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Ion mobility has become a ubiquitous tool in many aspects of Analytical Chemistry due to its ability to separate compounds in the gas phase prior to feeding them to a Mass Spectrometer. To understand how this complex separation occurs, it is necessary to thoroughly explain the ion-gas interaction. In particular, this manuscript aims to describe the physics behind the collisions at high fields using the two-temperature approximation. The two-temperature theory has been recently employed to describe the mobility of polyatomic ions quite successfully and thus a proper account is warranted. A concise description is provided along with rigorous mathematical arguments behind its success at predicting the ion's drift velocity. Moreover, a thorough procedure for obtaining the equations (including the matrix elements) for higher-order mobility approximations is also provided with high detail, making this work suitable for beginners and experts in ion mobility. In particular, a discussion is brought forth on the choice of the base temperature and its relation to both the effective temperature and the drift velocity of the ion. A comparison between a 12-6-4 potential and the Maxwell model is made, pointing at the possible errors of using the Maxwell model for low- and high-field calculations. Using our in-house algorithm IMoS, successive approximations up to the fourth are tested against previous ones and against experimental results, showing both, asymptotic convergence, as well as a good agreement for monoatomic gases and small ions.

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody‐like protein, using an anti‐HER2 VHH as a model. The total chemical synthesis of the 125 amino‐acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs—whether synthetic or recombinant ones—were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH‐drug conjugates. [ABSTRACT FROM AUTHOR]

Flavonoids are a large class of dietary molecules, among which quercetin is the most ubiquitous, which undergo an extensive intestinal phase-II metabolism. We compared the in vivo metabolism of quercetin in healthy volunteers with two in vitro models, HT29 cells and 3 D human intestinal tissues. Supernatants of the in vitro experiments and the human intestinal fluids (HIF) were analyzed by LC-IMS-MS and LC-HRMS in a qualitative way. Quercetin glucuronides, sulfates and their methyl conjugates were detected in all three systems. The metabolic profiles were found to be different, both in terms of the metabolites produced and their relative proportions. In particular, quercetin sulfates were almost absent in supernatants from HT29 cells incubations while they were a major metabolite in HIF and also found in 3 D intestinal tissues incubations. IMS provided structural information as well as a third dimension of characterization, while HRMS brought increased sensitivity and MS/MS confirmation. HT29 cells are a useful tool to generate phase-II metabolites but do not represent the in vivo situation. 3 D intestinal tissues appear as a more relevant tool to study the intestinal phase-II metabolism of flavonoids. [ABSTRACT FROM AUTHOR]

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central β-strands, β1 and β3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15N relaxation that, in addition to the N- and C-termini, the β2-β3 loop undergoes fast backbone dynamics (ps-ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI-IMS-MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding β-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer-dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR. [ABSTRACT FROM AUTHOR]

Cyclic oligosaccharides are well known to interact with various metals, able to form supramolecular complexes with distinct sizes and shapes. However, the presence of various isomers in a sample, including positional isomers and conformers, can significantly impact molecular recognition, encapsulation ability and chemical reactivity. Therefore, it is crucial to have tools for deep samples probing and correlation establishments. The emerging ion mobility mass spectrometry (IM-MS) has the advantages to be rapid and sensitive, but is still in its infancy for the investigation of supramolecular assemblies. In the herein study, it was demonstrated that IM-MS is suitable to discriminate several isomers of cyclodextrins (CD)-metals complexes, used as cyclic oligosaccharide models. In this sense, we investigated branched 6-O-α-glucosyl- or 6-O-α-maltosyl-β-cyclodextrins (G 1 -β-CD and G 2 -β-CD) and their purely cyclic isomers: CD8 (γ-CD) and CD9 (δ-CD). The corresponding collision cross section (CCS) values were deducted for the main positive singly and doubly charged species. Experimental CCS values were matched with models obtained from molecular modelling. The high mobility resolving power and resolution enabled discrimination of positional isomers, identification of various conformers and accurate relative content estimation. These results represent a milestone in the identification of carbohydrate conformers that cannot be easily reached by other approaches. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Structural analysis of C 5 H 7 + using ion-mobility spectrometry. • Structures of C 5 H 7 + derived from the precursors MPD and cyclohexene were identical. • Dissociation pathway DFT calculation of MPD to cyclopentenium cations. The formation pathways, intermediates , and products of alkadienes dissociation reactions are fundamental to combustion chemistry, yet their understanding is limited. Herein, the C 5 H 7 + fragment ion derived from 3-methyl-1,4-pentadiene (MPD) was investigated using an ion mobility/mass spectrometer. The reduced mobilities (K 0) of the C 5 H 7 + fragment ions generated by MPD and cyclohexene (used to generate the fragment ion standard) correlated with effective temperature in He buffer gas, and showed that these fragments were identical cyclopentenium cations. The dissociation of MPD to cyclopentenium cations was investigated using density functional theory calculations, which revealed the feasibility of this dissociation pathway over other fragments. [ABSTRACT FROM AUTHOR]

In this study, the size and shape of supramolecular assemblies between cyclo-oligosaccharides and proton, ammonium or a series of alkali metals by electrospray coupled to trapped ion mobility-mass spectrometry (ESI-TIMS) have investigated. Native cyclodextrins (CD) were selected as models, and collision cross section (CCS) values were deducted for the main positive singly and doubly charged species. Experimental CCS values were in good agreement with those obtained from molecular modeling. Due to the high mobility resolving power and resolution, it was possible to highlight the presence of various conformers. Also, TIMS allowed to discriminate and estimate the content of various orientations from non-covalent nanotubes-based CD, involving secondary/secondary rim hydroxyl groups (head-to head), primary/secondary rim (head-to-tail) hydroxyl groups or primary/primary rim (tail-to-tail) hydroxyl groups interactions. Such results pave the way for a better knowledge of the topology of cyclo-oligosaccharides based supramolecular complexes, demonstrating that TIMS can be a particularly attractive molecular descriptor. [Display omitted] [ABSTRACT FROM AUTHOR]

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn to the nine-residue peptide hormone oxytocin (OT, Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH, having a disulfide bond between Cys and Cys residues). Zn binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn, we find evidence for two primary OT conformations, which arise because the Cys-Pro peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn, we determine binding constants in water of K = 1.43 ± 0.24 and 0.42 ± 0.12 μM, for the trans- and cis-configured populations, respectively. The Zn bound form of OT, having a cross section of Ω = 235 Å, has Pro in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro-Leu-Gly-NH, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn. In this way, the proline residue regulates Zn binding to OT and, hence, is important in receptor binding. [ABSTRACT FROM AUTHOR]

Amylin amyloid deposition is a common feature in the pancreas of type-2 diabetic (T2D) patients, and may play a role in islet beta-cell death and disease development. Type-2 diabetes is also characterised by a dysregulation of systemic copper. While the relationship between these features is unclear, previous work has shown that copper can form a complex with amylin, which in turn prevents amylin misfolding, oligomerisation and fibril formation, and that this in turn decreases amylin-mediated cytotoxicity. To date, the nature of this interaction has not been determined. In this study, we have used ion mobility mass spectrometry to probe the effects of copper on amylin oligomer formation and to define the binding site(s) of copper on the amylin peptide. Here we show that there are two distinct sites to which copper can bind. The first is at a location in the C-terminal 5 amino acids of the peptide. The second is on Ala25, a residue which is known to be critical for the misfolding ability of human amylin. This interaction is not observed under standard mass spectrometry (acidic) conditions but is present at physiological pH values. These data further characterise the amylin–copper interaction, and may suggest a potential trigger for amylin misfolding in T2D. [ABSTRACT FROM AUTHOR]

Abstract: The substrate-free hydrolysis of Zn nanocrystals was investigated as the second step in a ZnO/Zn solar water-splitting thermochemical cycle. In this work, we combined two different ion-mobility schemes in series to study the hydrolysis kinetics of size-selected zinc nanocrystals (NCs). The first mobility characterization size selects particles with a differential mobility analyzer (DMA). The second mobility characterization employs an aerosol particle mass analyzer (APM) and measures changes in mass resulting from a controlled hydrolysis of the Zn NCs. A low temperature reaction mechanism is proposed to explain the mass change behavior of Zn NCs hydrolysis at 100–250°C. An Arrhenius law was used to extract the reaction kinetic parameters. The hydrolysis activation energy and the order of the reaction with respect to water mole fraction were found to be 24±2kJ/mol and 0.9±0.1, respectively. Complete conversion of 70nm Zn NCs was achieved at 175°C with a residence time of about 10s and water vapor mole fraction of 19%. [Copyright &y& Elsevier]

Abstract: The ion–neutral interaction potentials of HeO+ and HeO− are calculated from experimental gaseous ion-mobility data by using the direct inversion method. The obtained potentials are in good agreement with recent ab initio potentials, and they were used to obtain best parameters for the Morse–Spline–Van der Waals (MSV) potential model. The MSV potentials were then employed to compute the mobilities of O+ and O− ions in helium gas. Results show that the MSV model potentials reproduce the experimental ion mobilities within their experimental accuracy. [Copyright &y& Elsevier]

Abstract: We report ion-mobility measurements with a modified triple quadrupole mass spectrometer fitted with an ion molecule reactor (IMR) designed to investigate ion molecule reactivity in organic mass spectrometry. Functionalized pentacene ions, which are generally unreactive were chosen for study to decouple drift/diffusion effects from reactivity (including clustering). The IMR is equipped with a variable axial electrostatic drift field (ADF) and is able to trap ions. These capabilities were successfully employed in the measurement of ion mobilities in different modes of IMR operation. Theoretical modeling of the drift dynamics and the special localization of the large ion packet was successfully implemented. The contribution of the quadrupole RF field to the drift dynamics also was taken into consideration. [Copyright &y& Elsevier]

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology. Image 1 • VHH are small antibody-like proteins. They are entering the therapeutic arsenal. • VHH purity, homogeneity, structure and affinity towards their targets are described. • An analytical workflow to qualify those proteins is presented to validate the molecules. [ABSTRACT FROM AUTHOR]

In the continuous exploration of the VHH chemistry, biochemistry and therapeutic future use, we investigated two different production strategies of this small antibody-like protein, using an anti-HER2 VHH as a model. The total chemical synthesis of the 125 amino-acid peptide was performed with reasonable yield, even if optimization will be necessary to upgrade this kind of production. In parallel, we expressed the same sequence in two different hosts: Escherichia coli and Pichia pastoris. Both productions were successful and led to a fair amount of VHHs. The integrity and conformation of the VHH were characterized by complementary mass spectrometry approaches, while surface plasmon resonance experiments were used to assess the VHH recognition capacity and affinity toward its "antigen." Using this combination of orthogonal techniques, it was possible to show that the three VHHs-whether synthetic or recombinant ones-were properly and similarly folded and recognized the "antigen" HER2 with similar affinities, in the nanomolar range. This opens a route toward further exploration of modified VHH with unnatural amino acids and subsequently, VHH-drug conjugates., (© 2019 The Protein Society.)

Aims: There is limited knowledge about the association of lipoprotein particles and markers of coronary atherosclerosis such as coronary artery calcification (CAC) in relatively young high-risk persons. This study examines the association of lipoprotein subfractions and CAC in high cardiometabolic risk individuals., Methods: The study presents analysis from baseline data of a randomized trial targeted at high-risk workers. Employees of Baptist Health South Florida with metabolic syndrome or diabetes were recruited. At baseline, all 182 participants had lipoprotein subfraction analysis using the ion mobility technique and participants above 35 years (N=170) had CAC test done. Principal components (PC) were computed for the combination of lipoprotein subclasses. Multiple bootstrapped regression analyses (BSA) were conducted to assess the relationship between lipoprotein subfractions and CAC., Results: The study population (N=170) was largely female (84%) with a mean age of 58 years. Three PCs accounted for 88% variation in the sample. PC2, with main contributions from VLDL particles in the positive direction and large LDL particles in the negative direction was associated with a 22% increase in CAC odds (P value ＜0.05 in 100% of BSA). PC3, with main contributions from HDL lipoprotein particles in the positive direction and small/medium LDL and large IDL particles in the negative direction, was associated with a 9% reduction in CAC odds (P＜0.05 in 88% of BSA). PC1, which had approximately even contributions from HDL, LDL, IDL and VLDL lipoprotein subfractions in the positive direction, was not associated with CAC., Conclusion: In a relatively young but high-risk population, a lipoprotein profile predominated by triglyceride-rich lipoproteins was associated with increased risk of CAC, while one predominated by HDL lipoproteins offered modest protection. Lipoprotein sub-fraction analysis may help to further discriminate patients who require more intensive cardiovascular work-up and treatment.

A method is proposed to predict ions' collision cross-sectional area from properties that are already evaluated in standard quantum chemistry software. Computed molecular isodensity surface areas recover the predictions of existing projection approximations. Computed solvent cavity areas give comparable accuracy. This provides a simplified workflow for assigning ion-mobility mass spectra., (Copyright © 2018 John Wiley & Sons, Ltd.)