Correlation between the shape of the ion mobility signals and the stepwise folding process of polylactide ions

In the field of polymer characterization, the use of ion mobility mass spectrometry (IMMS) remains mainly devoted to the temporal separation of cationized oligomers according to their charge states, molecular masses and macromolecular architectures in order to probe the presence of different structures. When analyzing multiply charged polymer ions by IMMS, the most striking feature is the observation of breaking points in the evolution of the average collision cross sections with the number of monomer units. Those breaking points are associated to the folding of the polymer chain around the cationizing agents. Here, we scrutinize the shape of the arrival time distribution (ATD) of polylactide ions and associate the broadening as well as the loss of symmetry of the ATD signals to the coexistence of different populations of ions attributed to the transition from opened to folded stable structures. The observation of distinct distributions reveals the absence of folded/extended structure interconversion on the ion mobility time scale (1–10 ms) and then on the lifetime of ions within the mass spectrometer at room temperature. In order to obtain information on the possible interconversion between the different observed populations upon ion activation, we performed IM–IM–MS experiments (tandem ion mobility measurements). To do so, mobility‐selected ions were activated by collisions before a second mobility measurement. Interestingly, the conversion by collisional activation from a globular structure into a (partially) extended structure, i.e. the gas phase unfolding of the ions, was not observed in the energetic regime available with the used experimental setup. The absence of folded/extended interconversion, even upon collisional activation, points to the fact that the polylactide ions are ‘frozen’ in their specific 3D structure during the desolvation/ionization electrospray processes. Copyright © 2017 John Wiley & Sons, Ltd.     

Conformations of cationized linear oligosaccharides revealed by FTMS combined with in‐ESI H/D exchange

Previously (Kostyukevich et al. Anal Chem 2014, 86, 2595), we have reported that oligosaccharides anions are produced in the electrospray in two different conformations, which differ by the rate of gas phase hydrogen/deuterium (H/D) exchange reaction. In the present paper, we apply the in‐electrospray ionization (ESI) source H/D exchange approach for the investigation of the oligosaccharides cations formed by attaching of metal ions (Na, K) to the molecule. It was observed that the formation of different conformers can be manipulated by varying the temperature of the desolvating capillary of the ESI interphase. Separation of the conformers was performed using gas phase H/D approach. Because the conformers have different rates of the H/D exchange reaction, the deuterium distribution spectrum becomes bimodal. It was found that the conformation corresponding to the slow H/D exchange rate dominates in the spectrum when the capillary temperature is low (~200 °C), and the conformation corresponding to the fast H/D exchange rate dominates at high (~400 °C) temperatures. In the intermediate temperature region, two conformers are present simultaneously. It was also observed that large oligosaccharide requires higher temperature for the formation of another conformer. It was found that the presence of the conformers considerably depends on the solvent used for ESI and the pH. We have compared these results with the previously performed in‐ESI source H/D exchange experiments with peptides and proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

Structure and Dynamics of Oligonucleotides in the Gas Phase

By combining ion‐mobility mass spectrometry experiments with sub‐millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas‐phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi‐picosecond, nanosecond, and sub‐millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re‐evaluation on our view of the physics of nucleic acids upon vaporization is needed.     

Retention of Native Protein Structures in the Absence of Solvent: A Coupled Ion Mobility and Spectroscopic Study

Can the structures of small to medium‐sized proteins be conserved after transfer from the solution phase to the gas phase? A large number of studies have been devoted to this topic, however the answer has not been unambiguously determined to date. A clarification of this problem is important since it would allow very sensitive native mass spectrometry techniques to be used to address problems relevant to structural biology. A combination of ion‐mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β‐lactoglobulin, which are prototypical examples of helical and β‐sheet proteins, respectively. The results show that for low charge states under gentle conditions, aspects of the native secondary and tertiary structure can be conserved.     

“Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding,CEST‐NMR Experiments,and Molecular Dynamics Calculations

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two‐ and three‐state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide‐rich protein. In addition, sensitive ¹⁵N‐CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction.     

Reaction Dynamics and Solution Chemistry of Polyoxometalates by Electrospray Ionization Mass Spectrometry

Mass spectrometry both complements other analytical techniques and allows for types of analyses and experiments not possible with common analytical methods, such as NMR, IR, and UV/Vis spectroscopy. Electrospray constitutes one of the mildest forms of ionization, making it the preferred method for the analysis of large fragile or reactive ions. There is particular promise for mass spectrometry in aiding the characterization of polyoxometalates and their solutions, but caution must be taken in designing the experiments in order to yield reliable data and to avoid the temptation of over‐interpreting the relevance of gas‐phase data to solution chemistry.     

Mass Spectrometry Quantifies Protein Interactions—From Molecular Chaperones to Membrane Porins

Proteins possess an intimate relationship between their structure and function, with folded protein structures generating recognition motifs for the binding of ligands and other proteins. Mass spectrometry (MS) can provide information on a number of levels of protein structure, from the primary amino acid sequence to its three‐dimensional fold and quaternary interactions. Given that MS is a gas‐phase technique, with its foundations in analytical chemistry, it is perhaps counter‐intuitive to use it to study the structure and non‐covalent interactions of proteins that form in solution. Herein we show, however, that MS can go beyond simply preserving protein interactions in the gas phase by providing new insight into dynamic interaction networks, dissociation mechanisms, and the cooperativity of ligand binding. We consider potential pitfalls in data interpretation and place particular emphasis on recent studies that revealed quantitative information about dynamic protein interactions, in both soluble and membrane‐embedded assemblies.     

Conformational Distribution of Bradykinin [bk?+?2?H]2+ Revealed by Cold Ion Spectroscopy Coupled with FAIMS

We employ cold ion spectroscopy (CIS) in conjunction with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to study the peptide bradykinin in its doubly protonated charge state ([bk?+?2?H](2+)). Using FAIMS, we partially separate the electrosprayed [bk?+?2?H](2+) ions into two conformational families and selectively introduce one of them at a time into a cold ion trap mass spectrometer, where we probe them by UV photofragment spectroscopy. Although the two conformational families have distinct electronic spectra, some cross-conformer contamination can be observed under certain conditions. We demonstrate that this contamination comes from isomerization of ions energized during and/or after their separation and not from incomplete separation of the initially electrosprayed conformations in the FAIMS stage. By varying the injection voltage of the ions into our mass spectrometer, we can intentionally induce isomerization to produce what seems to be a gas phase equilibrium distribution of conformers. This distribution is different from the one produced initially by electrospray, indicating that some of the conformers are kinetically trapped and may be related to conformers that are more favored in solution.     

Polarization induced electrospray ionization mass spectrometry for the analysis of liquid,viscous and solid samples

In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.     

Uncovering the Stoichiometry of Pyrococcus furiosus RNase P,a Multi‐Subunit Catalytic Ribonucleoprotein Complex,by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry

We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg²⁺. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg²⁺‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21?RPP29 and POP5?RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21?RPP29 and (POP5?RPP30)₂ complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.     

The Role of the Detergent Micelle in Preserving the Structure of Membrane Proteins in the Gas Phase

Despite the growing importance of the mass spectrometry of membrane proteins, it is not known how their transfer from solution into vacuum affects their stability and structure. To address this we have carried out a systematic investigation of ten membrane proteins solubilized in different detergents and used mass spectrometry to gain physicochemical insight into the mechanism of their ionization and desolvation. We show that the chemical properties of the detergents mediate the charge state, both during ionization and detergent removal. Using ion mobility mass spectrometry, we monitor the conformations of membrane proteins and show how the surface charge density dictates the stability of folded states. We conclude that the gas‐phase stability of membrane proteins is increased when a greater proportion of their surface is lipophilic and is consequently protected by the physical presence of the micelle.     

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Biologically Relevant Metal-Cation Binding Induces Conformational Changes in Heparin Oligosaccharides as Measured by Ion Mobility Mass Spectrometry

Heparin interacts with many proteins and is involved in biological processes such as anticoagulation, angiogenesis, and antitumorigenic activities. These heparin-protein interactions can be influenced by the binding of various metal ions to these complexes. In particular, physiologically relevant metal cations influence heparin-protein conformations through electronic interactions inherent to this polyanion. In this study, we employed ion mobility mass spectrometry (IMMS) to observe conformational changes that occur in fully-sulfated heparin octasaccharides after the successive addition of metal ions. Our results indicate that binding of positive counter ions causes a decrease in collision cross section (CCS) measurements, thus promoting a more compact octasaccharide structure.     

Some critical aspects in the determination of binding constants by electrospray ionisation mass spectrometry at the example of arsenic bindings to sulphur‐containing biomolecules

The influences of reactant concentrations, solvent type, acid strength, pH conditions and ionic strength on the determination of apparent gas‐phase equilibrium constants K using electrospray ionisation mass spectrometry (ESI‐MS) were elucidated. As example serves the interaction of the tripeptide glutathione (GSH) with phenylarsine oxide (PAO). It was shown that rising initial concentrations of both reactants were not adequately compensated by increasing signal intensities of the reaction products in the mass spectra. The equilibrium constant for the formation of the phenylarsenic‐substituted peptide species decreased from 1.42 × 10⁵ ± 1.81 × 10⁴ l µmol^?1 to 1.54 × 10⁴ ± 1.5 × 10³ l µmol^?1 with rising initial GSH concentrations from 1 to 10 µM at fixed PAO molarity of 50 µM . K values resulting from a series with a fixed GSH molarity of 5 µM and a PAO molarity varied from 10 to 100 µM remained in a narrower range between 4.59 × 10⁴ ± 2.15 × 10⁴ l µmol^?1 and 1.07 × 10⁴ ± 4.0 × 10³ l µmol^?1. In contrast, consumption numbers calculated from the ion intensity ratios of reaction products to the unreacted peptide were not influenced by the initial reactant concentrations. In a water–acetonitrile–acetic acid mixture (48:50:2, v:v), the consumption of 5 µ M GSH increased from 8.3 ± 1.4% to 39.6 ± 1.6% with increased molar excess of PAO from 2 to 20, respectively. The GSH consumption was considerably enhanced in a changed solvent system consisting of 25% acetonitrile and 75% 10 mM ammonium formate, pH 5.0 (v:v) up to 80% of the original peptide amount at an only threefold molar arsenic excess. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous Determination 9 Anabolic Steroids Residues in Animal Muscle Tissues by Gas Chromatography‐Mass Spectrometry

Abstract

A method was developed for determining 9 anabolic steroids (ASs) residues in animal muscle tissues by gas chromatography‐mass spectrometry (GC/MS). After undergoing enzymolysis, being homogenized, and then being picked‐up by ultrasound the sample was extracted with tert‐Butyl methyl ether, cleaned up through solid‐phase extraction (SPE) based on the reverse phase (RP) principle, and after derivatization of the analyst of interest, analyzed by GC/MS under selective ion monitoring (SIM). The limits of the detection (LOD) of the GC/MS method used for testing epitestosterone (ETS), nandrolon (17β‐NT), 17α‐methyl‐testosterone (MTS), testosterone 17‐propionate (PTS), 17β‐estradiol 3‐benzoate (BES), estrone (ESN), 17β‐estradiol (17β‐ES), 17α‐ethynylestradiol (EES), and estriol (EST) in animal muscle ranged from 0.10 to 0.33 µg/kg and the limits of quantification (LOQ) were from 0.24 to 0.82 µg/kg. The experiments using spiked samples, such as pork, beef, chicken, and fish, made it clear that at addition level of 2.0 µg/kg, the average recovery of the ASs ranged from 62.5% to 80.5%, and the coefficient of variation ranged from 12.5% to 26.8%, while at an addition level of 5.0 µg/kg, the average recovery was from 70.8% to 86.4%, and the coefficient of variation was from 8.8% to 18.4%.     

Evaluation of different two‐dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large‐scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide‐level fractionation, the first dimension of separation was performed with high‐pH reversed‐phase chromatography (pH‐RP) and strong cation exchange chromatography (SCX). Regarding protein‐level fractionation, C₈ protein reversed‐phase (C₈‐RP Prot) and high‐recovery protein reversed‐phase (hr‐RP Prot) were used instead. The second dimension consisted of a reversed‐phase nano‐HPLC on‐Chip coupled to an electrospray ionization quadrupole time‐of‐flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide‐level pH‐RP and the hr‐RP Prot protein‐level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide‐level SCX, with 509 proteins identified, was the worst method. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐phase protomers of p‐(dimethylamino)chalcone investigated by travelling‐wave ion mobility mass spectrometry (TWIMS)

Results from ion‐mobility (IM) separation experiments demonstrate that O‐ and N‐protomers of p‐(dimethylamino)chalcone (p‐DMAC) can coexist in the gas phase. The relative populations of the two protomers strongly depend on the ion‐generating settings and the conditions the precursor ions experience from the point of their gas‐phase inception to the time of their detection. Under relatively dry source conditions, the ratio of the gas‐phase protomers generated under helium‐plasma ionization (HePI) conditions is biased towards the thermodynamically favored O‐protomer. However, when the humidity of the enclosed ion source was increased, the IM arrival‐time distribution profile of the mass‐selected protonated precursor of p‐DMAC changed rapidly to one dominated by the N‐protomer. Under spray‐ionization conditions, the formation of the thermodynamically less favored protomer has been generally attributed to a phenomenon called kinetic trapping. Herein, we demonstrate that the population of thermodynamically less favored N‐protomer can be dramatically increased simply by introducing water vapor to the HePI ion source.     

Ion mobility spectrometry – Mass spectrometry coupling for synthetic polymers

This review covers applications of ion mobility spectrometry (IMS) hyphenated to mass spectrometry (MS) in the field of synthetic polymers. MS has become an essential technique in polymer science, but increasingly complex samples produced to provide desirable macroscopic properties of high‐performance materials often require separation of species prior to their mass analysis. Similar to liquid chromatography, the IMS dimension introduces shape selectivity but enables separation at a much faster rate (milliseconds vs minutes). As a post‐ionization technique, IMS can be hyphenated to MS to perform a double separation dimension of gas‐phase ions, first as a function on their mobility (determined by their charge state and collision cross section, CCS), then as a function of their m/z ratio. Implemented with a variety of ionization techniques, such coupling permits the spectral complexity to be reduced, to enhance the dynamic range of detection, or to achieve separation of isobaric ions prior to their activation in MS/MS experiments. Coupling IMS to MS also provides valuable information regarding the 3D structure of polymer ions in the gas phase and regarding how to address the question of how charges are distributed within the structure. Moreover, the ability of IMS to separate multiply charged species generated by electrospray ionization yields typical IMS‐MS 2D maps that permit the conformational dynamics of synthetic polymer chains to be described as a function of their length.     

Determination of Volatile Disinfection Byproducts in Water by Gas Chromatography–Triple Quadrupole Mass Spectrometry

Chlorination disinfection byproducts have drawn significant attention in water quality research during the last decades, due to their adverse effects on public health. A method is reported for the determination of trihalomethanes, haloacetonitriles, haloketones, and chloropicrin based on liquid–liquid extraction and gas chromatography–tandem mass spectrometry. The optimum electron ionization energy was determined and precursor ions and product ions for thirteen volatile disinfection byproducts were identified. The method provides rapid chromatographic analysis (10 minutes) and good separation. The linear dynamic range extended from 0.05 to 100 µg L^?1 (r² = 0.9983 – 0.9997, n=10) and the limits of detection (LODs) were between 0.003 and 0.014 µg L ^?1 . The intra-day (n=5) and inter-day (n=6) relative standard deviations were in the ranges of 2.81–8.22% and 3.48–10.85%, respectively. The method was validated by measurement of the recoveries of fortified surface, ground, and wastewater to be 74.7–115.4%, 86.1–120.6%, and 81.6–126.1%, respectively.     

Synthesis of Organoselenium‐Modified β‐Cyclodextrins Possessing a 1,2‐Benzisoselenazol‐3(2H)‐one Moiety and Their Enzyme‐Mimic Study

Six novel H₂O‐soluble β‐cyclodextrin derivatives containing a 1,2‐benzisoselenazol‐3(2H)‐one moiety were synthesized by a convenient method in 25–60% yield and characterized by MS, elemental analysis, IR, ¹H‐NMR, and UV/VIS spectroscopy. The conformations of these β‐cyclodextrin derivatives 1 – 6 were analyzed by circular dichroism and fluorescence‐lifetime experiments. The superoxide dismutase (SOD) activities of 1 – 6 were determined by auto‐oxidation of pyrogallol at 25.0° in buffer solution (pH 8.2), giving relatively high SOD activities of up to 121–330 U/mg. Also, the glutathione peroxidase (GPX) activities of hosts 1 – 6 , determined by the method of Wilson at 37° in buffer solution (pH 7.0), show good GPX activities in the range of 0.34–0.86 U/μmol. The mimicking results of the bifunctional artificial enzyme models 1 – 6 were globally compared with regard to their structural and conformational difference.