Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. This phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established.     

Determination of micelle mass by electrospray ionization mass spectrometry

By electrospray ionization (ESI) mass spectrometry, micelle solutions of sodium cholate were investigated in detail in the presence and absence of ethanol. The average aggregation number could be evaluated from the spectra acquired under conditions where soft collisions adequate to measure the micelle solution were induced, and the value agreed well with that obtained previously by other methods. From the dependence on ethanol content, it was also found that the average aggregation number in aqueous solution without organic solvent could be reliably estimated. The ESI method proved to be a useful tool for determining the micelle mass in the original aqueous phase.     

Evaluation of flavonoids binding to DNA duplexes by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the binding interactions of ten flavonoid aglycones and ten flavonoid glycosides with DNA duplexes. Relative binding affinities of the flavonoids toward DNA duplexes were estimated based on the fraction of bound DNA. The results revealed that the 4'-OH group of flavonoid aglycones was essential for their DNA-binding properties. Flavonoid glycosides with sugar chain linked on ring A or ring B showed enhanced binding toward the duplexes over their aglycone counterparts, whereas glycosylation of the flavonol quercetin on ring C exhibited a less pronounced effect. The aglycone skeletons and other hydroxyl substitutions on the aglycone also have an effect on the fractions of bound DNA. Upon collision-induced dissociation, the complexes containing flavonoid aglycones underwent the predominant ejection of a neutral ligand molecule, suggesting an intercalative DNA-binding mode. However, for complexes containing flavonoid glycosides, the loss of nucleobase increased to different extents, indicating a stronger binding or different binding mode. The results may provide not only a deeper insight into the DNA-binding properties of flavonoids but also a useful guideline for the design of efficient DNA-binding agents for chemotherapy.     

Role of opposite charges in protein electrospray ionization mass spectrometry

The conformation dependence of protein spectra recorded by electrospray ionization mass spectrometry (ESI-MS) is an interesting and useful phenomenon, whose origin is still the object of debate. Different mechanisms have been invoked in the attempt to explain the lower charge state of folded versus unfolded protein ions in ESI-MS, such as electrostatic repulsions, solvent accessibility, charge availability, and native-like interactions. In this work we try to subject to direct experimental test the hypothesis that conformation-dependent neutralization of charges with polarity opposite to the net charge of the protein ion could play a critical role in such an effect. We present results of time-of-flight nano-ESI-MS on the peptide angiotensin II, indicating that negative charges of carboxylate groups can contribute to spectra recorded in positive-ion mode when stabilized by favorable electrostatic interactions, which is the central assumption of our hypothesis. Comparison of horse and spermwhale myoglobin (Mb) shows that changing the total number of basic residues within a given three-dimensional structure shifts the charge-state distribution (CSD) of the folded protein in positive-ion mode. This result appears to be in contrast to models in which electrostatic repulsions or availability of charges in the ESI droplets represent the limiting factor for the ionization of folded protein ions in ESI-MS. At the same time, it suggests a role of acidic residues in conformational effects in positive-ion mode. Furthermore, an attempt is made to rationalize those cases in which, in contrast, the main charge state observed in ESI-MS under non-denaturing conditions deviates considerably from the net charge expected on the basis of the amino-acid composition. These cases usually correspond to proteins with quite balanced content in basic and acidic residues, suggesting that this might be a factor influencing their charging behavior in ESI-MS. Experiments on mutants of ribonuclease Sa (RNase Sa) reveal that progressively reducing the excess of acidic residues, replacing them by lysine, causes almost no shift in the spectrum of the folded protein in negative-ion mode. Analogously, variants with an excess of three or five basic residues give similar spectra in positive-ion mode. These results indicate a lower limit to the extent of ionization observable by ESI-MS (6- or 8+ in the case of RNase Sa in water). Below such limit of net charge, changes in the relative amount of ionizable side chains do not affect the qualitative features of the observed CSDs. A progressive loss of signal intensity caused by the mutations in negative-ion mode suggests that low charge states might also be counterselected, even within the m/z range theoretically accessible to the instrument.     

Transformation of neutral rhenium compounds during electrospray ionization mass spectrometry

Four neutral rhenium compounds were examined by electrospray ionization mass spectrometry. Acetonitrile solutions of (Ind)Re(CO)₃ (Ind = indenyl) and (Cp)Re(CO)₃ (Cp = cyclopentadienyl) gave rise to [Re(CO)₃(CH₃CN)₃]⁺ ions. This is indicative of a reaction with the solvent, although these compounds do not react with acetonitrile under regular laboratory conditions. In contrast, (Ind)Re(CO)₂(butyne) and (Cp)Re(CO)₂(butyne) did not lose their aromatic hydrocarbon ligand upon ionization; the predominant product ions generated upon electrospray ionization were [(Ind)Re(CO)(CH₃CN)(butyne)]⁺ and [(Cp)Re(CO)(CH₃CN)(butyne)]⁺, respectively.     

Synchronized dual-polarity electrospray ionization mass spectrometry

This work describes the synchronized dual-polarity (DP) electrospray ionization (ESI) method and demonstrates the first DP ESI mass spectra obtained using two mass spectrometers. Stable double Taylor cones were produced by applying two counter electric voltages with opposite polarities to one electrosprayer. The development of double Taylor cones required higher extraction voltages than conventional ESI, but DP ESI worked effectively at liquid flow rate range three times wider than conventional ESI. Using pure methanol, the emission currents of the two cones were neutralized and no current was drawn from the sprayer. Synchronized DP mass spectra were obtained using electrospray calibrants dissolved in methanol solution of low water content. For bovine insulin with conventional electrospray solution, the gas-assisted electrospray delivered satisfactory sensitivity and stability for routine mass analyses.     

Evaluation of metal-mediated DNA binding of benzoxazole ligands by electrospray ionization mass spectrometry

The binding of a series of benzoxazole analogs with different amide- and ester-linked side chains to duplex DNA in the absence and presence of divalent metal cations is examined. All ligands were found to form complexes with Ni2+, Cu2+, and Zn2+, with 2:1 ligand/metal cation binding stoichiometries dominating for ligands containing shorter side chains (2, 6, 7, and 8), while 1:1 complexes were the most abundant for ligands with long side chains (9, 10, and 11). Ligand binding with duplex DNA in the absence of metal cations was assessed, and the long side-chain ligands were found to form low abundance complexes with 1:1 ligand/DNA binding stoichiometries. The ligands with the shorter side chains only formed DNA complexes in the presence of metal cations, most notably for 7 and 8 binding to DNA in the presence of Cu2+. The binding of long side-chain ligands was enhanced by Cu2+ and to a lesser degree by Ni2+ and Zn2+. The cytotoxicities of all of the ligands against the A549 lung cancer and MCF7 breast cancer cell lines were also examined. The ligands exhibiting the most dramatic metal-enhanced DNA binding also demonstrated the greatest cytotoxic activity. Both 7 and 8 were found to be the most cytotoxic against the A549 lung cancer cell line and 8 demonstrated moderate cytotoxicity against MCF7 breast cancer cells. Metal ions also enhanced the DNA binding of the ligands with the long side chains, especially for 9, which also exhibited the highest level of cytotoxicity of the long side-chain compounds.     

Evaluation of relative DNA binding affinities of anthrapyrazoles by electrospray ionization mass spectrometry

Binding interactions of a new series of anthrapyrazoles (APs) with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/anthrapyrazole mixtures, and they show a correlation to the shift in melting point of the DNA measured from a previous study. Minimal sequence specificity was observed for the series of anthrapyrazoles. Upon collisionally activated dissociation of the duplex/anthrapyrazole complexes, typically ejection of the ligand was the dominant pathway for most of the complexes. However, for complexes containing AP2 or mitoxantrone, strand separation with the ligand remaining on one of the single strands was observed, indicative of a different binding mode or stronger binding.     

Fragmentation studies of sartans by electrospray ionization mass spectrometry

Sartans and related analogues with 5‐oxo‐l, 2, 4‐oxadiazole ring and tetrazole ring are investigated in detail using collision‐induced dissociation (CID) method in positive ion mode by electrospray ionization tandem mass spectrometry (ESI‐MSⁿ). It is found that the protonated sartans and related analogues tend to form the N‐substituted‐3‐substituted phenanthridin‐6‐amine ion which has a large conjugative structure. The possible fragmentation pathways were proposed for the first time, and the key structure of product ions was confirmed by high resolution tandem mass spectrometry and theoretical calculation. It is very helpful for understanding the intriguing roles of sartans analogues in fragmentation reactions and enriching the knowledge of the gas‐phase chemistry of the oxadiazole and tetrazole ring. Copyright © 2017 John Wiley & Sons, Ltd.     

Identification of bismuth-thiolate-carboxylate clusters by electrospray ionization mass spectrometry

The known thermal and hydrolytic stability of bismuth-sulfur bonds indicates that biological targets for bismuth likely involve thiol or thiolate functionalities, such as in L-cysteine. Complexes of bismuth with cysteine or other thiol-carboxylic acid ligands have been isolated and characterized providing a preliminary view of the potential participation of these functional groups in the biochemical mechanisms involving bismuth. A broader assessment of bismuth-thiolate interactions has been possible using electrospray ionization mass spectrometry (ESI-MS). A wide range of complexes has been observed containing mercaptosuccinic acid, 2-mercaptopropionic acid, 3-mercaptopropionic acid, and/or 2-amino-3-mercaptopropionic acid (cysteine). The identification of various multibismuth multiligand cluster ions defines new chemistry for bismuth.     

Intact skin analysis by desorption electrospray ionization mass spectrometry

In vivo skin analysis by Desorption Electrospray Ionization was characterized on healthy human volunteers by directing pneumatically assisted electrospray directly onto their fingertips. In order to eliminate the risk of electric shock, a high ohmic resistor was built into the system. Positive ion DESI-MS analysis yields low intensity spectra, while negative ion spectra feature a number of various biogenic carboxylic acids. Compounds of external origin and excreted molecules were found to have different analysis kinetics, with the exception of highly hydrophobic species. The difference was demonstrated in the case of nicotine and cotinine. Pharmacokinetic studies were performed using a rat animal model. The kinetics of the anesthetic ketamine was followed by DESI, and results were in agreement with off-line HPLC-MS blood analysis. Using a similar approach for N,N'-dimethylthiourea (DMTU), a novel method was developed for the real-time quantification of oxidative stress. DMTU was administered to the animals, and the ratio of the molecule and its oxidized form was monitored from the skin surface. The ratio was found to be highly sensitive to experimentally induced diabetes mellitus type I and angiotensin-induced chronic oxidative stress. It was concluded that the method has a number of potential applications in the fields of forensics, pharmacology and clinical chemistry.     

High-throughput quantitative analysis by desorption electrospray ionization mass spectrometry

A newly developed high-throughput desorption electrospray ionization (DESI) source was characterized in terms of its performance in quantitative analysis. A 96-sample array, containing pharmaceuticals in various matrices, was analyzed in a single run with a total analysis time of 3 min. These solution-phase samples were examined from a hydrophobic PTFE ink printed on glass. The quantitative accuracy, precision, and limit of detection (LOD) were characterized. Chemical background-free samples of propranolol (PRN) with PRN-d₇ as internal standard (IS) and carbamazepine (CBZ) with CBZ-d₁₀ as IS were examined. So were two other sample sets consisting of PRN/PRN-d₇ at varying concentration in a biological milieu of 10% urine or porcine brain total lipid extract, total lipid concentration 250 ng/μL. The background-free samples, examined in a total analysis time of 1. 5 s/sample, showed good quantitative accuracy and precision, with a relative error (RE) and relative standard deviation (RSD) generally less than 3% and 5%, respectively. The samples in urine and the lipid extract required a longer analysis time (2. 5 s/sample) and showed RSD values of around 10% for the samples in urine and 4% for the lipid extract samples and RE values of less than 3% for both sets. The LOD for PRN and CBZ when analyzed without chemical background was 10 and 30 fmol, respectively. The LOD of PRN increased to 400 fmol analyzed in 10% urine, and 200 fmol when analyzed in the brain lipid extract.     

Antisense peptide interactions studied by electrospray ionization mass spectrometry

Non-covalent interactions between met- and leu-enkephalins and their antisense peptides were studied by electrospray ionization mass spectrometry. Mixtures of sense and antisense peptides gave both the corresponding homodimers and heterodimers. The relative abundance ratios of the heterodimer to that of the homodimer of the sense peptide and the relative stability constants of the heterodimers were compared with the corresponding values from mixtures of the sense peptides and a control peptide. The results show that there is a preferential interaction between the sense and antisense peptides compared with that between the sense and control peptides.     

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.     

Electrochemical processes in electrospray ionization mass spectrometry

Editorial Comment Last month we presented, as a Special Feature, a set of five articles that constituted a Commentary on the fundamentals and mechanism of electrospray ionization (ESI). These articles produced some lively discussion among the authors on the role of electrochemistry in ESI. Six authors participated in a detailed exchange of views on this topic, the final results of which constitute this month's Special Feature. We particularly hope that younger scientists will find value in this month's Special Feature, not only for the science that it teaches but also what it reveals about the processes by which scientific conclusions are drawn. To a degree, the contributions part the curtains on these processes and show science in action. We sincerely thank the contributors to this discussion. The give and take of intellectual debate is not always easy, and to a remarkable extent this set of authors has maintained good humor and friendships, even when disagreeing strongly on substance. Graham Cooks and Richard Caprioli Copyright 2000 John Wiley & Sons, Ltd.