Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections.

Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) to separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage the primary information obtained from IMS/MS, which is the characterization of the fragment ion structure by the measured momentum transfer cross sections. Here, we perform top-down analysis of intact proteins and assemblies using our tandem trapped ion mobility spectrometer/mass spectrometer (tTIMS/MS) and compile over 1400 cross section values of fragment ions. Our analysis reveals that most fragment ions exhibit multiple, stable conformations similar to those of intact polypeptides and proteins. The data further indicate that the conformational heterogeneity is strongly influenced by the amino acid sequences of the fragment ions. Moreover, time-resolved tTIMS/MS experiments reveal that conformations of top-down fragment ions can be metastable on the timescale of ion mobility measurements. Taken together, our analysis indicates that top-down fragment ions undergo a folding process in the gas phase and that this folding process can lead to kinetic trapping of intermediate states in ion mobility measurements. Hence, because the folding free energy surface of a polypeptide ion is encoded by its amino acid sequence and charge state, our analysis suggests that cross sections can be exploited as sequence-specific determinants of top-down fragment ions.

On the structural denaturation of biological analytes in trapped ion mobility spectrometry - mass spectrometry.

Key to native ion mobility/mass spectrometry is to prevent the structural denaturation of biological molecules in the gas phase. Here, we systematically assess structural changes induced in the protein ubiquitin during a trapped ion mobility spectrometry (TIMS) experiment. Our analysis shows that the extent of structural denaturation induced in ubiquitin ions is largely proportional to the amount of translational kinetic energy an ion gains from the applied electric field between two collisions with buffer gas particles. We then minimize the efficiency of the structural denaturation of ubiquitin ions in the gas phase during a TIMS experiment. The resulting "soft" TIMS spectra of ubiquitin are found largely identical to those observed on "soft" elevated-pressure ion mobility drift tubes and the corresponding calibrated cross sections are consistent with structures reported from NMR experiments for the native and A-state of ubiquitin. Thus, our analysis reveals that TIMS is useful for native ion mobility/mass spectrometry analysis.

Hybrid-virtual simulations for Canadian medical students during the COVID-19 pandemic.

Given the efficacy of simulations as a medical education tool, the inability to provide them during the COVID-19 pandemic may be detrimental to pre-clinical medical student learning. We developed hybrid simulations, where remote learner participants could direct an in-person assistant. This offered a learning opportunity that was more realistic than fully virtual simulations and abided by public health guidelines. Hybrid simulations provided an opportunity for medical students to practice real-time clinical decision making in a remote, high-fidelity, simulated environment. This approach could be adapted for rural healthcare students and professionals to participate in simulations without a local simulation centre.

BACKGROUND: Compte tenu de l'efficacité des simulations en tant qu'outil d'éducation médicale, l'impossibilité d'en proposer pendant la pandémie de la COVID-19 pourrait nuire à l'apprentissage préclinique des étudiants en médecine. Nous avons conçu des simulations hybrides, où les apprenants peuvent diriger à distance un assistant qui intervient en personne. Ces simulations permettent un apprentissage plus réaliste que celles qui sont entièrement virtuelles tout en respectant les directives en matière de santé publique. Les simulations hybrides ont permis aux étudiants en médecine de s'exercer à distance à la prise de décision clinique en temps réel dans un environnement simulé de haute fidélité. Cette approche pourrait être adaptée pour permettre aux étudiants et aux professionnels de la santé en milieu rural, qui ne disposent pas d'un centre de simulation local, de néanmoins participer à des simulations.

On the Preservation of Non-covalent Peptide Assemblies in a Tandem-Trapped Ion Mobility Spectrometer-Mass Spectrometer (TIMS-TIMS-MS).

Ion mobility spectrometry-mass spectrometry (IMS-MS) has demonstrated the ability to characterize structures of weakly-bound peptide assemblies. However, these assemblies can potentially dissociate during the IMS-MS measurement if they undergo energetic ion-neutral collisions. Here, we investigate the ability of tandem-trapped ion mobility spectrometry-mass spectrometry (TIMS-TIMS-MS) to retain weakly-bound peptide assemblies. We assess ion heating and dissociaton in the tandem-TIMS instrument using bradykinin and its assemblies as reference systems. Our data indicate that non-covalent bradykinin assemblies are largely preserved in TIMS-TIMS under carefully selected operating conditions. Importantly, we observe quadruply-charged bradykinin tetramers, which attests to the "softness" of our instrument. Graphical Abstract.