Collision Cross Sections of Charge-Reduced Proteins and Protein Complexes: A Database for Collision Cross Section Calibration

Abstract

The use of charge-reducing reagents to generate lower-charge ions has gained popularity in the field of native mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS). This is because the lower number of charged sites decreases the propensity for Coulombic repulsions and unfolding/restructuring, helping to preserve the native-like structure. Furthermore, lowering the charge state consequently increases the mass-to-charge values (m/z), effectively increasing spacing between signals originating from small mass differences, such as different proteoforms or protein–drug complexes. IM-MS yields collision cross section (CCS, Ω) values that provide information about the three-dimensional structure of the ion. Traveling wave IM (TWIM) is an established and expanding technique within the native MS field. TWIM measurements require CCS calibration, which is achieved via the use of standard species of known CCS. Current databases for native-like proteins and protein complexes provide CCS values obtained using normal (i.e., non-charge-reducing) conditions. Herein, we explored the validity of using “normal” charge calibrants to calibrate for charge-reduced proteins and show cases where it is not appropriate. Using a custom linear field drift cell that enables the determination of ion mobilities from “first principles”, we directly determined CCS values for 19 protein calibrant species under three solution conditions (yielding a broad range of charge states) and two drift gases. This has established a database of CCS and reduced-mobility (K 0) values, along with their associated uncertainties, for proteins and protein complexes over a large m/z range. TWIM validation of this database shows improved accuracy over existing methods in calibrating CCS values for charge-reduced proteins.

Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry

“…In linear cell drift tube IMS, a potential is applied along the ion mobility cell while ions are accelerated into a quasi-stationary bath gas, providing a drag force that opposes the ion motion along the potential gradient. [13][14][15] As a result, larger or more expanded ions undergo a greater number of collisions, therefore taking longer to traverse the IMS cell compared to more rigid or compact conformations. For ions of the same m/z, more compact ions, described as having higher mobility, will elute from the IMS cell sooner compared to expanded ions.…”

Surface-Induced Dissociation of Anionic vs Cationic Native-like Protein Complexes

“…40 Hence, the addition of TEAA and dissociation of lowercharge states is advantageous in SID structural studies. However, the addition of TEAA can often result in increased adduction and hence increased peak width, 22 which can be problematic in ligand binding studies when high mass accuracy is required. Given the previous reports of lower average charge states for proteins when analyzed in negative ionization mode, we next studied CRP prepared in 200mM AmAc but ionized in negative ionization mode.…”

“…[19][20][21] Charge reduction is typically achieved using solution-phase additives such as triethylammonium acetate (TEAA), which is an efficient charge reduction reagent; however, TEAA can lead to peak broadening due to increased adduction on the ion. 22 Here we seek to determine if the lower charge state protein complex anions generated through negative ESI can be used as an alternative to solution-phase charge reduction in nMS protein structural studies, offering the advantage of lower charge states without the increased adduction. nMS studies commonly employ gas-phase dissociation methods to obtain substructural information.…”

Experimental Determination of Activation Energies for Covalent Bond Formation via Ion/Ion Reactions and Competing Processes

“…Therefore, the higher energy conformer family likely also reflects a slightly compacted solution state. The robustness of the lowest charge states of proteins in the gas-phase to unfolding 23,[39][40][41] makes them particularly good candidates to probe via our ion/ion covalent chemistry, and native MS methods in general.…”

“…Since reduction in overall charge stabilizes the compact conformers of gas-phase proteins 39,[41][42] , The first major difference between activation thermochemistry of CIU (Table 2) and covalent bond formation via ion/ion reactions is the positive activation entropy for CIU. This is consistent with previous measurements of activation entropies during CIU 29 , and indicates that the transition state between the folded and partially folded structures is more disordered than the more compact, native-like state.…”