A Modified Drift Tube Ion Mobility-Mass Spectrometer for Charge-Multiplexed Collision-Induced Unfolding.

Collision-induced unfolding (CIU) of protein ions and their noncovalent complexes offers relatively rapid access to a rich portfolio of biophysical information, without the need to tag or purify proteins prior to analysis. Such assays have been characterized extensively for a range of therapeutic proteins, proving exquisitely sensitive to alterations in protein sequence, structure, and post-translational modification state. Despite advantages over traditional probes of protein stability, improving the throughput and information content of gas-phase protein unfolding assays remains a challenge for current instrument platforms. In this report, we describe modifications to an Agilent 6560 drift tube ion mobility-mass spectrometer in order to perform robust, simultaneous CIU across all precursor ions detected. This approach dramatically increases the speed associated with typical CIU assays, which typically involve mass selection of narrow m/z regions prior to collisional activation, and thus their development requires a comprehensive assessment of charge-stripping reactions that can unintentionally pollute CIU data with chemical noise when more than one precursor ion is allowed to undergo simultaneous activation. By studying the unfolding and dissociation of intact antibody ions, a key analyte class associated with biotherapeutics, we reveal a predictive relationship between the precursor charge state, the amount of buffer components bound to the ions of interest, and the amount of charge stripping detected. We then utilize our knowledge of antibody charge stripping to rapidly capture CIU data for a range of antibody subclasses and subtypes across all charge states simultaneously, demonstrating a strong charge state dependence on the information content of CIU. Finally, we demonstrate that CIU data collection times can be further reduced by scanning fewer voltage steps, enabling us to optimize the throughput of our improved CIU methods and confidently differentiate antibody variant ions using similar to 20% of the data typically collected during CIU. Taken together, our results characterize a new instrument platform for biotherapeutic stability measurements with dramatically improved throughput and information content.