Electrospray Ionization Process Research Articles

Complexation of diserinol isophthalamide with phosphorylated biomolecules in electrospray ionization mass spectrometry

Electrospray ionization (ESI) enables gentle transfer of biomolecules from solution to vacuum, facilitating the study of biomolecular structure under highly controlled conditions. However, biomolecules are desolvated during the ESI process, and the loss of ionic hydrogen bonds to solvent molecules can drive structural rearrangement, most prominently at solvent-exposed charge sites. Microsolvation reagents can bind to these bare charge sites in ESI mass spectrometry (ESI–MS) experiments, providing alternative intermolecular interaction partners. Previously, 18-crown-6 was shown to be an effective reagent for binding to cationic monoalkylammonium residues. More recently, diserinol isophthalamide (DIP) was reported as an analogous anionic microsolvation reagent, primarily for carboxylate residues of small model peptides. Herein, we expand upon this work to examine the complexation of DIP, 1,1’-(1,2-phenylene)bis(3-phenylurea) (PBP), and triclocarban (TCC) with molecules featuring a terminal or linking phosphate moiety. Specifically, using ESI–MS, we assess the binding of these reagents with dimethyl phosphate (DMP), cyclic adenosine monophosphate (cAMP), dibutyryl cAMP, RNA dinucleotides ApU and CpG, and angiotensin II phosphate (DRVpYIHPF). For DMP, the smallest target molecule, reagents TCC, PBP and DIP showed favorable adduction. However, for larger systems, PBP and TCC showed reduced complexation, which was attributed to steric hindrance from the terminal aromatic moieties of PBP and the limited hydrogen bonding network of TCC. Overall, of the three reagents, DIP showed the most consistent performance for anionic microsolvation of phosphate groups, facilitating future studies of gas-phase biomolecular structure and the effects of microsolvation.

Is Native Mass Spectrometry in Ammonium Acetate Really Native? Protein Stability Differences in Biochemically Relevant Salt Solutions.

Ammonium acetate is widely used in native mass spectrometry to provide adequate ionic strength without adducting to protein ions, but different ions can preferentially stabilize or destabilize the native form of proteins in solution. The stability of bovine serum albumin (BSA) was investigated in 50 mM solutions of a variety of salts using electrospray emitters with submicron tips to desalt protein ions. The charge-state distribution of BSA is narrow (+14 to +18) in ammonium acetate (AmmAc), whereas it is much broader (+13 to +42) in solutions containing sodium acetate (NaAc), ammonium chloride (AmmCl), potassium chloride (KCl), and sodium chloride (NaCl). The average charge state and percent of unfolded protein increase in these respective solutions, indicating greater extents of protein destabilization and conformational changes. In contrast, no high charge states of either bovine carbonic anhydrase II or IgG1 were formed in AmmAc or NaCl despite their similar melting temperatures to BSA, indicating that the presence of unfolded BSA in some of these solutions is not an artifact of the electrospray ionization process. The charge states formed from the nonvolatile salt solutions do not change significantly for up to 7 min of electrospray, but higher charging occurs after 10 min, consistent with solution acidification. Formation of unfolded BSA in NaAc but not in AmmAc indicates that the cation identity, not acidification, is responsible for structural differences in these two solutions. Temperature-dependent measurements show both increased charging and aggregation at lower temperatures in NaCl:Tris than in AmmAc, consistent with lower protein stability in the former solution. These results are consistent with the order of these ions in the Hofmeister series and indicate that data on protein stability in AmmAc may not be representative of solutions containing nonvolatile salts that are directly relevant to biology.

Probing the Architecture of Multisubunit Protein Complexes with In-line Disulfide Reduction and Native MS Analysis.

Native mass spectrometry (MS) continues to enjoy growing popularity as a means of providing a wealth of information on noncovalent biopolymer assemblies ranging from composition and binding stoichiometry to characterization of the topology of these assemblies. The latter frequently relies on supplementing MS measurements with limited fragmentation of the noncovalent complexes in the gas phase to identify the pairs of neighboring subunits. While this approach has met with much success in the past two decades, its implementation remains difficult (and the success record relatively modest) within one class of noncovalent assemblies: protein complexes in which at least one binding partner has multiple subunits cross-linked by disulfide bonds. We approach this problem by inducing chemical reduction of disulfide bonds under nondenaturing conditions in solution followed by native MS analysis with online buffer exchange to remove unconsumed reagents that are incompatible with the electrospray ionization process. While this approach works well with systems comprised of thiol-linked subunits that remain stable upon reduction of the disulfide bridges (such as immunoglobulins), chemical reduction frequently gives rise to species that are unstable (prone to aggregation). This problem is circumvented by taking advantage of the recently introduced cross-path reactive chromatography platform (XPRC), which allows the disulfide reduction to be carried out in-line, thereby minimizing the loss of metastable protein subunits and their noncovalent complexes with the binding partners prior to MS analysis. The feasibility of this approach is demonstrated using hemoglobin complexes with haptoglobin 1-1, a glycoprotein consisting of four polypeptide chains cross-linked by disulfide bonds.

Observation of Solvent-Dependence in the Mechanism of Neutral-Catalyzed Isomerization of para-Aminobenzoic Acid Protomers.

Proton-transfer reactions are commonplace during electrospray ionization (ESI) mass spectrometry experiments and are often responsible for imparting charge to analyte molecules. Multiple protonation-site isomers (protomers) can arise for polyfunctional molecules and these isomers can interconvert via solvent-mediated proton transfer reactions during various stages of the ESI process. Studying the populations and interconversion of protonation isomers provides key insight into the ESI process, ion-molecule interactions, and ion dissociation mechanisms. An archetype molecule to study protomer interconversion fundamentals in this context is para-aminobenzoic acid (pABA), where both the amino and carboxylic acid protomers are typically formed under ESI and the mechanisms for interconversion are still under refinement. Using ion-trap mass spectrometry reaction kinetics (2.5 mTorr, 300 K), this study examines gas-phase interconversion catalysis of pABA protomers by seven neutral species, which are commen solvents and additives used for ESI: water, formic acid, methanol, ethanol, propanol, ammonia, and acetonitrile. Three distinct reaction cases are reported: (i) formic acid, methanol, ethanol, propanol, and ammonia each catalyze the interconversion between the amino and carboxylic acid protomers via a n = 1 solvent-molecule vehicle mechanism; (ii) for water, however, a n = 6 adduct complex is detected and this suggests that the observed protomer interconversion occurs through a Grotthuss mechanism, in accord with literature reports; (iii) acetonitrile inhibits proton transfer by the formation of particularly stable n = 1 and 2 adduct complexes. The second-order rate constants for the protomer interconversion are observed to increase in the following order: H2O < HCO2H < MeOH < EtOH < PrOH < NH3. Potential energy schemes are reported for all neutral-catalyzed proton transfer reactions using the DSD-PBEP86-D3(BJ)/aug-cc-pVDZ level of theory. A central transition state, which connects the protonation site adducts, is shown to be the key rate-limiting step. The energy of this transition state is sensitive to the proton affinity of the neutral solvent, and this is supported by the correlation between the reaction rate and the solvent proton affinity.

Encapsulation in Charged Droplets Generates Distorted Host-Guest Complexes.

The ability of various hydrogen-bonded resorcinarene-based capsules to bind α,ω-alkylbisDABCOnium (DnD) guests of different lengths was investigated in solution and in the gas-phase. While no host-guest interactions were detected in solution, encapsulation could be achieved in the charged droplets formed during electrospray ionisation (ESI). This included guests, which are far too long in their most stable conformation to fit inside the cavity of the capsules. A combination of three mass spectrometric techniques, namely, collision-induced dissociation, hydrogen/deuterium exchange, and ion-mobility mass spectrometry, together with computational modelling allow us to determine the binding mode of the DnD guests inside the cavity of the capsules. Significant distortions of the guest into horseshoe-like arrangements are required to optimise cation-π interactions with the host, which also adopt distorted geometries with partially open hydrogen-bonding seams when binding longer guests. Such quasi "spring-loaded" capsules can form in the charged droplets during the ESI process as there is no competition between guest encapsulation and ion pair formation with the counterions that preclude encapsulation in solution. The encapsulation complexes are sufficiently stable in the gas-phase - even when strained - because non-covalent interactions significantly strengthen in the absence of solvent.

On the Chemistry of Aqueous Ammonium Acetate Droplets during Native Electrospray Ionization Mass Spectrometry.

Ammonium acetate (NH4Ac) is a widely used solvent additive in native electrospray ionization (ESI) mass spectrometry. NH4Ac can undergo proton transfer to form ammonia and acetic acid (NH4+ + Ac- → NH3 + HAc). The volatility of these products ensures that electrosprayed ions are free of undesired adducts. NH4Ac dissolution in water yields pH 7, providing "physiological" conditions. However, NH4Ac is not a buffer at pH 7 because NH4+ and Ac- are not a conjugate acid/base pair (Konermann, L. J. Am. Soc. Mass Spectrom. 2017, 28, 1827-1835.). In native ESI, it is desirable that analytes experience physiological conditions not only in bulk solution but also while they reside in ESI droplets. Little is known about the internal milieu of NH4Ac-containing ESI droplets. The current work explored the acid/base chemistry of such droplets, starting from a pH 7 analyte solution. We used a two-pronged approach involving evaporation experiments on bulk solutions under ESI-mimicking conditions, as well as molecular dynamics simulations using a newly developed algorithm that allows for proton transfer. Our results reveal that during droplet formation at the tip of the Taylor cone, electrolytically generated protons get neutralized by Ac-, making NH4+ the net charge carriers in the weakly acidic nascent droplets. During the subsequent evaporation, the droplets lose water as well as NH3 and HAc that were generated by proton transfer. NH3 departs more quickly because of its greater volatility, causing the accumulation of HAc. Together with residual Ac-, these HAc molecules form an acetate buffer that stabilizes the average droplet pH at 5.4 ± 0.1, as governed by the Henderson-Hasselbalch equation. The remarkable success of native ESI investigations in the literature implies that this pH drop by ∼1.6 units relative to the initially neutral analyte solution can be tolerated by most biomolecular analytes on the short time scale of the ESI process.

Recent Developments in the Methods and Applications of Electrostatic Theory.

ConspectusThe review improves our understanding of how electrostatic interactions in the electrolyte, gas phase, and on surfaces can drive the fragmentation and assembly of particles. This is achieved through the overview of our advanced theoretical and computational modeling toolbox suitable for interpretation of experimental observations and discovery of novel, tunable assemblies and architectures. In the past decade, we have produced a significant, fundamental body of work on the development of comprehensive theories based on a rigorous mathematical foundation. These solutions are capable of accurate predictions of electrostatic interactions between dielectric particles of arbitrary size, anisotropy, composition, and charge, interacting in solvents, ionized medium, and on surfaces. We have applied the developed electrostatic approaches to describe physical and chemical phenomena in dusty plasma and planetary environments, in Coulomb fission and electrospray ionization processes, and in soft matter, including a counterintuitive but widespread attraction between like-charged particles.Despite its long history, the search for accurate methods to provide a deeper understanding of electrostatic interactions remains a subject of significant interest, as manifested by a constant stream of theoretical and experimental publications. While major international effort in this area has focused predominantly on the computational modeling of biocatalytic and biochemical performance, we have expanded the boundaries of accuracy, generality, and applicability of underlying theories. Simple solvation models, often used in calculating the electrostatic component of molecular solvation energy and polarization effects of solvent, rarely go beyond the induced dipole approximation because of computational costs. These approximations are generally adequate at larger separation distances; however, as particles approach the touching point, more advanced charged-induced multipolar descriptions of the electrostatic interactions are required to describe accurately a collective behavior of polarizable neutral and charged particles. At short separations, the electrostatic forces involving polarizable dielectric and conducting particles become nonadditive which necessitates further developments of quantitatively accurate many-body approaches. In applications, the electrostatic response of materials is commonly controlled by externally applied electric fields, an additional complex many-body problem that we have addressed most recently, both theoretically and numerically.This review reports on the most significant results and conclusions underpinning these recent advances in electrostatic theory and its applications. We first discuss the limitations of classical approaches to interpreting electrostatic phenomena in electrolytes and complex plasmas, leading to an extended analytical theory suitable for accurate estimation of the electrostatic forces in a dilute solution of a strong electrolyte. We then introduce the concept and numerical realization of many-body electrostatic theory focusing on its performance in selected experimental cases. These experiments underpin, among other applications, electrostatic self-assembly of two-dimensional lattice structures, melting of ionic colloidal crystals in an external electric field, and coalescence of charged clusters.

In searching of optimum electrospray ionization using both spray image and electric current measurement

Electrospray image and spray electric current measurement were used to experimentally characterize the optimum operating condition of the electrospray ionization process under different operating conditions, including electrospray flow rate (Q), solution electric conductivity (k) and the organic solvent content in the sample solution (Ro). It was first observed that a special cone-jet mode electrospray with extremely high stability could be obtained when the electrospray voltage was operated in a specific range (the optimum voltage range). In this mode, the spray current remains independent of spray voltage and the angle of electrospray (the optimum spray angle) was much larger than the ones in other modes of electrospray. The experimental results also showed that the optimum voltage range was dependent on multiple factors. The average value of the optimum voltage range (the optimum voltage) increased slightly as Q was increased and decreased significantly as Ro was increased. In addition, the optimum voltage would increase initially with the increase of k and become essentially independent of k at a sufficiently high solution electric conductivity. In contrast to a rather complicated parametric dependence of the optimum voltage range, the optimum spray angle was found to only depend on Q. It would increase initially as the increase of Q and remain to be constant when Q was higher than a specific value. The experimental results in this study allow one to maintain an optimum electrospray ionization under different operating conditions as required by the actual method of mass spectrometry analysis.

Gas-Phase Internal Proton-Transfer of Protonated para-Aminobenzoic Acid Catalyzed by One Methanol Molecule.

Electrospray ionization (ESI) is used to deliver analytes for mass analysis across a huge range of mass spectrometry applications. Despite its ubiquitous application and many mechanistic investigations, it remains that a fundamental understanding of ESI processes is not complete. In particular, all the factors that influence the populations of protonation isomers are elusive such that it remains a challenge to optimize experimental conditions to favor one isomer over another. The molecule para-aminobenzoic acid has emerged as an archetype for the study of protonation isomers, with both amino and carboxylic acid protonation site isomers (protomers) typically formed upon ESI, with the isomer ratio shown to be sensitive to several physical and chemical parameters. Here we report an ion-trap mass spectrometry study of the time-resolved methanol-catalyzed proton transfer between the amine and carboxylic acid moieties of para-aminobenzoic acid. The experimental and computational results presented are consistent with a bimolecular mechanism where isomerization is mediated by a single methanol rather than a multimolecular Grotthuss proton transfer process. Pseudo-first-order rate constants for protomer specific product ions are reported and confirm the depletion of the amino protomer is correlated to the growth of the carboxylic acid protomer. Under the controlled conditions of a low-pressure ion-trap mass spectrometer (2.5 mTorr, 300 K), the number of methanol molecules required to isomerize para-aminobenzoic acid is determined to be one, and the second-order rate constant for methanol-catalyzed isomerization is (1.9 ± 0.1) × 10-11 cm3 molecule-1 s-1. The para-aminobenzoic acid vehicle mechanism is explored computationally at the DSD-PBEP86-D3BJ/aug-cc-pVDZ level of theory and reveals that the transition state for proton transfer is submerged (-10 kJ mol-1) relative to the separated reactant energies. The findings from this paper show that single-solvent catalyzed intramolecular proton transfer reactions are possible and must be considered during the late stages of ESI to predict the site(s) of protonation and the ion's stability in the presence of solvent molecules.

Inter-Domain Repulsion of Dumbbell-Shaped Calmodulin during Electrospray Ionization Revealed by Molecular Dynamics Simulations.

The mechanisms whereby protein ions are released from nanodroplets at the liquid-gas interface have continued to be controversial since electrospray ionization (ESI) mass spectrometry was widely applied in biomolecular structure analysis in solution. Several viable pathways have been proposed and verified for single-domain proteins. However, the ESI mechanism of multi-domain proteins with more complicated and flexible structures remains unclear. Herein, dumbbell-shaped calmodulin was chosen as a multi-domain protein model to perform molecular dynamics simulations to investigate the structural evolution during the ESI process. For [Ca4CAM], the protein followed the classical charge residue model. As the inter-domain electrostatic repulsion increased, the droplet was found to split into two sub-droplets, while stronger-repulsive apo-calmodulin unfolded during the early evaporation stage. We designated this novel ESI mechanism as the domain repulsion model, which provides new mechanistic insights into further exploration of proteins containing more domains. Our results suggest that greater attention should be paid to the effect of domain-domain interactions on structure retention during liquid-gas interface transfer when mass spectrometry is used as the developing technique in gas phase structural biology.

New Insights into the Intrinsic Electron-Based Dissociation Behavior of Cytochrome c Oligomers.

Between 2003 and 2017, four reports were published that demonstrated the intrinsic ability of the native iron-containing proteins cytochrome c and ferritin to undergo radical-based backbone fragmentation in the gas phase without the introduction of exogenous electrons. For cytochrome c in particular, this effect has so far only been reported to occur in the ion source, preventing the in-depth study of reactions occurring after gas-phase isolation of specific precursors. Here, we report the first observation of this intrinsic native electron capture dissociation behavior after quadrupole isolation of specific charge states of the cytochrome c dimer and trimer, providing direct experimental support for key aspects of the mechanism proposed 20 years ago. Furthermore, we provide evidence that, in contrast to some earlier proposals, these oligomeric states are formed in bulk solution rather than during the electrospray ionization process and that the observed fragmentation site preferences can be rationalized through the structure and interactions within these native oligomers rather than the monomer. We also show that the observed fragmentation pattern─and indeed, whether or not fragmentation occurs─is highly sensitive to the provenance and history of the protein samples, to the extent that samples can show distinct fragmentation behavior despite behaving identically in ion mobility experiments. This rather underexplored method therefore represents an exquisitely sensitive conformational probe and will hopefully receive more attention from the biomolecular mass spectrometry community in the future.

Stabilization of Labile Lysozyme-Ligand Interactions in Native Electrospray Ionization Mass Spectrometry.

Flavonoids are polyphenolic secondary metabolites with extensive biological activities and pharmacological effects. Exploring the interactions of flavonoids with proteins may be helpful for understanding their biological processes. Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool to characterize the noncovalent protein-ligand (PL) complexes. However, some protein-flavonoid complexes are labile during electrospray ionization. Here, the labile lysozyme-flavonoid (rutin, icariin, and naringin) complexes were determined by direct ESI-MS without derivation. It has been found that low amounts of N-methylpyrrolidinone and dimethylformamide can protect labile lysozyme-flavonoid complexes away from dissociation during electrospray ionization process. The intact lysozyme-flavonoid complexes were specifically observed in mass spectra, and the measured binding affinities by ESI-MS were matched with the fluorescence data. The effects of additives on the analysis of lysozyme-flavonoid complexes were investigated by ESI-MS, combined with the molecular docking and fluorescence. This strategy was helpful to investigate the labile PL interactions by direct ESI-MS.

Release of nanodiscs from charged nano-droplets in the electrospray ionization revealed by molecular dynamics simulations

Electrospray ionization (ESI) is essential for application of mass spectrometry in biological systems, as it prevents the analyte being split into fragments. However, due to lack of a clear understanding of the mechanism of ESI, the interpretation of mass spectra is often ambiguous. This is a particular challenge for complex biological systems. Here, we focus on systems that include nanodiscs as membrane environment, which are essential for membrane proteins. We performed microsecond atomistic molecular dynamics simulations to study the release of nanodiscs from highly charged nano-droplets into the gas phase, the late stage of ESI. We observed two distinct major scenarios, highlighting the diversity of morphologies of gaseous product ions. Our simulations are in reasonable agreement with experimental results. Our work provides a detailed atomistic view of the ESI process of a heterogeneous system (lipid nanodisc), which may give insights into the interpretation of mass spectra of all lipid-protein systems.

Optimization of Mobile Phase Modifiers for Fast LC-MS-Based Untargeted Metabolomics and Lipidomics.

Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for the untargeted profiling of biological samples. A multiplatform LC-MS-based approach is needed to screen polar metabolites and lipids comprehensively. Different mobile phase modifiers were tested to improve the electrospray ionization process during metabolomic and lipidomic profiling. For polar metabolites, hydrophilic interaction LC using a mobile phase with 10 mM ammonium formate/0.125% formic acid provided the best performance for amino acids, biogenic amines, sugars, nucleotides, acylcarnitines, and sugar phosphate, while reversed-phase LC (RPLC) with 0.1% formic acid outperformed for organic acids. For lipids, RPLC using a mobile phase with 10 mM ammonium formate or 10 mM ammonium formate with 0.1% formic acid permitted the high signal intensity of various lipid classes ionized in ESI(+) and robust retention times. For ESI(-), the mobile phase with 10 mM ammonium acetate with 0.1% acetic acid represented a reasonable compromise regarding the signal intensity of the detected lipids and the stability of retention times compared to 10 mM ammonium acetate alone or 0.02% acetic acid. Collectively, we show that untargeted methods should be evaluated not only on the total number of features but also based on common metabolites detected by a specific platform along with the long-term stability of retention times.

Investigating Electrosprayed Droplets Using Particle-into-Liquid Sampling for Nanoliter Electrochemical Reactions

Electrospray ionization (ESI) is a powerful ionization technique that can generate charged solvent droplets and bare analyte ions from sample solutions. Despite seeing extensive use in mass spectrometry due in part to the low internal energy deposited into the ions formed during ionization, some unknowns persist regarding the exact dynamics of droplet breakup and molecule behavior during spray, and research is still underway regarding how various types of molecules acquire charge during the ESI process. Previously, the authors introduced a novel aerosol measurement technique, particle-into-liquid sampling for nanoliter electrochemical reactions (PILSNER). The current work introduces a new method utilizing PILSNER for the examination of the particles generated during ESI using simple analysis techniques with a commercially available potentiostat. This technique is applied in this work for the detection of charges on electrosprayed droplets, including the estimation of the number of charges on individual ESI droplets using a fluorescent proxy. This technique provides an additional tool for the exploration of the complex process of droplet generation and ion liberation during ESI.

Coated Blade Spray with a Barrier: Improving Negative Electrospray Ionization and Sample Preparation.

Substrate-based electrospray ionization (ESI) techniques like paper, wooden tip, plastic tip, and metal-needle-based spray suffer from corona discharge, high background noise, and unstable spray in negative ionization mode, especially for the analysis of complex biological matrices, such as blood and urine. Coated blade spray coupled with mass spectrometry (CBS-MS) combines solid-phase microextraction's (SPME) efficient sample clean-up and enrichment and ambient MS's fast analysis and has proven to be an appealing alternative tool for the fast screening of target analytes in complex matrices. This paper documents the development of a new CBS blade design that features a barrier at the far end of the ESI tip. The findings of this work show that the addition of this simple barrier enabled the total RSD% to be reduced to less than 10% for sample preparation, ionization, and the MS detection of several drugs of abuse in negative mode, without compensation using internal standards. The improved stability of ESI in negative mode was investigated by observing the ESI process with a microscope camera and testing via CBS-MS. The new design was applied for the analysis of three drugs of abuse in urine, with the calibration curve correlation coefficient (R2 ≥ 0.9997) being calculated without the use of internal standards. The overall RSD% of the peak area for one compound in 42 samples was 6.9%, which highlights the method's incredible reproducibility compared to other ambient MS techniques for analyzing real samples. The CBS device with a barrier was also applied for the on-blade sampling of 14 drugs of abuse in 20 μL of plasma spot in positive ionization mode. The results of these tests yielded a calibration curve correlation coefficient of R2 ≥ 0.9883 and limits of quantification (LOQs) between 0.25 and 25 ng/mL. The obtained results provide guidance on CBS device design optimization and the effective automation of the protocol.

Electrospray ionization in-source decay of N-glycans and the effects on N-glycan structural identification.

Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are soft ionization techniques commonly used in mass spectrometry. Although in-source and post-source decays of MALDI have been investigated extensively, the analogous decays of ESI have received little attention. Previous studies regarding the analogous decays of ESI focus on the dissociation of multiply charged proteins and peptides. The decay of carbohydrates in ESI has not been investigated yet, and it may have interference in carbohydrate structural determination. Commercial apparatus, including a high-performance liquid chromatography (HPLC), an ESI source, and a linear ion trap mass spectrometer, were used to investigate the fragmentation of several N-glycans during the ESI process. About 0.2%-3% of neutral N-glycans and more than 50% of N-glycans consisting of a sialic acid are dissociated into small N-glycans by ESI in-source decay in typical ESI operating conditions. The efficiencies of most dissociation channels increase as the temperature of ion transfer capillary increases, indicating that part of the energy deposited into the precursor ions for cracking is from the heated capillary. The cracking patterns of ESI in-source decay are slightly different from those of gaseous phase collision-induced dissociation. Large N-glycans are dissociated into small N-glycans in ESI in-source decay that may result in the interference of the structural identification of small N-glycans. Separation of large N-glycans from small N-glycans, for example, using HPLC, prior to ESI ionization is necessary to eliminate the interference. This is particularly important when N-glycans consist of sialic acid or large N-glycans have much higher concentration than that of small N-glycans in ESI solution.

Characterization of the Internal Energy of Ions Produced by Electrospray Ionization Using Substituted Benzyl Ammonium Thermometer Ions.

We propose the use of substituted benzyl ammonium species as thermometer ions to characterize the internal energy distribution of the ions produced by electrospray ionization (ESI). Crucially, we found that the activation of the benzyl ammonium species preferentially provided a benzyl cation via N-Cα bond cleavage. In addition, calculations at the CCSD(T)/cc-PVTZ//M06-2X-D3/6-311++G(d,p) level of theory revealed that the threshold energies of fragmentation of the tested model ions ranged from 86 to 192 kJ mol-1, significantly lower than those of conventional 4-substituted benzylpyridinium thermometer ions. Thus, the substituted benzyl ammoniums are suitable for the characterization of the ESI process under typical experimental conditions. Further, the internal energies of the ions were found to depend on the radiofrequency voltage of the ion funnel, which is used to increase the transport efficiency of the ions from atmospheric to vacuum conditions. Our reported method will aid the determination of the optimum ion-funnel radiofrequency voltage for the analysis of small molecules such as metabolites. Furthermore, benzyl ammoniums are commercially available, which will facilitate the routine and widespread measurement of the internal energy distributions of ions.

Tautomers and Rotamers of Curcumin: A Combined UV Spectroscopy, High-Performance Liquid Chromatography, Ion Mobility Mass Spectrometry, and Electronic Structure Theory Study.

The structures of tautomers and rotameric forms of curcumin, the bioactive compound present in spice plant turmeric, have been investigated using ion mobility mass spectrometry (IMMS) in conjunction with high-performance liquid chromatography (HPLC) and UV-visible spectroscopy. Two tautomeric forms of this β-diketone compound, keto-enol and diketo, have been chromatographically separated, and the electronic absorption spectra for these two tautomeric forms in methanol solution have been recorded separately for the first time. The molecular identity of the HPLC-separated solution fractions is established unambiguously by recording the mass and fragmentation spectra simultaneously. The ion mobility spectrum for the deprotonated curcumin anion, [Cur-H]-, corresponding to the diketo tautomer, displays only one peak (P), and the collision cross-section (CCS) value is measured to be 185.9 Å2. However, the ion mobility spectrum corresponding to the HPLC-separated keto-enol tautomer shows three distinctly separated peaks, P, Q, and R, with CCS values of 185.9, 194.8, and 203.4 Å2, respectively, whereby peak R appears to be the most intense one, followed by peaks P and Q. The theoretically calculated CCS values of different isomers of [Cur-H]-, optimized by electronic structure theory methods, display satisfactory correlation with the experimentally observed values, corroborating our assignments. The spectral attributes also indicate the occurrence of structural rearrangements in the electrospray ionization process. With the aid of the electronic structure calculation, low-energy pathways for the occurrence of the structural isomerization to surpass the energy barrier are suggested, which are consistent with the assignments of the peaks observed in the IM spectra.

Impact of instrumental settings in electrospray ionization ion trap mass spectrometry on the analysis of multi-CH3-/CD3-isotopologs in cellulose ether analysis: a quantitative evaluation

Exact quantification of the molar ratios of isotopologous mixed O-methyl-O-methyl-d3-cellooligosaccharides (COS) comprising all combinations from fully methylated to fully deuteromethylated constituents within an individual degree of polymerization (DP) is the key step in the analysis of the substituent distribution over the polymer chains in methyl celluloses (MC). Deuteromethylation of MC is performed to level chemical differences, but due to a m/z range of 3 DP·ΔMe/Me-d3, bias during MS measurement cannot certainly be excluded. Therefore, ionization, ion transportation, and ion storage were studied with an electrospray ionization ion trap mass spectrometer (ESI-IT-MS) using binary equimolar mixtures of per-O-Me- and per-O-Me-d3-COS, defining the border cases of a particular Me/Me-d3-profile. Reference data of their molar ratio were determined after reductive amination with m-amino benzoic acid by HPLC-UV. COS of DP2–6 were measured as their sodium adducts at c = 10−6 M by syringe pump infusion. The impact of the RF voltage of the ion trap (TD), the octopole RF and DC voltages, and the Cap Exit potential on absolute and relative ion intensities were studied. Adapting the Cap Exit voltage was essential for correct quantification of DP2, while all COS of higher DP behaved insensitive with respect to bias. To check whether any bias occurs in the electrospray ionization process of the isotopologs, concentration-dependent measurements were performed with optimized instrumental settings for each DP. Intensity ratios IR = I (Me-d3)/I (Me) did not show any concentration-dependent trend and no selective ion suppression. Its decrease with DP observed under usually applied standard conditions (smart mode) is a consequence of discrimination according to m/z and can be overcome by appropriate instrumental settings of Oct 2 DC and TD. IR between 0.971 ± 0.008 and 1.040 ± 0.009 with no trend for DP (2-6) were obtained by averaging all measurements in the range 2 · 10−7 to 2 · 10−5 M total concentration. The DP-related optimized settings were applied to two MCs and compared with the results obtained under so far applied standard conditions.Graphical abstract