A Flexible, Modular Platform for Multidimensional Ion Mobility of Native-like Ions.

Native ion mobility (IM) mass spectrometry (MS) is used to probe the size, shape, and assembly of biomolecular complexes. IM-IM-MS can increase the amount of information available in structural studies by isolating subpopulations of structures for further analysis. Previously, IM-IM-MS has been implemented using the Structures for Lossless Ion Manipulations (SLIM) architecture to probe the structural stability of gas-phase protein ions. Here, a new multidimensional IM instrument constructed from SLIM devices is characterized using multiple operational modes. In this new design, modular devices are used to perform all ion manipulations, including initial accumulation, injection, separation, selection, and trapping. Using single-dimension IM, collision cross section (Ω) values are determined for a set of native-like ions. These Ω values are within 3% of those reported previously based on measurements using RF-confining drift cells. Tandem IM experiments are performed on a sample of ubiquitin ions that contains both compact and partially unfolded structures, demonstrating that this platform can isolate subpopulations of structures. Finally, additional modes of analysis, including multiplexed IM and inverse IM, are demonstrated using this platform. The ability of this platform to quickly switch between different modes of IM analysis makes it a highly flexible tool for studying protein structures and dynamics.

Principles of Ion Selection, Alignment, and Focusing in Tandem Ion Mobility Implemented Using Structures for Lossless Ion Manipulations (SLIM).

Tandem ion mobility (IM) enables the characterization of subpopulations of ions from larger ensembles, including differences that cannot be resolved in a single dimension of IM. Tandem IM consists of at least two IM regions that are each separated by an ion selection region. In many implementations of tandem IM, ions eluting from a dimension of separation are filtered and immediately transferred to the subsequent dimension of separation (selection-only experiments). We recently reported a mode of operation in which ions eluting from a dimension are trapped prior to the subsequent dimension (selection-trapping experiments), which was implemented on an instrument constructed using the structures for lossless ion manipulations (SLIM) architecture. Here, we use a combination of experiments and trajectory simulations to characterize aspects of the selection, trapping, and separation processes underlying these modes of operation. For example, the actual temporal profile of filtered ions can be very similar to the width of the waveforms used for selection, but depending on experimental parameters, can differ by up to ± 500 µs. Experiments and simulations indicate that ions in selection-trapping experiments can be spatially focused between dimensions, which removes the broadening that occurred during the preceding dimension. During focusing, individual ions are thermalized, which aligns and establishes common initial conditions for the subsequent dimension. Therefore, selection-trapping experiments appear to offer significant advantages relative to selection-only experiments, which we anticipate will become more pronounced in future experiments that make use of longer IM separations, additional dimensions of analysis, and the outcomes of this study. Graphical Abstract.

Structural Dynamics of Native-Like Ions in the Gas Phase: Results from Tandem Ion Mobility of Cytochrome c.

Ion mobility (IM) is a gas-phase separation technique that is used to determine the collision cross sections of native-like ions of proteins and protein complexes, which are in turn used as restraints for modeling the structures of those analytes in solution. Here, we evaluate the stability of native-like ions using tandem IM experiments implemented using structures for lossless ion manipulations (SLIM). In this implementation of tandem IM, ions undergo a first dimension of IM up to a switch that is used to selectively transmit ions of a desired mobility. Selected ions are accumulated in a trap and then released after a delay to initiate the second dimension of IM. For delays ranging from 16 to 33 231 ms, the collision cross sections of native-like, 7+ cytochrome c ions increase monotonically from 15.1 to 17.1 nm2. The largest products formed in these experiments at near-ambient temperature are still far smaller than those formed in energy-dependent experiments (∼21 nm2). However, the collision cross section increases by ∼2% between delay times of 16 and 211 ms, which may have implications for other IM experiments on these time scales. Finally, two subpopulations from the full population were each mobility selected and analyzed as a function of delay time, showing that the three populations can be differentiated for at least 1 s. Together, these results suggest that elements of native-like structure can have long lifetimes at near-ambient temperature in the gas phase but that gas-phase dynamics should be considered when interpreting results from IM.

Analysis of Native-Like Ions Using Structures for Lossless Ion Manipulations.

Ion mobility separation of native-like protein and protein complex ions expands the structural information available through native mass spectrometry analysis. Here, we implement Structures for Lossless Ion Manipulations (SLIM) for the analysis of native-like ions. SLIM has been shown previously to operate with near lossless transmission of ions up to 3000 Da in mass. Here for the first time, SLIM was used to separate native-like protein and protein complex ions ranging in mass from 12 to 145 kDa. The resulting arrival-time distributions were monomodal and were used to determine collision cross section values that are within 3% of those determined from radio-frequency-confining drift cell measurements. These results are consistent with the retention of native-like ion structures throughout these experiments. The apparent resolving powers of native-like ions measured using SLIM are as high as 42, which is the highest value reported directly from experimental data for the native-like ion of a protein complex. Interestingly, the apparent resolving power depends strongly on the identity of the analyte, suggesting that the arrival-time distributions of these ions may have contributions from an ensemble of structures in the gas phase that is unique to each analyte. These results suggest that the broad range of emerging SLIM technologies may all be adaptable to the analysis of native-like ions, which will enable future applications in the areas of structural biology, biophysics, and biopharmaceutical characterization.

Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing.

The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process-and not only its endpoint-to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach. Graphical Abstract A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.