The charge states of biomolecular ions in ESI-MS can be significantly increased by the addition of low-vapor supercharging (SC) reagents into the spraying solution. Despite the considerable interest from the community, the mechanistic aspects of SC are not well understood and are hotly debated. Arguments that denaturation accounts for the increased charging observed in proteins sprayed from aqueous solutions containing SC reagent have been published widely, but often with incomplete or ambiguous supporting data. In this work, we explored ESI MS charging and SC behavior of several biopolymers including proteins and DNA oligonucleotides. Analytes were ionized from 100 mM ammonium acetate (NH4Ac) aqueous buffer in both positive (ESI+) and negative (ESI–) ion modes. SC was induced either with m-NBA or by the elevated temperature of ESI capillary. For all the analytes studied we, found striking differences in the ESI MS response to these two modes of activation. The data suggest that activation with m-NBA results in more extensive analyte charging with lower degree of denaturation. When working solution with m-NBA was analyzed at elevated temperatures, the SC effect from m-NBA was neutralized. Instead, the net SC effect was similar to the SC effect achieved by thermal activation only. Overall, our observations indicate that SC reagents enhance ESI charging of biomolecules via distinctly different mechanism compared with the traditional approaches based on analyte denaturation. Instead, the data support the hypothesis that the SC phenomenon involves a direct interaction between a biopolymer and SC reagent occurring in evaporating ESI droplets.
Understanding the charging mechanism of electrospray ionization is central to overcoming shortcomings such as ion suppression or limited dynamic range, and explaining phenomena such as supercharging. Towards that end, we explore what accumulated observations reveal about the mechanism of electrospray. We introduce the idea of an intermediate region for electrospray ionization (and other ionization methods) to account for the facts that solution charge state distributions (CSDs) do not correlate with those observed by ESI-MS (the latter bear more charge) and that gas phase reactions can reduce, but not increase, the extent of charging. This region incorporates properties (e.g., basicities) intermediate between solution and gas phase. Assuming that droplet species polarize within the high electric field leads to equations describing ion emission resembling those from the equilibrium partitioning model. The equations predict many trends successfully, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for sprays employing smaller emitter opening diameters. From this view, a single mechanism can be formulated to explain how reagents that promote analyte charging (“supercharging”) such as m-NBA, sulfolane, and 3-nitrobenzonitrile increase analyte charge from “denaturing” and “native” solvent systems. It is suggested that additives’ Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI, as are Brønsted acidities for negative ESI. Because supercharging agents reduce an analyte’s solution ionization, excess spray charge is bestowed on evaporating ions carrying fewer opposing charges. Brønsted basicity (or acidity) determines how much ESI charge is lost to the agent (unavailable to evaporating analyte). Graphical Abstract
Manipulation for simplifying or increasing the observed charge state distributions of proteins can be highly desirable in mass spectrometry experiments. In the present work, we implemented a vapor introduction technique to an Agilent Jet Stream ESI (Agilent Technologies, Santa Clara, CA, USA) source. An apparatus was designed to allow for the enrichment of the nitrogen sheath gas with basic vapors. An optical setup, using laser-induced fluorescence and a pH-chromic dye, permits the pH profiling of the droplets as they evaporate in the electrospray plume. Mechanisms of pH droplet modification and its effect on the protein charging phenomenon are elucidated. An important finding is that the enrichment with basic vapors of the nitrogen sheath gas, which surrounds the nebulizer spray, leads to an increase in the spray current. This is attributed to an increase in the electrical conductivity of water-amine enriched solvent at the tip exit. Here, the increased current results in a generation of additional electrolytically produced OH(-) ions and a corresponding increase in the pH at the tip exit. Along the electrospray plume, the pH of the droplets increases due to both droplet evaporation and exposure to basic vapors from the seeded sheath gas. The pH evolution in the ESI plume obtained using pure and basic seeded sheath gas was correlated with the evolution of the charge state distribution observed in mass spectra of proteins, in the negative ion mode. Taking advantage of the Agilent Jet Stream source geometry, similar protein charge state distributions and ion intensities obtained with basic initial solutions, can be obtained using native solution conditions by seeding the heated sheath gas with basic vapors. 相似文献
The ability of polyprotic acids to give doubly charged ions in negative mode electrospray was studied and related to physicochemical properties of the acids via linear discriminant analysis (LDA). It was discovered that the compound has to be strongly acidic (low pKa1 and pKa2) and to have high hydrophobicity (logPow) to become multiply charged. Ability to give multiply charged ions in ESI/MS cannot be directly predicted from the solution phase acidities. Therefore, for the first time, a quantitative model to predict the charge state of the analyte in ESI/MS is proposed and validated for small anions. Also, a model to predict ionization efficiencies of these analytes was developed. Results indicate that acidity of the analyte, its octanol-water partition coefficient, and charge delocalization are important factors that influence ionization efficiencies as well as charge states of the analytes. The pH of the solvent was also found to be an important factor influencing the ionization efficiency of doubly charged ions.
Recently, dozens of new psychoactive substances have appeared on the European drug market every year. The most abundant group of these compounds is synthetic cannabinoids. In the first few years of the “legal highs” phenomenon, JWH (John W. Huffman) compounds were especially popular among drug users. However, the group of synthetic cannabinoids is constantly expanding, as new compounds are created by replacing known structural elements with different chemical groups. The problem with the identification of novel substances in forensic laboratories results from the structural similarity of the compounds and the rapid introduction of newer designer drugs on the black market. In this study, the fragmentation patterns of 29 new-type synthetic cannabinoids using electrospray ionization were investigated. The analysis was performed using quadrupole time-of-flight mass spectrometry. Based on measurements carried out under various conditions, the way of fragmentation of the tested compounds that were divided into groups due to their chemical structure was established. The study showed that the bond between the carbon atom of the carbonyl group and the ring or NH group attached to the ring was mainly cleaved. This mechanism was adequate for the fragmentation of first-generation synthetic cannabinoids. This paper presents characteristic ions formed by synthetic cannabinoids (i.e., ions originating from an indole/indazole ring and an adamanyl/naphthalene/quinoline ring) using electrospray ionization. Knowledge of these specific fragments can be used in forensic laboratories to determine the structure of novel compounds from the group of synthetic cannabinoids.
Electrospray ionization mass spectrometry (ESI-MS) was used for the study of cyclization of organic chelating compounds (chelators). Four chelating compounds were studed: Symmetrical ethylenediaminediacetic acid (s-EDDA), Unsymmetrical ethylenediaminediacetic acid (u-EDDA), N-(2-hydroxyethyl) ethylenediaminetriacetic acid (HEDTA), and N-(2-hydroxyethyl)iminodiacetic acid (HEIDA). The chelators were cyclized with treatments of acids and heating. The open and cyclized form of the chelators were semi-quantified by both positive and negative ion modes ESI-MS. The kinetics of chelator cyclization was studied as a function of reaction temperature and the pH of the matrix. The cyclization of s-EDDA was found to be a pseudo-first order reaction in s-EDDA and overall second order. The cyclizations of HEIDA and HEDTA are reversible reactions. Higher temperature and lower pH favors cyclization. 相似文献
The connection between charge state distributions, protein structure, and mechanistic details of electrospray are discussed
in relation to the emerging field of gas phase structural biology. Comparisons are drawn with the established area of enzymatic
catalysis in organic solvents, which shares many similar challenges. Charge solvation emerges as a dominant force in both
systems that must be dealt with to enable kinetic trapping of native structures in foreign environments. Potential methods
for mediating unfavorable charge solvation effects are discussed and, ironically, do not include partial solvation by water.
The importance of timescale in relation to the evolution of protein structure during the process of electrospray ionization
is discussed. Finally several prospects for future endeavors are highlighted. 相似文献
Growth process information and molecular structure identification are very important for characterization of self-assembled films. Here, we explore the possible application of desorption electrospray ionization mass spectrometry (DESI-MS) that provides the assembled information of rhodamine B (Rh B) and rhodamine 123 (Rh 123) films. With the help of lab-made DESI source, two characteristic ions [Rh B]+ and [Rh 123]+ are observed directly in the open environment. To evaluate the reliability of this technique, a comparative study of ultraviolet-visible (UV-vis) spectroscopy and our method is carried out, and the result shows good correlation. According to the signal intensity of characteristic ions, the layer-by-layer adsorption process of dyes can be monitored, and the thicknesses of multilayer films can also be comparatively determined. Combining the high sensitivity, selectivity, and speed of mass spectrometry, the selective adsorption of similar structure molecules under different pH is recognized easily from extracted ion chronograms. The variation trend of dyes signalling intensity with concentration of polyelectrolyte is studied as well, which reflects the effect of surface charge on dyes deposition. Additionally, the desorption area, surface morphology, and thicknesses of multilayer films are investigated using fluorescence microscope, scanning electron microscope (SEM), and atomic force microscopy (AFM), respectively. Because the desorption area was approximately as small as 2 mm2, the distribution situation of organic dyes in an arbitrary position could be gained rapidly, which means DESI-MS has advantages on in situ analysis.
The site of protonation in a molecule can greatly affect the fragments observed in product ion MS/MS spectra. In electrospray
positive ionization mass spectra, protonation usually occurs predominantly on the most basic site on the molecule to produce
the thermodynamically favored protonated species. However, the literature is unclear whether liquid phase or gas phase thermodynamics
has the greater influence. This paper describes the protonation and fragmentation behavior of crizotinib and two of its impurities.
Crizotinib has two possible protonation sites, a pyridine nitrogen and a secondary amine, piperidine nitrogen; the former is the favored site in the gas phase and the latter the more favored site in the liquid phase. The impurities
contain alkyl substitution on the piperidine nitrogen, producing tertiary amine species. Literature precedence suggests that
in the liquid phase, the piperidine nitrogen is still the most basic site but, in the gas phase, the pyridine nitrogen and
the piperidine nitrogen have very similar basicities. Fragmentation data for the three molecules suggest that the secondary
and tertiary amines protonate preferentially and almost exclusively on different sites. We propose that the secondary amine
protonates on the piperidine nitrogen (influenced by solution thermodynamics) and the two tertiary amine structures protonate
on the pyridine nitrogen because of steric hindrance at the most basic site of the molecule, allowing kinetic control of the
protonation process. 相似文献
The gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from isobutanol or aqueous solutions of GA-containing nanodiscs (NDs), are investigated using electrospray ionization-ion mobility separation-mass spectrometry (ESI-IMS-MS) and molecular dynamics (MD) simulations. The IMS arrival times measured for (2GA + 2Na)2+ ions from isobutanol reveal three different conformations, with collision cross-sections (Ω) of 683 Å2 (conformation 1, C1), 708 Å2 (C2), and 737 Å2 (C3). The addition of NH4CH3CO2 produced (2GA + 2Na)2+ and (2GA + H + Na)2+ ions, with Ω similar to those of C1, C2, and C3, as well as (2GA + 2H)2+, (2GA + 2NH4)2+, and (2GA + H + NH4)2+ ions, which adopt a single conformation with a Ω similar to that of C2. These results suggest that the nature of the charging agents, imparted by the ESI process, can influence dimer conformation in the gas phase. Notably, the POPC NDs produced exclusively (2GA + 2NH4)2+ dimer ions; the DMPC NDs produced both (2GA + 2H)2+ and (2GA + 2NH4)2+ dimer ions. While the Ω of (2GA + 2H)2+ is similar to that of C2, the (2GA + 2NH4)2+ ions from NDs adopt a more compact structure, with a Ω of 656 Å2. It is proposed that this compact structure corresponds to the ion conducting single stranded head-to-head helical GA dimer. These findings highlight the potential of NDs, combined with ESI, for transferring transmembrane peptide complexes directly from lipid bilayers to the gas phase.
Abstract The negative ion mass spectrometry of N-benzyloxycarbonyl and N-ethoxycarbonyl 1-aminoarylmethylphosphonic methyl, ethyl, and phenyl monoesters was investigated under electrospray ionization conditions. Their fragmentation pathways are proposed and supported by collisional activated dissociation product-ion spectrometry. All of the deprotonated molecules preferentially eliminate a molecule of benzyl alcohol or ethanol first to yield isocyanato-alkylphosphonate anions, which further generate phosphonate ions by the loss of carbon monoxide, phenol or alcohol, carbon monoxide plus arenes. The isocyanato-sulfonate anions can further cyclize to generate 3-aryl-2-phenoxy-1,2,4-oxaphosphazolidine-2,5-dione amide anions, which can undergo rearrangements by the loss of carbon dioxide, metaphosphorous acid phenyl ester, or carbon dioxide plus metaphosphorous acid phenyl ester, respectively, to give rise to nitrogen-containing anions. The title compounds show an obviously different fragmentation in the negative ion mode from that in the positive ion mode under the electrospray ionization conditions. 相似文献
