Generation of four postmortem dura-derived iPS cell lines from four control individuals with genotypic and brain-region-specific transcriptomic data available through the BrainSEQ consortium.

In this study, we established induced pluripotent stem (iPS) cell lines from postmortem dura-derived fibroblasts of four control individuals with low polygenic risk score for psychiatric disorders including schizophrenia and bipolar disorder. The fibroblasts were reprogrammed into iPS cells using episomal vectors carrying OCT3/4, SOX2, KLF4, L-Myc, LIN28 and shRNA-p53. All iPS cell lines showed the same genotype with parental postmortem brain tissues, expressed pluripotency markers, and exhibited the differentiation potency into three embryonic germ layers.

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.

Surgery Website as a 24/7 Adjunct to a Surgical Curriculum.

OBJECTIVE: Successfully teaching duty hour restricted trainees demands engaging learning opportunities. Our surgical educational website and its associated assets were assessed to understand how such a resource was being used. DESIGN: Our website was accessible to all Mayo Clinic employees via the internal web network. Website access data from April 2015 through October 2016 were retrospectively collected using Piwik. SETTING: Academic, tertiary care referral center with a large general surgery training program. Mayo Clinic, Rochester, MN. PARTICIPANTS: A total of 257 Mayo Clinic employees used the website. RESULTS: The website had 48,794 views from 6313 visits by 257 users who spent an average of 14 ± 11 minutes on the website. Our website houses 295 videos, 51 interactive modules, 14 educational documents, and 7 flashcard tutorials. The most popular content type was videos, with a total of 30,864 views. The most popular visiting time of the day was between 8 pm and 9 pm with 6358 views (13%), and Thursday was the most popular day with 17,907 views (37%).  A total of 78% of users accessed content beyond the homepage. Average visits peaked in relation to 2 components of our curriculum: a 240% increase one day before our biannual intern simulation assessments, and a 61% increase one day before our weekly conducted Friday simulation sessions. Interns who rotated on the service of the staff surgeon who actively endorses the website had 93% more actions per visit as compared to other users. The highest clicks were on the home banner for our weekly simulation session pre-emptive videos, followed by "groin anatomy," and "TEP hernia repair" videos. CONCLUSIONS: Our website acted as a "just-in-time" accessible portal to reliable surgical information. It supplemented the time sensitive educational needs of our learners by serving as a heavily used adjunct to 3 components of our surgical education curriculum: weekly simulation sessions, biannual assessments, and clinical rotations.

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.

Radio-Frequency (rf) Confinement in Ion Mobility Spectrometry: Apparent Mobilities and Effective Temperatures.

Ion mobility is a powerful tool for separating and characterizing the structures of ions. Here, a radio-frequency (rf) confining drift cell is used to evaluate the drift times of ions over a broad range of drift field strengths (E/P, V cm-1 Torr-1). The presence of rf potentials radially confines ions and results in excellent ion transmission at low E/P (less than 1 V cm-1 Torr-1), thereby reducing the dependence of ion transmission on the applied drift voltage. Non-linear responses between drift time and reciprocal drift voltages are observed for extremely low E/P and high rf amplitudes. Under these conditions, pseudopotential wells generated by the rf potentials dampen the mobility of ions. The effective potential approximation is used to characterize this mobility dampening behavior, which can be mitigated by adjusting rf amplitudes and electrode dimensions. Using SIMION trajectories and statistical arguments, the effective temperatures of ions in an rf-confining drift cell are evaluated. Results for the doubly charged peptide GRGDS suggest that applied rf potentials can result in a subtle increase (2 K) in effective temperature compared to an electrostatic drift tube. Additionally, simulations of native-like ions of the protein complex avidin suggest that rf potentials have a negligible effect on the effective temperature of these ions. In general, the results of this study suggest that applied rf potentials enable the measurement of drift times at extremely low E/P and that these potentials have negligible effects on ion effective temperature. Graphical Abstract ᅟ.

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.

Ion mobility mass spectrometry of peptide, protein, and protein complex ions using a radio-frequency confining drift cell.

Ion mobility mass spectrometry experiments enable the characterization of mass, assembly, and shape of biological molecules and assemblies. Here, a new radio-frequency confining drift cell is characterized and used to measure the mobilities of peptide, protein, and protein complex ions. The new drift cell replaced the traveling-wave ion mobility cell in a Waters Synapt G2 HDMS. Methods for operating the drift cell and determining collision cross section values using this experimental set up are presented within the context of the original instrument control software. Collision cross sections for 349 cations and anions are reported, 155 of which are for ions that have not been characterized previously using ion mobility. The values for the remaining ions are similar to those determined using a previous radio-frequency confining drift cell and drift tubes without radial confinement. Using this device under 2 Torr of helium gas and an optimized drift voltage, denatured and native-like ions exhibited average apparent resolving powers of 14.2 and 16.5, respectively. For ions with high mobility, which are also low in mass, the apparent resolving power is limited by contributions from ion gating. In contrast, the arrival-time distributions of low-mobility, native-like ions are not well explained using only contributions from ion gating and diffusion. For those species, the widths of arrival-time distributions are most consistent with the presence of multiple structures in the gas phase.

Collision cross section calibrants for negative ion mode traveling wave ion mobility-mass spectrometry.

Unlike traditional drift-tube ion mobility-mass spectrometry, traveling-wave ion mobility-mass spectrometry typically requires calibration in order to generate collision cross section (CCS) values. Although this has received a significant amount of attention for positive-ion mode analysis, little attention has been paid for CCS calibration in negative ion mode. Here, we provide drift-tube CCS values for [M - H](-) ions of two calibrant series, polyalanine and polymalic acid, and evaluate both types of calibrants in terms of the accuracy and precision of the traveling-wave ion mobility CCS values that they produce.

Effects of polarity on the structures and charge states of native-like proteins and protein complexes in the gas phase.

Native mass spectrometry and ion mobility spectrometry were used to investigate the gas-phase structures of selected cations and anions of proteins and protein complexes with masses ranging from 6 to 468 kDa. Under the same solution conditions, the average charge states observed for all native-like anions were less than those for the corresponding cations. Using an rf-confining drift cell, similar collision cross sections were measured in positive and negative ion mode suggesting that anions and cations have very similar structures. This result suggests that for protein and protein complex ions within this mass range, there is no inherent benefit to selecting a specific polarity for capturing a more native-like structure. For peptides and low-mass proteins, polarity and charge-state dependent structural changes may be more significant. The charged-residue model is most often used to explain the ionization of large macromolecules based on the Rayleigh limit, which defines the upper limit of charge that a droplet can hold. Because ions of both polarities have similar structures and the Rayleigh limit does not depend on polarity, these results cannot be explained by the charged-residue model alone. Rather, the observed charge-state distributions are most consistent with charge-carrier emissions during the final stages of analyte desolvation, with lower charge-carrier emission energies for anions than the corresponding cations. These results suggest that the observed charge-state distributions in most native mass spectrometry experiments are determined by charge-carrier emission processes; although the Rayleigh limit may determine the gas-phase charge states of larger species, e.g., virus capsids.

HPLC method for rapidly following biodiesel fuel transesterification reaction progress using a core-shell column.

There are a wide and growing variety of feedstocks for biodiesel fuel. Most commonly, these feedstocks contain triglycerides which are transesterified into the fatty acid alkyl esters (FAAEs) which comprise biodiesel fuel. While the tranesterification reaction itself is simple, monitoring the reaction progress and reaction products is not. Gas chromatography-mass spectrometry is useful for assessing the FAAE products, but does not directly address either the tri-, di-, or monoglycerides present from incomplete transesterification or the free fatty acids which may also be present. Analysis of the biodiesel reaction mixture is complicated by the solubility and physical property differences among the components of the tranesterification reaction mixture. In this contribution, we present a simple, rapid HPLC method which allows for monitoring all of the main components in a biodiesel fuel transesterification reaction, with specific emphasis on the ability to monitor the reaction as a function of time. The utilization of a relatively new, core-shell stationary phase for the HPLC column allows for efficient separation of peaks with short elution times, saving both time and solvent.