Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Multiple and cooperative binding of fluorescence light-up probe thioflavin t with human telomere DNA G-quadruplex

Thioflavin T (ThT), a typical probe for protein fibrils, also binds human telomeric G-quadruplexes with a fluorescent light-up signal change and high specificity against DNA duplexes. Cell penetration and low cytotoxicity of fibril probes having been widely established, modifying ThT and other fibril probes is an attractive means of generating new G-quadruplex ligands. Thus, elucidating the binding mechanism is important for the design of new drugs and fluorescent probes based on ThT. Here, we investigated the binding mechanism of ThT with several variants of the human telomeric sequence in the presence of monovalent cations. Fluorescence titrations and electrospray ionization mass spectrometry (ESI-MS) analyses demonstrated that each G-quadruplex unit cooperatively binds to several ThT molecules. ThT brightly fluoresces when a single ligand is bound to the G-quadruplex and is quenched as ligand binding stoichiometry increases. Both the light-up signal and the dissociation constants are exquisitely sensitive to the base sequence and to the G-quadruplex structure. These results are crucial for the sensible design and interpretation of G-quadruplex detection assays using fluorescent ligands in general and ThT in particular. © 2013 American Chemical Society

Electronic Spectroscopy of Isolated DNA Polyanions

In solution, UV-vis spectroscopy is often used to investigate structural changes in biomolecules (i.e., nucleic acids), owing to changes in the environment of their chromophores (i.e., the nucleobases). Here we address whether action spectroscopy could achieve the same for gas-phase ions, while taking the advantage of additional spectrometric separation of complex mixtures. We therefore systematically studied the action spectroscopy of homo-base 6-mer DNA strands (dG6, dA6, dC6, dT6) and discuss the results in light of gas-phase structures validated by ion mobility spectrometry and infrared ion spectroscopy, of electron binding energies measured by photoelectron spectroscopy, and of calculated electronic photo-absorption spectra. When UV photons interact with oligonucleotide polyanions, two main actions may take place: (1) fragmentation and (2) electron detachment. The action spectra reconstructed from fragmentation follow the absorption spectra well, and result from multiple cycles of absorption and internal conversion. The action spectra reconstructed from the electron photodetachment (ePD) efficiency reveal interesting phenomena: ePD depends on the charge state because it depends on electron binding energies. We illustrate with the G-quadruplex [dTG4T]4 that the ePD action spectrum shifts with the charge state, pointing to possible caveats when comparing the spectra of systems having different charge densities to deduce structural parameters. Moreover, ePD is particularly efficient for purines but not pyrimidines. ePD thus reflects not only absorption, but also particular relaxation pathways of the electronic excited states. As these pathways lead to photo-oxidation, their investigation on model gas-phase systems may prove useful to elucidate mechanisms of photo-oxidative damages, which are linked to mutations and cancers

Compaction of RNA Hairpins and Their Kissing Complexes in Native Electrospray Mass Spectrometry

When electrosprayed from typical native MS solution conditions, RNA hairpins and kissing complexes acquire charge states at which they get significantly more compact in the gas phase than their initial structure in solution. Here we show the limits of using force field molecular dynamics to interpret the gas-phase structures of nucleic acid complexes in the gas phase, and we suggest that higher-level calculation levels should be used in the future.<br /

Distinctive structural motifs of RNA G-quadruplexes composed of AGG, CGG and UGG trinucleotide repeats

International audienceTrinucleotide repeats are microsatellite sequences that are polymorphic in length. Their expansion in specific genes underlies a number of neurode-generative disorders. Using ultraviolet-visible, circular dichroism, nuclear magnetic resonance (NMR) spectroscopies and electrospray ionization mass spectrometry, the structural preferences of RNA molecules composed of two and four repeats of AGG, CGG and UGG in the presence of K + , Na + and NH 4 + were analysed. (AGG) 2 A, (AGG) 4 A, p(UGG) 2 U and p(UGG) 4 U strongly prefer folding into G-quadruplexes, whereas CGG-containing sequences can adopt different types of structure depending on the cation and on the number of repeats. In particular, the two-repeat CGG sequence folds into a G-quadruplex in potassium buffer. We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG) 2 A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG) 2 C and p(UGG) 2 U, respectively. Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellu-lar environment on the folding. Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K + ions

Native Ion Mobility Mass Spectrometry: when Gas Phase Ion Structures Depend on the Electrospray Charging Process

Ion mobility spectrometry (IMS) has become popular to characterize biomolecule folding. Numerous studies have shown that proteins that are folded in solution remain folded in the gas phase, whereas proteins that are unfolded in solution adopt more extended conformations in the gas phase. Here, we discuss how general this tenet is. We studied single-stranded DNAs (human telomeric cytosine-rich sequences with CCCTAA repeats), which fold into an intercalated motif (i-motif) structure in a pH-dependent manner, thanks to the formation of C‒H+‒C base pairs. As i-motif formation is favored at low ionic strength, we could investigate the ESI-IMS-MS behavior of i-motif structures at pH ~5.5 over a wide range of ammonium acetate concentrations (15 mM to 100 mM). The control experiments consisted of either the same sequence at pH ~7.5, wherein the sequence is unfolded, or sequence variants that cannot form i-motifs (CTCTAA repeats). The surprising results came from the control experiments. We found that the ionic strength of the solution had a greater effect on the compactness of the gas-phase structures than the solution folding state. This means that electrosprayed ions keep a memory of the charging process, which is influenced by the electrolyte concentration. We discuss these results in light of the analyte partitioning between the droplet interior and droplet surface, which in turn influences the probability of being ionized via a charged residue pathway or a chain extrusion pathway.<br /

DNA-based nanostructures: the effect of the base sequence on octamer formation from d(XGGYGGT) tetramolecular G-quadruplexes

In a previous work we have demonstrated that the DNA sequence CGGTGGT folds into a higher order G-quadruplex structure (2Q), obtained by the 5'-5' stacking of two unusual G(:C):G(:C):G(:C):G(:C) planar octads belonging to two identical tetra-stranded parallel quadruplexes, when annealed in the presence of ammonium or potassium ions. In the present paper, we discuss the role played by the title nucleosides X and Y (where X and Y stand for A, C, G, or T) on the formation and stability of 2Q structures formed by the XGGYGGT oligodeoxynucleotides. We found that the above mentioned dimerization pathway is not peculiar to the CGGTGGT sequence, but is possible for all the remaining CGGYGGT sequences (with Y = A, C, or G). Furthermore, we have found that the TGGAGGT sequence, despite the absence of the 5'-ending C, is also capable of forming a 2Q-like higher order quadruplex by using a slightly different dimerization interface, as characterized by NMR spectroscopy. To the best of our knowledge, this is the first characterization of a quadruplex multimer formed by an oligodeoxynucleotide presenting a thymine at its 5'-end. Examples of such structures were observed previously only in crystals and in the presence of non-physiological cations. Our results expand the repertoire of DNA quadruplex nanostructures of chosen length and add further complexity to the structural polymorphism of G-rich DNA sequences

Thiosugar naphthalene diimide conjugates: G-quadruplex ligands with antiparasitic and anticancer activity

International audienceGlycosyl conjugation to drugs is a strategy being used to take advantage of glucose transporters (GLUT) overexpression in cancer cells in comparison with non-cancerous cells. Its extension to the conjugation of drugs to thiosugars tries to exploit their higher biostability when compared to O-glycosides. Here, we have synthesized a series of thiosugar naphthalene diimide conjugates as G-quadruplex ligands and have explored modifications of the amino sidechain comparing dimethyl amino and morpholino groups. Then, we studied their antiproliferative activity in colon cancer cells, and their antiparasitic activity in T. brucei and L. major parasites, together with their ability to bind quadruplexes and their cellular uptake and location. We observed higher toxicity for the sugar-NDI-NMe2 derivatives than for the sugar-NDI-morph compounds, both in mammalian cells and in parasites. Our experiments indicate that a less efficient binding to quadruplexes and a worse cellular uptake of the carb-NDI-morph derivatives could be the reasons for these differences. We found small variations in cytotoxicity between O-carb-NDIs and S-carb-NDIs, except against non-cancerous human fibroblasts MRC-5, where thiosugar-NDIs tend to be less toxic. This leads to a notable selectivity for β-thiomaltosyl-NDI-NMe212 (9.8 fold), with an IC50 of 0.3 μM against HT-29 cells. Finally, the antiparasitic activity observed for the carb-NDI-NMe2 derivatives against T. brucei was in the nanomolar range with a good selectivity index in the range of 30- to 69- fold

Software for the Analysis and Interpretation of Native Mass Spectrometry Data

The last few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalysed by the availability of commercial instrumentation capable of carrying out such analyses. Like in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realise the full potential of the datasets generated. Here we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS datasets, and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardised processing approaches and serve as a location for the deposition of data for this emerging field.</div