Traveling-wave Ion Mobility Mass Spectrometry Research Articles

Salt-Bridged Peptide Anion Gaseous Structures Enable Efficient Negative Ion Electron Capture Dissociation.

We previously discovered that electron attachment to gaseous peptide anions can occur within a relatively narrow electron energy range. The resulting charge-increased radical ions undergo dissociation analogous to conventional cation electron capture/transfer dissociation (ECD/ETD), thus enabling a novel tandem mass spectrometry (MS/MS) technique that we termed negative ion electron capture dissociation (niECD). We proposed that gaseous zwitterionic structures are required for niECD with electron capture either occurring at or being directed by a positively charged site. Here, we further evaluate this zwitterion mechanism by performing niECD of peptides derivatized to alter their ability to form zwitterionic gaseous structures. Introduction of a fixed positive charge tag, a highly basic guanidino group, or a highly acidic sulfonate group to promote zwitterionic structures in singly charged anions, rescued the niECD ability of a peptide refractory to niECD in its unmodified form. We also performed a systematic study of five sets of synthetic peptides with decreasing zwitterion propensity and found that niECD efficiency decreased accordingly, further supporting the zwitterion mechanism. However, traveling-wave ion mobility-mass spectrometry experiments, performed to gain further insight into the gas-phase structures of peptides showing high niECD efficiency, exhibited an inverse correlation between the orientationally averaged collision cross sections and niECD efficiency. These results indicate that compact salt-bridged structures are also a requirement for effective niECD.

Gas-phase Ni(II) affinities of alternative metal binding peptides from competitive threshold collision-induced dissociation

The Ni(II) affinity of the polyhistidine tag is used in the purification of recombinant proteins by immobilized metal affinity chromatography. Here we measured the relative gas-phase Ni(II) affinities of four alternative metal binding (amb) peptides and the 7xHis tag using the competitive threshold collision-induced dissociation (TCID) technique. The general primary structure of the four amb peptides was acetyl-Aa1-Aa2-Gly3-Pro4-Aa5-Gly6-Cys7, designed to test whether the His1-Cys2 or Asp1-His2 would exhibit the higher Ni(II) affinity and whether the Tyr5 with its ability of forming long-range π-nickel interaction and hydrogen bonding would contribute to the Ni(II) affinity. The Cys7 is retained in all the amb sequences because previous research has shown that both the thiolate side group and carboxylate terminus simultaneously coordinate the metal ion. The Ni(II) affinity was measured using the dissociation of the [amb + Ni(II) + NTA]− complex, where NTA = nitrilotriacetic acid, which is a commonly used ligand for Ni(II) inside the IMAC column. The dissociation of [amb + Ni(II) + NTA]− produced two main product channels; [amb + Ni(II)]− + NTA and [NTA + Ni(II)]− + amb, whose competition was modeled by TCID to extract the relative gas-phase Ni(II) affinities. Extensive molecular modeling using PM6 located the low-energy structures whose molecular parameters were used in the TCID analyses if their collision cross sections agreed with those measured by traveling-wave ion mobility mass spectrometry and they were compatible with a concerted reaction. We compare the final results by conducting the TCID analyses using alternative PM6 parameters and by making the ternary complexes in acidic and basic solutions.

Automated Structural Activity Screening of β-Diketonate Assemblies with High-Throughput Ion Mobility-Mass Spectrometry.

Embracing complexity in design, metallo-supramolecular self-assembly presents an opportunity for fabricating materials of economic significance. The array of accessible supramolecules is alluring, along with favourable energy requirements. Implementation is hampered by an inability to efficiently characterise complex mixtures. The stoichiometry, size, shape, guest binding properties and reactivity of individual components and combinations thereof are inherently challenging to resolve. A large combinatorial library of four transition metals (Fe, Cu, Ni and Zn), and six β-diketonate ligands at different molar ratios and pH was robotically prepared and directly analysed over multiple timepoints with electrospray ionisation travelling wave ion mobility-mass spectrometry. The dataset was parsed for self-assembling activity without first attempting to structurally assign individual species. Self-assembling systems were readily categorised without manual data-handling, allowing efficient screening of self-assembly activity. This workflow clarifies solution phase supramolecular assembly processes without manual, bottom-up processing. The complex behaviour of the self-assembling systems was reduced to simpler qualities, which could be automatically processed.

Multi-functionalized Metallo-Supramolecular Spoked Wheels: Their Emission-Tunable Hierarchically Assembled System with Dye Molecules.

Supramolecular architectures with multiple emissive units are especially appealing due to their desired properties, such as artificial light harvesting and white-light emission. But fully achieving multi-wavelength photoluminescence in a single supramolecular architecture remains a challenge. In this paper, functionalized supramolecular architectures containing twelve metal centers and six pyrene moieties were nearly quantitatively synthesized by multi-component self-assembly and fully characterized by 1D and 2D nuclear magnetic resonance, dynamic light scattering, electrospray ionization mass spectrometry, traveling-wave ion mobility mass spectrometry, and transmission electron microscopy. Moreover, the hierarchical nano-assemblies were prepared by introducing anionic dyes to the positively charged self-assembled framework, which contained three luminescence centers, namely, pyrene, tpy-Cd coordination parts, and Sulforhodamine B anions. Such a hierarchically assembled system displayed tunable emission by taking full advantage of aggregation-induced emission enhancement, aggregation-caused quenching, and fluorescence resonance energy transfer effects and showed the diverse emission colors. This research provides a new insight for constructing multiple emissive metallo-supramolecular assemblies.

Extending the competitive threshold collision-induced dissociation of Zn(II) ternary complexes using traveling-wave ion mobility-mass spectrometry

The competitive threshold collision-induced dissociation (TCID) of the ternary complex [amb5+Zn(II)+NTA]− → [amb5+Zn(II)]− + NTA or → [NTA+Zn(II)]− + amb5, where amb5 = His-Cys-Gly-Pro-Gly-Gly-Cys or Asp-His-Gly-Pro-Gly-Gly-Cys and NTA = nitrilotriacetic acid, were studied using traveling-wave ion mobility-mass spectrometry (TWIMMS). The analyses included calculating the reaction cross sections as a function of center-of-mass collision energy at three pressures of the argon collision gas, which covers the pressure range available in the TWIMMS instrument. The ternary complex contained one of the amb5 peptides, whose His, Cys and/or Asp substituent groups could compete with the NTA carboxylate sites for binding Zn(II). A new molecular modeling procedure was developed, initially using PM6 geometry-optimizations and thermochemical analyses to locate low-energy conformers of the ternary complex and its products that also had collision cross sections (ΩHe), measured using the ion size scaled Lennard-Jones (L-J) method, that were close to the TWIMMS measured ΩHe. A selection of these conformers were further geometry-optimized at the DFTB/3OB_D4 level of theory and using these as starting structures, molecular dynamics (MD) simulations were conducted to sample the ensemble of structures L-J ΩHe at 300 K. From these analyses, a selection of reactant and product conformers whose L-J ΩHe best matched the TWIMMS ΩHe were used in the TCID fitting of the center-of-mass energy-dependent, reaction cross sections extrapolated to zero pressure. The TCID fitting determined the threshold energies of the two product channels which in the absence of an activation barrier in excess of the energy of the products equates to the 0 K dissociation enthalpies (ΔH0). The difference in ΔH0 for the two competing reactions allowed for a relative gas-phase Zn(II) affinity scale to be constructed for the amb5 peptides studied here and from our previous published amb5 research.

Physicochemical property evaluation of modified oligonucleotides by traveling-wave ion mobility mass spectrometry.

Therapeutic oligonucleotides have molecular weights of more than 6000 Da. They typically contain chemically modified structures such as phosphorothioate (PS) and a locked nucleic acid (LNA). To determine the effect of the length and chemical modification on the physicochemical properties, various nucleic acids with different lengths and modified structures were analyzed using traveling-wave ion mobility mass spectrometry (TWIMS). The physicochemical characteristics of the modified oligonucleotides were determined using IM-MS. Each oligonucleotide was evaluated by confirming the multivalent charge state drift times, collision cross-section (CCS) values, and CCS widths. By plotting the m/z for oligonucleotides of different lengths and the CCS values at each charge state, a bottoming-out shape plot at one charge per 4.0-3.5 bases was confirmed. Moreover, significant differences were observed in the CCS values between the PS-modified and unmodified oligonucleotides. The PS-modified oligonucleotide showed a wider CCS range that was proportional to the PS modification ratio of the oligonucleotide sequence. The TWIMS results showed a correlation between the length and modification of oligonucleotides and the CCS values. In addition, it suggested that each charge state of the oligonucleotide ion has different physicochemical properties.

Analysis of Protein-Glycosaminoglycan Interactions Using Traveling Wave Ion-Mobility Mass Spectrometry.

Traveling wave ion-mobility mass spectrometry (TWIMS) combined with native mass spectrometry (MS) has emerged as a powerful tool for analyzing biomolecules, including complexes of protein and heparan sulfate (HS). This technique allows determination of the stoichiometry of the protein-HS interaction and information on the overall 3D molecular envelope.

Identification of N-glycans with GalNAc-containing antennae from recombinant HIV trimers by ion mobility and negative ion fragmentation

Negative ion collision-induced dissociation (CID) of underivatized N-glycans has proved to be a simple, yet powerful method for their structural determination. Recently, we have identified a series of such structures with GalNAc rather than the more common galactose capping the antennae of hybrid and complex glycans. As part of a series of publications describing the negative ion fragmentation of different types of N-glycan, this paper describes their CID spectra and estimated nitrogen cross sections recorded by travelling wave ion mobility mass spectrometry (TWIMS). Most of the glycans were derived from the recombinant glycoproteins gp120 and gp41 from the human immunodeficiency virus (HIV), recombinantly derived from human embryonic kidney (HEK 293T) cells. Twenty-six GalNAc-capped hybrid and complex N-glycans were identified by a combination of TWIMS, negative ion CID, and exoglycosidase digestions. They were present as the neutral glycans and their sulfated and α2→3-linked sialylated analogues. Overall, negative ion fragmentation of glycans generates fingerprints that reveal their structural identity.

Ion Mobility Mass Spectrometry Uncovers Guest-Induced Distortions in a Supramolecular Organometallic Metallosquare.

The encapsulation of the tetracationic palladium metallosquare with four pyrene‐bis‐imidazolylidene ligands [1]4+ with a series of organic molecules was studied by Electrospray ionization Travelling Wave Ion‐Mobility Mass Spectrometry (ESI TWIM‐MS). The method allowed to determine the Collision Cross Sections (CCSs), which were used to assess the size changes experienced by the host upon encapsulation of the guest molecules. When fullerenes were used as guests, the host is expanded ΔCCS 13 Å2 and 23 Å2, for C60 or C70, respectively. The metallorectangle [1]4+ was also used for the encapsulation of a series of polycyclic aromatic hydrocarbons (PAHs) and naphthalenetetracarboxylic diimide (NTCDI), to form complexes of formula [(NTCDI)2(PAH)@1]4+. For these host:guest adducts, the ESI IM‐MS studies revealed that [1]4+ is expanded by 47–49 Å2.. The energy‐minimized structures of [1]4+, [C60@1]4+, [C70@1]4+, [(NTCDI)2(corannulene)@1]4+ in the gas phase were obtained by DFT calculations.Introduction

Revealing Fatty Acid Heterogeneity in Staphylococcal Lipids with Isotope Labeling and RPLC-IM-MS.

Up to 80% of the fatty acids in Staphylococcus aureus membrane lipids are branched, rather than straight-chain, fatty acids. The branched fatty acids (BCFAs) may have either an even or odd number of carbons, and the branch position may be at the penultimate carbon (iso) or the antepenultimate (anteiso) carbon of the tail. This results in two sets of isomeric fatty acid species with the same number of carbons that cannot be resolved by mass spectrometry. The isomer/isobar challenge is further complicated when the mixture of BCFAs and straight-chain fatty acids (SCFAs) are esterified into diacylated lipids such as the phosphatidylglycerol (PG) species of the S. aureus membrane. No conventional chromatographic method has been able to resolve diacylated lipids containing mixtures of SCFAs, anteiso-odd, iso-odd, and iso-even BCFAs. A major hurdle to method development in this area is the lack of relevant analytical standards for lipids containing BCFA isomers. The diversity of the S. aureus lipidome and its naturally high levels of BCFAs present an opportunity to explore the potential of resolving diacylated lipids containing BCFAs and SFCAs. Using our knowledge of lipid and fatty acid biosynthesis in S. aureus, we have used a stable-isotope-labeling strategy to develop and validate a 30 min C18 reversed-phase liquid chromatography method combined with traveling-wave ion mobility-mass spectrometry to provide resolution of diacylated lipids based on the number of BCFAs that they contain.

Construction of Macromolecular Pinwheels Using Predesigned Metalloligands.

Developing a methodology to build target structures is one of the major themes of synthetic chemistry. However, it has proven to be immensely challenging to achieve multilevel elaborate molecular architectures in a predictable way. Herein, we describe the self-assembly of a series of pinwheel-shaped starlike supramolecules through three rationally preorganized metalloligands L1-L3. The key octa-uncomplexed terpyridine (tpy) metalloligand L3, synthesized with an 8-fold Suzuki coupling reaction to metal-containing complexes, has four different types of terpyridines connected with three ⟨tpy-Ru2+-tpy⟩ units, making this the most subunits known so far for a preorganized module. Based on the principle of geometric complementation and the high "density of coordination sites", these metalloligands were assembled with Zn2+ ions to form a pinwheel-shaped star trigon P1, pentagram P2, and hexagram P3 with precisely controlled shapes in nearly quantitative yields. With molecular weights ranging from 16756 to 56053 Da and diameters of 6.7-13.6 nm, the structural composition, shape, and rigidity of these pinwheel-shaped architectures have been fully characterized by 1D and 2D (NMR), electrospray ionization mass spectrometry, traveling-wave ion mobility mass spectrometry, and transmission electron microscopy.

Travelling Wave Ion Mobility-Derived Collision Cross Section for Mycotoxins: Investigating Interlaboratory and Interplatform Reproducibility.

Parent and modified mycotoxin analysis remains a challenge because of their chemical diversity, the presence of isomeric forms, and the lack of analytical standards. The creation and application of a collision cross section (CCS) database for mycotoxins may bring new opportunities to overcome these analytical challenges. However, it is still an open question whether common CCS databases can be used independently from the instrument type and ion mobility mass spectrometry (IM-MS) technologies, which utilize different methodologies for determining the gas-phase mobility. Here, we demonstrated the reproducibility of CCS measurements for mycotoxins in an interlaboratory study (average RSD 0.14% ± 0.079) and across different traveling wave IM-MS (TWIMS) systems commercially available (ΔCCS% < 2). The separation in the drift time dimension of critical pairs of isomers for modified mycotoxins was also achieved. In addition, the comparison of measured and predicted CCS values, including regulated and emerging mycotoxins, was addressed.

From Dimeric to Octameric Metallo-Supramolecular Macrocycles Based on Sterically Congested Ligand-assisted Self-Assembly with Zn(II), Cd(II), and Fe(II).

Two sterically congested 2,2':6',2″-terpyridine-based ligands LA and LB, composed of asymmetrically contiguous terpyridine units, are designed and synthesized for metallo-supramolecular architectures. The significant advantage of this design is that the terpyridines in these ligands have different chemical environments and show a selective coordination ability with each other. For ditopic ligand LA, the self-assembly with Zn(II), Cd(II), and Fe(II) gave the rhombic dimers, which have the same sets of terpyridine signals as ligand LA. The self-assembly behavior of tritopic ligand LB with Cd(II) and Zn(II) are observed for discrete tetramers under thermodynamic control, whereas ligand LB and Fe(II) are assembled to generate a mixture of tetramer, hexamer, and octamer, which are successfully isolated using regular chromatographic separation. Moreover, the sterically congested ligands and metal ions formed stable intermediates to drive the formation of discrete structures, which is also proved by mixing LB and Cd(II) with a precise stoichiometric ratio of 1:1. These supramolecular complexes are thoroughly characterized by NMR spectroscopy, electrospray ionization-mass spectrometry, and 2D traveling-wave ion mobility-mass spectrometry. This conceptually new design of sterically congested ligands provides a novel strategy for precisely controlled supramolecular complexes with diverse architectures.

Traveling Wave Ion Mobility-Mass Spectrometry to Enhance the Detection of Low Abundance Features in Untargeted Lipidomics.

Ion mobility has become a valuable tool in mass spectrometry-based lipidomics workflows, thanks to its ability to separate ions in the gas phase based on their size and conformation. Over the last years, it was demonstrated that the comparative analysis of ion mobility data has the potential to highlight the presence of low abundance species in untargeted lipidomics. The present chapter illustrates the background of the topic and guides the reader from the sample preparation to the data analysis of an untargeted lipidomics experiment performed using ion mobility.

Ditopic Chiral Pineno-Fused 2,2':6',2″-Terpyridine: Synthesis, Self-Assembly, and Optical Properties.

The syntheses of 4'-substituted chiral 2,2':6',2″-terpyridine (tpy) ligands with predetermined configurations and directionalities are rather limited in the supramolecular chemistry field. In this study, a carbazole-linked ditopic chiral ligand L was synthesized using 4'-bromo-substituted pineno-fused tpy 5 as the precursor. Upon complexation with Cd(NO3)2·4H2O and Zn(NO3)2·6H2O, two enantiomerically pure metallosupramolecules, [Cd3L3] and [Zn4L4], have been self-assembled and characterized by NMR, electrospray ionization-mass spectrometry, traveling wave ion mobility-mass spectrometry, and DOSY analysis. In addition, their optical properties are characterized by UV-vis, fluorescence, circular dichroism, and circularly polarized luminescence, suggesting an efficiency transmission and amplification of chirality from the ligand to metal center via self-assembly.

Order from Chaos: Self-Assembly of Nanoprism from a Mixture of Tetratopic Terpyridine-Porphyrin Conformers.

Porphyrins have been widely used in the self-assembly of metallo-supramolecules. In this study, we introduced 2,2':6,2"-terpyridine (tpy) into a porphyrin core to synthesize a tetratopic building block with multiple conformers. During the self-assembly with Zn(II), such a mixture of conformers was able to form a discrete nanoprism with all building blocks in one conformation. Detailed characterizations, including NMR, ESI-MS and traveling-wave ion mobility-mass spectrometry (TWIM-MS), all supported the formation of the desired assemblies. AFM and TEM further confirmed the dimensions of assembled nanoprisms. Moreover, the photophysical properties of the ligands and complexes were noticeably different depending upon size and metal ion center.

Metal-Organic Pt(II) Hexagonal-Prism Macrocycles and Their Photophysical Properties.

In this work, we present the formation of two open-ended hexagonal-prism tubular macrocycles by the [6 + 12] self-assembly of the symmetric ∼120° organic ligand donor with ∼90° Pt(II) acceptor in a 1:2 ratio. The assembled structures were characterized by multinuclear NMR (1H NMR, 31P{1H} NMR, and 1H-1H COSY NMR), electrospray ionization mass spectrometry (ESI-TOF-MS), traveling wave ion mobility-mass spectrometry (TWIM-MS), and transmission electron microscopy. Molecular modeling was further conducted to get insight into their structured characteristics. We also examined their photophysical properties.

Structure Determination of Large Isomeric Oligosaccharides of Natural Origin through Multipass and Multistage Cyclic Traveling-Wave Ion Mobility Mass Spectrometry.

Carbohydrate isomers with identical atomic composition cannot be distinguished by mass spectrometry. By separating the ions according to their conformation in the gas phase, ion mobility (IM) coupled to mass spectrometry is an attractive approach to overcome this issue and extend the limits of mass spectrometry in structural glycosciences. Recent technological developments have significantly increased the resolving power of ion mobility separators. One such instrument features a cyclic traveling-wave IM separator integrated in a quadrupole/time-of-flight mass spectrometer. This system allows for multipass ion separations and for pre-, intra-, and post-IM fragmentation. In the present study, we utilize this system to explore a complex mixture of oligoporphyrans derived from the enzymatic digestion of the cell wall of the red alga P. umbilicalis. We are able to deduce their complete structure using IM arrival times and the m/z of specific fragments. This approach was successfully applied for sequencing of oligoporphyrans of up to 1500 Da and included the positioning of the methyl ether and sulfate groups. The structures defined in this study by IM-MS/MS agree with those found in the past but use much more time-consuming analytical approaches. This study also revealed some so far undescribed structures, present at very low abundance. In addition, the results made it possible to compare the abundance of the different isomers released by the enzyme and to draw further conclusions on the specificity of β-porphyranase and more particularly on its accommodation tolerance of anhydro-bridges in subsites. Finally, a separation of two isomers with very similar mobility was obtained after 58 passes around the cIM, with an estimated resolving power of 920 for these triply charged species, confirming the structures attributed to these two isomers.

A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry: Application to the characterisation of metabolites in rat urine

A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1–2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.

Route to Useful Metallomonomers: Step-Wise Construction of Bimetallic Triangles by Site-Specific Metalation

Three bimetallic triangles were constructed via a step-wise assembly of tailored terpyridine building blocks. Oligomeric trimer 2 was obtained by reacting ligand 1 with Ru(II). Subsequent cyclization of trimer 2 with Zn(II), Cd(II), and Fe(II) resulted in the formation of bimetallic triangles 3, 4, and 5 in high yield, respectively. All triangles were characterized by NMR spectroscopy, ESI and travelling-wave ion mobility-mass spectrometry (MS), as well as gradient MS, which provided insight into their stabilities.