Tandem Ion mobility coupled with mass spectrometry for gas phase protein unfolding studies

Aims/Introduction (600 chars)

Collision-induced unfolding (CIU) has gained interest in the fields of life science and biopharmaceutical research. The technique subjects gas phase protein ions to elevated activation energies in order to induce structural changes that are monitored by ion mobility-mass spectrometry. Published work has shown that unfolding profiles relate closely to protein domain architecture. Here, we use a cyclic ion mobility (cIM)-enabled quadrupole time-of-flight mass spectrometer to perform novel tandem ion mobility (tandem-IMS) experiments, probing protein unfolding pathways in greater detail.

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Experimental and results (1500 chars)

Studies were performed using a Cyclic IMS mass spectrometer which enables both mass and mobility precursor selection and tandem-IMS functionality. Human transthyretin (TTR) was used as a model protein and was introduced using ESI and conductive-coated glass capillaries at a concentration of 4 micromolar in 200 mM ammonium acetate. Initially focussing on the 16+ charge state, at low activation voltage a single species was observed in the arrival time distribution. This finding did not change when six passes of the cIM device were performed (approximately 6m pathlength). Protein unfolding was induced by increasing the acceleration voltage into the trap ion guide situated before the cIM device. Increasing the voltage to 40 V resulted in a minimum of five species (1-5 from most compact to most extended) being observed after a single pass of the cIM device. The multifunctional array was used to isolate and eject each of the five species to the pre-array store. The protein ions were subsequently reinjected into the cIM device at elevated energies to induce further unfolding. Briefly, after selecting Species 1, Species 2-4 were formed; on selecting Species 2, Species 3 and 4 were formed; upon selecting Species 3, Species 4 was formed. Interestingly, no compacted intermediates were observed for TTR. If required, multiple mobility selections were made per round of IMS.

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Conclusions (600 chars)

Human TTR was investigated as a model system for CIU. The geometry of the Cyclic IMS is both selective and highly flexible and allowed multi-stage gas phase separations. IMS2 experiments with activation demonstrated a reproduction of the stepwise TTR unfolding pathway and IMS3 offers further opportunities for delineating gas phase protein unfolding pathways.