin and 100 kDa F(ab)2 subunit of monoclonal antibodies) by combining ion-ion and ion-photon reactions in the gas-phase on a tribrid Orbitrap mass spectrometer. First, we show that spectral simplification via proton transfer charge reduction (PTCR) following different ion fragmentations can significantly improve the number of matched product ions by reducing signal overlap. Second, we demonstrate that this PTCR MS3-based approach can be effectively implemented under the time constraints dictated by liquid chromatography (LC) separation and, at the same time, it is possible to further improve spectral signal-to-noise ratio by averaging mass spectra derived from multiple, consecutive LC runs. Finally, we present evidence that gas-phase denaturation of protein ions using IR photons prior to electron transfer dissociation, known as activated ion ETD (AI-ETD), is particularly beneficial to improve the sequencing of disulfide protected regions of whole, large proteins.

Speaker: Luca Fornelli - University of Oklahoma