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Background. Study of properties and intermolecular interactions of biologically active compounds which can be used for the purposes of transmembrane drugs delivery is a topical task of modern molecular biophysics. Ascorbyl Palmitate (AP) as a fat-soluble form of vitamin C has recently attracted attention as a promising agent for formation of nanosomes for the “fat insoluble” drug molecules transfer through membranes. However, AP is not sufficiently characterized by up-to-date soft ionization mass spectrometric techniques. Objectives. The aim of the present work is to characterize AP and its intermolecular interactions by a number of mass spectrometric techniques: Electrospray Ionization (ESI), Laser Desorption/Ionization (LDI) and Matrix-Assisted Laser Desorption/Ionization (MALDI). The comparison of these techniques applicability to the study of AP intermolecular interactions as a drug delivery assisting agent is scheduled. Methods. ESI mass spectra are obtained with triple quadrupole Micromass Quattro mass spectrometer. LDI and MALDI experiments are performed by Autoflex II mass spectrometer. Results. In the ESI experiments in the positive ion mode abundant peaks of protonated and cationized AP molecules as well as the peaks of AP clusters nAP•H+ and nAP•Na+ (n=2÷4) are revealed in the mass spectra. This result testifies to the formation of stable noncovalent complexes of the AP molecules in the polar media and confirms the AP ability of formation nanosomes for drug delivery. Analysis of LDI and MALDI mass spectra of AP in positive and negative ion modes shows that in the presence of molecular ions of AP, the peaks of AP dimers or larger AP clusters are not recorded. The ESI probing of the model system containing AP and dipalmitoylphosphatidylcholine (DPPC) reveals stable AP•DPPC•H+ complex which models the AP intermolecular interactions with the phospholipid components of biomembranes and/or liposomes under AP functioning as a drug delivery assisting agent. Conclusions. The current study demonstrates the applicability of all tested mass spectrometric techniques for AP identification in solutions and solid phase, while for the purpose of examining of the AP noncovalent complexes formation and study of AP interactions with biomolecules the ESI is defined as the most effective technique.

Native electrospray ionization-mass spectrometry, with gas-phase activation and solution compositions that partially release subcomplexes, can elucidate topologies of macromolecular assemblies. That so much complexity can be preserved in gas-phase assemblies is remarkable, although a long-standing conundrum has been the differences between their gas- and solution-phase decompositions. Collision-induced dissociation of multimeric noncovalent complexes typically distributes products asymmetrically (i.e., by ejecting a single subunit bearing a large percentage of the excess charge). That unexpected behavior has been rationalized as one subunit "unfolding" to depart with more charge. We present an alternative explanation based on heterolytic ion-pair scission and rearrangement, a mechanism that inherently partitions charge asymmetrically. Excessive barriers to dissociation are circumvented in this manner, when local charge rearrangements access a lower-barrier surface. An implication of this ion pair consideration is that stability differences between high- and low-charge state ions usually attributed to Coulomb repulsion may, alternatively, be conveyed by attractive forces from ion pairs (salt bridges) stabilizing low-charge state ions. Should the number of ion pairs be roughly inversely related to charge, symmetric dissociations would be favored from highly charged complexes, as observed. Correlations between a gas-phase protein's size and charge reflect the quantity of restraining ion pairs. Collisionally-facilitated salt bridge rearrangement (SaBRe) may explain unusual size "contractions" seen for some activated, low charge state complexes. That some low-charged multimers preferentially cleave covalent bonds or shed small ions to disrupting noncovalent associations is also explained by greater ion pairing in low charge state complexes. Graphical Abstract ᅟ.

Background: While antibiotic cycloserine (CYS) is widely applied in the treatment of tuberculosis, our knowledge of the drug intermolecular interactions with targeting biomolecules and other drugs remains incomplete. It is believed that the CYS antibacterial activity is related to inhibiting the bacterial cell wall biosynthesis. On the other hand, intermolecular interactions of CYS with ascorbic acid (ASC) molecules is worth of studying taking into account that ASC can be used as supporting vitamin preparation or can be affiliated with the patients nutrition. Objectives: The purpose of the current model study are to examine biologically significant intermolecular interactions of CYS with N-acetyl-D-glucosamine (NAG) as one of the main component of peptidoglycan of bacterial cell wall and to verify the possibility of noncovalent complexes formation between CYS and ASC molecules using electrospray ionization mass spectrometry (ESI MS) technique. Materials and methods: The objects of the study are model systems composed of CYS and NAG or CYS and ASC prepared in a polar methanol solvent for the ESI MS probing. ESI mass spectra are obtained using the approach earlier developed by us for investigation of the noncovalent complexation of drugs with targeting biomolecules. Results: The experiments reveal that the ESI mass spectrum of (CYS–NAG) model system contains peaks of protonated molecular clusters of CYS with NAG: [CYS•NAG•H]+ and [CYS•2NAG•H]+. Existing of such peaks in the spectrum testifies to formation of stable noncovalent complexes between CYS and NAG in the studied solution. ESI MS examining of (CYS–ASC) system reveals the noncovalent pair complexation of CYS and ASC molecules confirmed by the recording of intensive peak of [CYS•ASC•H]+ cluster in the spectrum. Conclusions: The ESI MS findings point to the possibility of noncovalent complexation of CYS with NAG in the polar media including biological systems. Such complexation between the antibiotic and NAG as component of peptidoglycan of bacterial cell wall is considered to be biologically significant for the process of the cell wall biosynthesis inhibiting by CYS. Stable noncovalent complexes formation between the CYS and ASC molecules is suggested as a potential molecular mechanism of the drugs activity modulation under their joint usage.

Biochemistry and structural biology have benefited tremendously from the development of electrospray ionization and matrix-assisted laser desorption/ionization, the two most important soft ionization methods used in modern mass spectrometry. Of importance in this context is that noncovalently bound complexes can be maintained in the gas phase using appropriate sample preparation conditions and instrumental settings in ESI-MS and MALDI-MS. This allows one to determine the architecture and stoichiometry of large noncovalent complexes, the binding of small molecule ligands and cofactors, and often even noncovalent binding affinities, since bound and unbound states can be clearly resolved and quantified using MS. This article presents an overview of the research in this particular area undertaken by my research group at ETH Zurich. I will introduce a number of specialized technologies that we have available in our laboratory, including MALDI mass spectrometry with high-mass detection for measuring complexes with molecular weights in the 100s of kDa range; the so-called “gap sampler” technology for high-throughput experiments; and a temperature programmed spray source for ESI. A number of selected applications will then be presented, including the study of GPCRs and other membrane proteins and their complexes, the determination of the melting behavior of non-canonical nucleic acid structures, the investigation of heterogeneous protein complexes and protein-based capsids, and the investigation of the influence of protein post-translational modifications on noncovalent binding profiles.

In dialysis patients, the protein β2-microglobulin (β2m) forms amyloid fibrils in a condition known as dialysis-related amyloidosis. To understand the early stages of the amyloid assembly process, we have used native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble preamyloid oligomers. ESI-IM-MS reveals the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, results which are distinct from β2m oligomers formed at low pH. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers—one that is compact and another that is more expanded. Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems in which conformational heterogeneity is often associated with different amyloid morphologies or off-pathway events, conformational heterogeneity in the tetramer seems to be a necessary aspect of Cu(II)-induced amyloid formation by β2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of Cu(II) release in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies. Image 1 • Characterizing amyloid formation in vitro is complicated by structural heterogeneity. • Multiple conformational isomers of β2m oligomers have been identified by IM-MS. • Preamyloid tetramers are heterogeneous and release Cu(II) for amyloid formation. • Structural models of transformed Cu(II)-free tetramers are derived from modeling. • The structural models allow further studies of mechanisms and inhibitory strategies. [ABSTRACT FROM AUTHOR]

Chiral molecules frequently remain undistinguishable using ion mobility mass spectrometry (IM‐MS), due to insufficient differences of their collision cross sections at the available mobility resolution of the ion mobility drift tubes. The influence of the complexation with organic acids on the ion mobility separation of peptide epimers is evaluated using traveling‐wave ion mobility (TWIMS). The examined epimeric tripeptides containing Arg residue with the sequence: Ac‐Phe‐Arg‐Trp‐NH2 formed stable complexes in the gas phase, and under the increased pressure in ion mobility drift tube, noncovalent associates formed with carboxylic or sulfonic monoacids and diacids with chiral variation of certain acids. Overall, the complexation with an acid leads to the improvement in stereodifferentiation among epimeric peptides, in comparison to the analysis of pure epimers. Detailed characterization of peptide epimer‐acid associates obtained for dibenzoyl‐D‐tartaric acid by theoretical calculations and collisional dissociation studies revealed that the presence of multiple hydrogen bonding interactions between carboxylate anions and hydrogens from N―H of both the guanidinium group of arginine and the indole of tryptophan, as well as the amide backbone hydrogens in the peptide, is responsible for stability of acid‐peptide complexes and for their differentiation in the ion mobility drift tube. The specificity of complex formation toward Arg was determined in terms of complex stability. Based on the reported results, we present general conclusions regarding the utility of the acid‐based complexation in the separation of peptide isomers. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Phthalocyanines form noncovalent complexes with Sc 3 N@C 80. • Interaction pattern depends on phthalocyanine structure (free base or metal complex). • The values of binding energies span from −24.33 to −42.89 kcal/mol. Abstract We performed systematic theoretical analysis of noncovalent interactions of free-base phthalocyanine H 2 Pc and its 3 d transition metal(II) complexes (MnPc, singlet and triplet FePc, CoPc, NiPc, CuPc and ZnPc) with endohedral fullerene Sc 3 N@ I h –C 80. In terms of the optimized geometries and variability of bonding pattern, electrostatic potential plots, frontier orbital features and spin density distribution, the calculated characteristics of noncovalent MPc + Sc 3 N@C 80 conjugates are generally more similar to the corresponding properties of MPc + C 60 than of MPc + C 80 dyads studied previously. However, the magnitude of binding strength for MPc + Sc 3 N@C 80 dyads matches more closely the interaction strength in MPc + C 80 series than the one for MPc + C 60 dyads. [ABSTRACT FROM AUTHOR]

Native mass spectrometry (MS) encompasses methods to keep noncovalent interactions of biomolecular complexes intact in the gas phase throughout the instrument and to measure the mass-to-charge ratios of supramolecular complexes directly in the mass spectrometer. Electrospray ionization (ESI) in nondenaturing conditions is now an established method to characterize noncovalent systems. Matrix-assisted laser desorption/ionization (MALDI), on the other hand, consumes low quantities of samples and largely tolerates contaminants, making it a priori attractive for native MS. However, so-called native MALDI approaches have so far been based on solid deposits, where the rapid transition of the sample through a solid state can engender the loss of native conformations. Here we present a new method for native MS based on liquid deposits and MALDI ionization, unambiguously detecting intact noncovalent protein complexes by direct desorption from a liquid spot for the first time. To control for aggregation, we worked with HUαβ, a heterodimer that does not spontaneously rearrange into homodimers in solution. Screening through numerous matrix solutions to observe first the monomeric protein, then the dimer complex, we settled on a nondenaturing binary matrix solution composed of acidic and basic organic matrices in glycerol, which is stable in vacuo. The role of temporal and spatial laser irradiation patterns was found to be critical. Both a protein-protein and a protein-ligand complex could be observed free of aggregation. To minimize gas-phase dissociation, source parameters were optimized to achieve a conservation of complexes above 50% for both systems.ᅟ [ABSTRACT FROM AUTHOR]

Spectral-fluorescent and photochemical properties of meso -substituted thiacarbocyanine dyes 3,3′-dimethyl-9-phenylthiacarbocyanine and 3,3′-diethyl-9-(2-hydroxy-4-methoxyphenyl)thiacarbocyanine in solutions and their interaction with DNA were studied. The dyes form noncovalent complexes with DNA, which is accompanied by changes in the absorption spectra and an increase in the fluorescence intensity of the dyes. The data obtained suggest that the dyes are in the form of trans -isomers both in solvents of different polarity and in complexes with DNA. It was shown that the interaction of the dyes with DNA is a complex process involving monomeric dye molecules and aggregates of the dyes. The primary photochemical processes of the dyes in solutions and in complexes with DNA were studied by flash photolysis technique. Upon flash photoexcitation in solutions, the formation and decay of the photoisomers of the dyes were observed, with no generation of the triplet states. In the complex with DNA, no signal of photoisomers was detected; in the absence of oxygen, the formation of the triplet state of the dyes was observed. The decay kinetics of the triplet state of the dyes were two-exponential. The process of quenching of the triplet state of the dyes by oxygen in a complex with DNA was studied, the respective quenching rate constants were estimated, being lower than the diffusion-controlled value. [ABSTRACT FROM AUTHOR]

Chemical cross-linking combined with MALDI- MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (Aa LS-13) was investigated that self-assembles into a capsid-like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI- MS and native ESI- MS, were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo-electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross-linking with glutaraldehyde followed by MALDI- MS. The same approach was used to study protein-protein interactions during encapsulation of GFP(+36) by the Aa LS-13 capsid. Heterocomplexes between GFP(+36) and Aa LS-13 multimers were not observed when mixed at maximal loading capacity (AalS-13 monomer: GFP(+36) 4:1). This is in agreement with the known fast encapsulation of GFP(+36) by the protein capsid, which essentially removes any free GFP(+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP(+36) results in aggregation. [ABSTRACT FROM AUTHOR]

Ion mobility-mass spectrometry (IM-MS) is a powerful tool for the simultaneous analysis of mass, charge, size, and shape of ionic species. It allows the characterization of even low-abundant species in complex samples and is therefore particularly suitable for the analysis of proteins and their assemblies. In the last few years even complex and intractable species have been investigated successfully with IM-MS and the number of publications in this field is steadily growing. This trend article highlights recent advances in which IM-MS was used to study protein-ligand complexes and in particular focuses on the catch and release (CaR) strategy and collision-induced unfolding (CIU). [ABSTRACT FROM AUTHOR]

Complexes of the anthracycline antitumor antibiotic daunomycin with biocompatible polymer carriers, poly(vinylpyrrolidone) and poly(ethylene glycol), have been studied. According to the UV-visible absorption spectroscopy and dynamic light scattering data, variation of the weight ratio of the components leads to the formation of antibiotic-polymer complexes in which the polymer is linked through hydrophobic and electrostatic interactions to certain functional groups of the antibiotic molecules. The concentration constants for the complex formation have been calculated. [ABSTRACT FROM AUTHOR]

Ion mobility-mass spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, native-like or denatured proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a 'trajectory method' (TM) based CCS calculator, named 'Collidoscope,' which utilizes parallel processing and optimized trajectory sampling, and implements both He and N as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared with those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using X-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions. [ABSTRACT FROM AUTHOR]

The formation of zinc finger peptide−Zn2+ complexes in electrospray ionization mass spectrometry (ESI-MS) was examined using three different metal ion sources: ZnCl2, Zn(CH3COO)2, and Zn(OOC(CHOH)2COO). For the four zinc finger peptides (Sp1-1, Sp1-3, CF2II-4, and CF2II-6) that bind only a single Zn2+ in the native condition, electrospray of apo-zinc finger in solution containing ZnCl2 or Zn(CH3COO)2 resulted in the formation of zinc finger−Zn2+ complexes with multiple zinc ions. This result suggests the formation of nonspecific zinc finger−Zn2+ complexes. Zn(tartrate), Zn(OOC(CHOH)2COO),mainly produced specific zinc finger−Zn2+ complexes with a single zinc ion. This study clearly indicates that tartrate is an excellent counter ion in ESI-MS studies of zinc finger−Zn2+ complexes, which prevents the formation of nonspecific zinc finger−Zn2+ complexes.

Haptoglobin (Hp) is a plasma glycoprotein that generates significant interest in the drug delivery community because of its potential for delivery of antiretroviral medicines with high selectivity to macrophages and monocytes, the latent reservoirs of human immunodeficiency virus. As is the case with other therapies that exploit transport networks for targeted drug delivery, the success of the design and optimization of Hp-based therapies will critically depend on the ability to accurately localize and quantitate Hp-drug conjugates on the varying and unpredictable background of endogenous proteins having identical structure. In this work, we introduce a new strategy for detecting and quantitating exogenous Hp and Hp-based drugs with high sensitivity in complex biological samples using gallium as a tracer of this protein and inductively coupled plasma mass spectrometry (ICP MS) as a method of detection. Metal label is introduced by reconstituting hemoglobin (Hb) with gallium(III)-protoporphyrin IX followed by its complexation with Hp. Formation of the Hp/Hb assembly and its stability are evaluated with native electrospray ionization mass spectrometry. Both stable isotopes of Ga give rise to an abundant signal in ICP MS of a human plasma sample spiked with the metal-labeled Hp/Hb complex. The metal label signal exceeds the spectral interferences' contributions by more than an order of magnitude even with the concentration of the exogenous protein below 10 nM, the level that is more than adequate for the planned pharmacokinetic studies of Hp-based therapeutics. [ABSTRACT FROM AUTHOR]

Data on intermolecular noncovalent interactions of nitrogen base components of nucleic acid with organic polyethers are of interest both for fundamental molecular biophysics and for applications in the production of pharmaceutical organic nanoparticles. In the present work, systems composed of pyrimidine nitrogen base cytosine (Cyt) or its methyl derivatives and polyethylene glycol PEG-400 oligomers M have been examined by electrospray ionization ( ESI) and fast atom bombardment mass spectrometry. Detection of a set of associates of the M · Cyt · H type ( n = 3-17) in ESI mass spectra evidences the formation of protonated complexes of the base with oligomers of different chain lengths in all of the studied systems. Computer modeling by molecular dynamics gave answers to the questions concerning the stability and structure of these complexes under different conditions. It has been shown that M · Cyt · H clusters in the gas phase (vacuum) adopt a compact structure with a quasi-cyclic or quasi-helical self-organization of the polymer chain around the protonated base. It has been found that the compact structure of the M · Cyt · H complex characteristic for its state in the gas phase is preserved in a liquid medium of methanol (used as a solvent in ESI). The results of simulation of the evolution of a methanol droplet in vacuum, which mimics the droplet disintegration under ESI conditions, have shown that the M · Cyt · H complex initially present in the droplet does not disintegrate in the course of droplet evaporation. After the completion of desolvation, the complex returns to the conformation characteristic for the gas phase. Thus, the evidence has been found confirming that the M · Cyt · H clusters recorded in ESI mass spectra adequately reflect the presence of corresponding noncovalent complexes in the analyzed liquid solution. [ABSTRACT FROM AUTHOR]

Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase. [ABSTRACT FROM AUTHOR]

Ion mobility coupled with mass spectrometry provides a fast and repeatable method to separate catechin epimers by previous complexation with selected chiral modifiers and transition metals. Several combinations with chiral ligands such as D- and L-amino acids and/or additional metal cations, chiral crown ethers, tartaric acid and heptakis(2,6-di-O-methyl)- β-cyclodextrin were screened for their ability to affect the separation efficiency. The clusters having the form of [2M + D-amino acid + Cu2+ − 3H]− (M stands for (−)-epicatechin or (+)-catechin) showed improvement in stereodifferentiation between two epimeric catechins in comparison to the analysis of pure epimers, where no separation was observed or the separation was hampered by the formation of mixed dimer complexes. Among various examined D-amino acids only those possessing hydrophobic side chains induced the improvement of separation efficiency. The best peak-to-peak resolution (Rp-p) was determined to be 0.71 for [2M + D-Leucine + Cu2+ − 3H]− clusters. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Cationic polymers can bind DNA to form polyplexes, which are noncovalent complexes used for gene delivery into the targeted cells. For more insight on such biologically relevant systems, the noncovalent complexes between the cationic polymer poly(ethylene imine) (PEI) and the nucleotide mimicking dye Cibacron Blue F3G-A (CB) were investigated using mass spectrometry methods. Two PEIs of low molecular weight were utilized (Mn ≈ 423 and 600 Da). The different types of CB anions produced by Na+/H+ exchanges on the three sulfonic acid groups of CB and their dehydrated counterparts were responsible for complex formation with PEI. The CB anions underwent noncovalent complex formation with protonated, but not with sodiated PEI. A higher proportion of cyclic oligomers were detected in PEI423 than PEI600, but both architectures formed association products with CB. Tandem mass spectrometry studies revealed a significantly stronger noncovalent interaction between PEI and dehydrated CB than between PEI and intact CB. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the β ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. β ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa. [ABSTRACT FROM AUTHOR]

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Proton-bound [M ⋅H ⋅G]+ diastereomeric complexes between some chiral aromatic amino acids or dipeptides (G) and a chiral multifunctional macrocyclic receptor (M=Chirabite-A) undergo, in the gas phase, highly selective substitution and addition reactions by amines, such as 2-aminobutane and piperidine. All the [M ⋅H ⋅G]+ complexes follow time-dependent monoexponential decays. In some cases, the kinetic curves exhibit a plateau revealing the presence of unreactive [M ⋅H ⋅G]+ structures. In them, the amino acid is accommodated in the macrocycle cavity in the zwitterionic form by sharing its acidic hydrogen atoms with the pyridine nitrogen atoms of the host. The same interactions are structurally inaccessible to G=dipeptides or monofunctional amines, which then can be readily released from [M ⋅H ⋅G]+. When the amino acid interacts with the amidocarbonyl oxygen atoms pointing outside the macrocycle cavity, it saves the canonical structure and can be readily displaced by the amine. The Chirabite-A may act as an efficient template for aromatic amino acids by releasing them or not depending upon the amino acid configuration and the basicity of the amine. These unique properties confer to the gas-phase diastereomeric [M ⋅H ⋅G]+ complexes the features of multi-input multi-output chemo-'logic gates'. [ABSTRACT FROM AUTHOR]

The most common protocols for the quantitative determination of the enantiomeric excess ( ee) of raw mixtures by ESI-MS reveal inadequate in cases where the distribution of diastereomeric derivatives diverges from the ee of original solutions. This phenomenon is attributable to a matrix effect, i.e., to the stereospecific formation of high order noncovalent adducts in the ESI droplets, which alters the actual availability of the diastereomeric species under MS analysis. In this frame, the assumption of classic protocols that the ionization correction factor q is independent on the composition of the mixture submitted to analysis is questionable. An alternative methodology is presented in this paper, which is aimed at circumventing the problem by excluding any chemical derivatization of the original raw mixture. It is based on the measurement of the actual distribution of ESI-formed proton-bound diastereomeric complexes from the enantiomeric mixture through a careful analysis of their reaction kinetics with a suitable reactant. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A time-of-flight (TOF) mass spectrometer intended particularly for the study of noncovalent complexes and large proteins is described. In this device, a heated capillary couples an electrospray or nanospray source to an RF-only quadrupole ion guide. The ion guide provides collisional cooling, and injects the ion beam into a TOF mass spectrometer. 16kV TOF acceleration potential improves transmission and detection efficiency, and also increases the m/z range for a given extraction frequency. A two-stage reflector, together with collisional cooling in the ion guide, provides resolving power up to 18,000 (FWHM) for small molecules, and somewhat improved resolution for larger ones, although the widths of the noncovalent peaks are mainly determined by their intrinsic spreads. Examples of m/z spectra obtained with this instrument in our laboratory are presented. [Copyright &y& Elsevier]

Characterizing intact multiprotein complexes in terms of both their mass and size by ion mobility-mass spectrometry is becoming an increasingly important tool for structural biology. Furthermore, the charge states of intact protein complexes can dramatically influence the information content of gas-phase measurements performed. Specifically, protein complex charge state has a demonstrated influence upon the conformation, mass resolution, ion mobility resolution, and dissociation properties of protein assemblies upon collisional activation. Here we present the first comparison of charge-reduced multiprotein complexes generated by solution additives and gas-phase ion-neutral reaction chemistry. While the charge reduction mechanism for both methods is undoubtedly similar, significant gas-phase activation of the complex is required to reduce the charge of the assemblies generated using the solution additive strategy employed here. This activation step can act to unfold intact protein complexes, making the data difficult to correlate with solution-phase structures and topologies. We use ion mobility-mass spectrometry to chart such conformational effects for a range of multi-protein complexes, and demonstrate that approaches to reduce charge based on ion-neutral reaction chemistry in the gas-phase consistently produce protein assemblies having compact, 'native-like' geometries while the same molecules added in solution generate significantly unfolded gas-phase complexes having identical charge states. [ABSTRACT FROM AUTHOR]

Abstract: In order to evaluate the essence of the interactions of ginsenosides and proteins which are composed by α-amino acids, electrospray ionization mass spectrometry was employed to study the noncovalent interactions between ginsenosides (Rb2, Rb3, Re, Rg1 and Rh1) and 18 kinds of α-amino acids (Asp, Glu, Asn, Phe, Gln, Thr, Ser, Met, Trp, Val, Gly, Ile, Ala, Leu, Pro, His, Lys and Arg). The 1:1 and 2:1 noncovalent complexes of ginsenosides and amino acids were observed in the mass spectra. The dissociation constants for the noncovalent complexes were directly calculated based on peak intensities of ginsenosides and the noncovalent complexes in the mass spectra. Based on the dissociation constants, it can be concluded that the acidic and the basic amino acids, Asp, Glu, Lys and Arg, bound to ginsenosides more strongly than other amino acids. The experimental results were verified by theoretical calculations of parameters of noncovalent interaction between ginsenoside Re and Arg which served as a representative example. Two kinds of binding forms, “head–tail” (“H–T”) and “head–head” (“H–H”), were proposed to explain the interaction between ginsenosides and amino acids. And the interaction in “H–T” form was stronger than that in “H–H” form. [Copyright &y& Elsevier]

The article presents a study which compared three label-free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. These methods include isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and nanoelectrospray mass spectrometry (nESI-MS). The performance, limitations and disadvantages of the three methods are discussed. It is concluded that label-free methods to determine the binding strengths of protein-ligand systems are important in life sciences.

[Display omitted] • Meso-aryl-substituted thiacarbocyanines I and II interact with dsDNA and ssDNA. • Complexes of both monomeric molecules and unstructured aggregates of the dyes are formed. • Dye – dsDNA docking and melting tests confirm intercalation. • Dye II with a bulky substituent interacts with ssDNA less efficiently. • Interaction with ssDNA gives an increase in fluorescence and triplet yield of dyes. The noncovalent interaction of meso -aryl-substituted thiacarbocyanine dyes I and II with dsDNA and ssDNA in aqueous solutions has been studied by spectral-fluorescent methods. Complexation with DNA is accompanied by both aggregation of the dyes and the formation of monomeric strongly fluorescent complexes. Experiments on molecular docking of dyes I and II with dsDNA confirm the previous assumption about the possibility of the formation of complexes of different types: intercalation between base pairs and in the grooves of the double helix of the biopolymer. The possibility of intercalation of the dyes in the complex is confirmed by experiments on thermal dissociation of dsDNA in the presence of dyes I and II, as well as experiments on the interaction of the dyes with ssDNA. An increase in the melting temperatures T m of dsDNA is obtained in the presence of I and II, similar to that observed for the classical intercalator ethidium bromide. The limits of detection and quantification of DNA, which are important for the use of the dyes as probes for DNA, have been determined. The primary photochemical processes of the dyes in complexes with ssDNA were studied by flash photolysis technique. Complexation with ssDNA hinders photoisomerization and creates favorable conditions for the dye triplet state formation. The decay kinetics of the triplet state of the dyes were monoexponential. The rate constant of quenching of the triplet state by air oxygen was estimated for dye I complexed with ssDNA and was found to be less than the diffusion-controlled limit. This is probably a consequence of the shielding effect of the complex on the triplet quenching process. [ABSTRACT FROM AUTHOR]

Abstract: We report a mass spectrometric study of ruthenium(II) sensitizer ((C4H9)4N)2[Ru(4-carboxy-4′-carboxylate-2,2′bipyridine)2(NCS)2] (N719) in various organic and aqueous solvents. At the equilibrium different species were found, depending on solution properties. After stoichiometry confirmation through high-resolution mass spectra and isotopic pattern analysis, the formation of these complexes was found to strongly depend from the polarity and the ionic strength of solvent medium. Mass spectra obtained in acetonitrile/tert-butanol (1:1) mixture, used for the absorption of N719 on TiO2, suggests that the main species involved in the adsorption process is the dicarboxylate form of N719 with one (C4H9)4N as counter ion. The photovoltaic data of the N719 sensitizer adsorbed on TiO2 films exhibited a remarkable power conversion efficiency, 11.20% at 1 sun. [Copyright &y& Elsevier]

Abstract: A theoretical study of the interaction of tetramethylammonium cation with chlorine anion and 2,5-dihydroxybenzoic acid modeling a carboxylic group and an aromatic ring of side radicals of proteins has been performed by the DFT/B3LYP/6-31++G∗∗ and MP2/6-31++G∗∗ theoretical methods. To evaluate how the solvation affects the interaction, the Polarizable Continuum Models method has been employed. The calculated interaction energies of the noncovalent complexes were compared with the experimental data describing the stability of some noncovalent complexes of a dication of bisquaternary ammonium salt decamethoximun with Cl− and 2,5-dihydroxybenzoic acid obtained with the mass-spectrometric technique. [Copyright &y& Elsevier]

The fact that ions of macromolecular complexes produced by electrospray ionization can be maintained intact in a mass spectrometer has stimulated exciting new lines of research. In this review we chart the progress of this research from the observation of simple homo-oligomers to complex heterogeneous macromolecular assemblies of mega-Dalton proportions. The applications described herein not only confirm the status of mass spectrometry (MS) as a structural biology approach to complement X-ray analysis or electron microscopy, but also highlight unique attributes of the methodology. This is exemplified in studies of the biogenesis of macromolecular complexes and in the exchange of subunits between macromolecular complexes. Moreover, recent successes in revealing the overall subunit architecture of complexes are set to promote MS from a complementary approach to a structural biology tool in its own right. [ABSTRACT FROM AUTHOR]

Abstract: Organometallic assemblies, containing the bis-terpyridinyl-Ru(II) connectivity (symbolized as [–〈Ru〉–]) in a range of small bismetal complexes to macrocycles possessing up to 12 metal ions, have been studied by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). ESI MS is found to be a more suitable characterization method than MALDI MS, because (1) it can directly detect multiply charged ions, (2) does not alter the connectivity of the complexes through disassembly/reassembly sequences and (3) causes essentially no or very little fragmentation. Requirements for the optimum signal-to-noise ratio in ESI mass spectra include: (1) a low ionization source temperature (30°C), (2) low skimmer voltage (10–15V), (3) moderate flow rates of both the nebulizing and drying gases and (4) a pre-cooled sample. Tandem mass spectrometry experiments on intact complex ions indicate that the noncovalent interactions between the Ru(II) center and the two terpyridine ligands, which hold the supramolecular assembly together, are very strong. [Copyright &y& Elsevier]

Abstract: Proton-bound complexes between underivatized monosaccharides and hydrazine have been generated in an ion trap mass spectrometer by electrospray ionization (ESI) and their collision induced decomposition (CID) investigated. The CID spectra of the selected complexes are invariably characterized by losses of hydrazine and several water molecules in proportions and at collision energies which markedly depend upon the nature and the structural features of the specific monosaccharide. Structural analysis of the proton-bound monosaccharide/hydrazine complexes at the B3LYP/6-31G(d,p) level of theory allowed to interpret the mass spectrometric results in terms of the specific intracomplex interactions between the amine and the various functional groups of the monosaccharide. [Copyright &y& Elsevier]

The high mass accuracy and resolution of Fourier transform (FT)-ion cyclotron resonance (ICR) mass spectrometry are making it an increasingly useful tool in drug discovery and development. The basics of FT-ICR are described here, including modern ion sources and fragmentation methods. Although FT-ICR is not a high-throughput method in the traditional sense, previously difficult and complex problems are being efficiently approached using steadily improving instruments and magnets. Applications are surveyed in fields such as proteomics, metabonomics, natural product analysis and non-covalent complexes. [Copyright &y& Elsevier]

Abstract: Ribonucleic acids (RNA) are an attractive target for drug discovery since they play critical roles in cellular functions. Because small structured subdomains are known to mimic the behavior of the entire RNA, it is possible to design RNA drug targets that are amenable to interrogation by high performance mass spectrometry. We have developed a high throughput drug discovery platform that uses electrospray ionization Fourier transform ion cyclotron mass spectrometry to investigate ligand binding to structured RNA drug targets. This assay is called multitarget affinity/specificity screening (MASS). Using MASS, we show that it is possible to screen synthetic and natural product libraries in a high throughput and robust manner. [Copyright &y& Elsevier]

The dianionic noncovalent complex between vancomycin and the bacterial cell-wall precursor peptide Ac2-l-Lys-d-Ala-d-Ala (DD) has previously been shown to retain its structural specific interactions from solution into the gas phase [Int. J. Mass Spectrom. Ion Processes 188 (1999) 63]. We have now determined the destruction cross sections for this specific complex and for the nonspecific complex between vancomycin and Ac2-d-Lys-l-Ala-l-Ala (LL). Although the gaseous nonspecific complex is only very loosely bound compared to the specific complex, their destruction cross sections are identical. This indicates that there is no difference in the compactness of their gas phase structures. Upon high-energy collisional activation (ELAB=100 keV) of the specific dianionic complex, electron detachment occurs and covalent bond cleavage with retainment of noncovalent bonds is observed; these reactions were not observed at low collision energy (ELAB=300 eV). Furthermore, we now report how small changes in the peptide binding pocket of vancomycin affect gas phase stability in a manner which parallels known solution binding affinity. The results show that Ac2-l-Lys-d-Ala-d-Ala cannot form as strong electrostatic interactions with CDP-1 as with vancomycin (or dechlorovancomycin) in the gas phase. [Copyright &y& Elsevier]

Both transient and stable dimers of metallothionein have been characterized, based on earlier studies using NMR, circular dichroism and size-exclusion chromatography. Here additional characterization is provided by nanospray mass spectrometry. Rapid redistribution of metal ions between monomeric Cd7– and Zn7–metallothionein 2a is monitored by nanospray. An experiment in which theses two forms of the monomeric protein are separated by a dialysis membrane, which will pass metal ions but not proteins, confirms that a transient dimer must form for metal ions to be redistributed. On the other hand, size-exclusion chromatography of reconstituted Zn7– or Cd7–metallothionein revealed the presence of monomeric and dimeric species. These dimers do not equilibrate readily to form monomers and they are shown to be covalent. [ABSTRACT FROM AUTHOR]

[Display omitted] • Increase in fluorescence and triplet yield of dyes K1, K2 with DNA. • Interaction with DNA interferes with the process of photoisomerization of dyes. • Aggregation and cis–trans isomerization make dye-DNA interaction multidimensional. • Docking and DNA melting confirm partial intercalation of dyes in a complex with DNA. • The K2 LOD of dsDNA reaches a level of about 15 ng mL−1. Spectral-fluorescent and photochemical properties (photoisomerization and generation of the triplet state) of meso -substituted cationic carbocyanine dyes, 3,3′-di-(β-hydroxyethyl)-5,5′-dimethoxy-9-ethylthiacarbocyanine iodide (K1) and 3,3′-di-(β-hydroxyethyl)-9-methylthiacarbocyanine iodide (K2), have been studied in solutions and in the presence of DNA. In solutions, on passing from acetonitrile to dioxane, a growth of fluorescence of the dyes is observed due to a shift of the equilibrium of cis/trans isomers toward the fluorescent trans -isomer. Upon flash photolysis of dye solutions in dioxane, the formation and subsequent decay of the cis -photoisomers of the dyes are observed. In aqueous solutions, the interaction with DNA leads to the formation of noncovalent complexes of K1 and K2 with DNA, which is accompanied by a significant increase in the fluorescence intensity. The results of the molecular docking experiments showed the possibility of several types of binding, which was confirmed by the data obtained from other experiments. The effects of temperature and additions of NaCl on the stability of the dye–DNA complexes were studied. The spectral-fluorescent data were used to estimate the binding constants of the dyes with DNA and other characteristics of the dyes that are important for their use as probes. Upon flash photolysis of the dyes in complexes with DNA, photoisomerization is not observed, but the quantum yield of intersystem crossing to the triplet state increases. The decay of the triplet states occurs by a two-exponential law. The rate constants for quenching of the triplet states of the dyes complexed with DNA by oxygen were found to be lower than the expected values for diffusion-controlled quenching (taking into account the spin statistical factor 1/9), which is explained by the steric factor of complexation. [ABSTRACT FROM AUTHOR]

Biochemistry and structural biology have benefited tremendously from the development of electrospray ionization and matrix-assisted laser desorption/ionization, the two most important soft ionization methods used in modern mass spectrometry. Of importance in this context is that noncovalently bound complexes can be maintained in the gas phase using appropriate sample preparation conditions and instrumental settings in ESI-MS and MALDI-MS. This allows one to determine the architecture and stoichiometry of large noncovalent complexes, the binding of small molecule ligands and cofactors, and often even noncovalent binding affinities, since bound and unbound states can be clearly resolved and quantified using MS. This article presents an overview of the research in this particular area undertaken by my research group at ETH Zurich. I will introduce a number of specialized technologies that we have available in our laboratory, including MALDI mass spectrometry with high-mass detection for measuring complexes with molecular weights in the 100s of kDa range; the so-called "gap sampler" technology for high-throughput experiments; and a temperature programmed spray source for ESI. A number of selected applications will then be presented, including the study of GPCRs and other membrane proteins and their complexes, the determination of the melting behavior of non-canonical nucleic acid structures, the investigation of heterogeneous protein complexes and protein-based capsids, and the investigation of the influence of protein post-translational modifications on noncovalent binding profiles., (Copyright 2022 Renato Zenobi. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

Noncovalent complexes between cyclodextrin (CD) and divalent metal cations drew growing attentions due to their applications in the pharmaceutical industry for molecular recognition. In this study, gas-phase binding of noncovalent complexes between α-, or γ-CD and divalent metal cations was investigated by electrospray ionization mass spectrometry (ESI-MS), demonstrating the formation of 1:1 stoichiometric noncovalent complexes. The binding of the complexes were furtherly confirmed by collision-induced dissociation (CID) with tandem mass spectrometry. The CID revealed the fragmentation pattern were strongly dependent on the electronic configuration of the cations and the charge separation reaction frequently took place in the cyclodextrin-complexes with transition metal cations. For the non-covalent complexes of α-CD with Mg2+, Ca2+, Sr2+ or Ba2+ at a collision energy of 25 eV, the fragments attributed to [α-CD + cation– n Glucose unit]2+ were observed (named series A). However, for the γ-CD complexes with transition metal cations Co2+, Ni2+, Cu2+ or Zn2+, apart from fragments of series A, it were observed fragment ions of [γ-CD + cation– n Glucose unit]+ (named series B), together with the Glucose unit (m/z 163.2) and its products with loss of H 2 O (m/z 145.2 and 126.8). The CID performed at a collision energy from 10 to 50 eV showed that the binding strength of complexes increase in the order of [α-CD + Mg]2+, [α-CD + Ca]2+, [α-CD + Sr]2+ and [α-CD + Ba]2+. Through mass spectrometric titrations, the values of dissociation constant K d (in μmol•L−1) for the complexes of α-CD with Ca2+ or Ni2+ were obtained, which were 4.30 and 4.26, respectively. Image 1 • Fragment pattern for the complexes of cyclodextrin with alkaline-earth metals differed from transition metals. • The complexes of cyclodextrin with alkaline-earth metals were more stable with the increasing of radius of metals. • The complexes of cyclodextrin with dications occurred charge separation reaction when increasing declustering voltage. • Dissociation constant for the complexes of α-CD with Ca2+ or Ni2+ were obtained through mass spectrometric titrations. [ABSTRACT FROM AUTHOR]

Electrostatic interactions are one of the main factors influencing biomolecular conformation. The formation of noncovalent complexes by electrostatic interactions is governed by certain amino acid residues and post-translational modifications. It has been demonstrated that adjacent arginine forms noncovalent complex with phosphate; however, histidine noncovalent complexes have rarely been investigated. In the present work, we compare the interaction between basic epitopes (NLRRITRVN, SHHGLHSTPD) and diverse acidic and aromatic-rich peptides using both MALDI and ESI Mass spectrometry. We show that adjacent histidines can also form stable noncovalent bonds and that those bonds are probably formed by a salt bridge between the phosphate or the acid residues and the histidines. However, noncovalent complexes with the arginine epitopes form more readily and are stronger than those with histidine-containing epitopes.