Carbonation and Other Super Saturated Gases as Solution Modifiers for Improved Sensitivity in Solvent Assisted Ionization Inlet (SAII) and ESI

Solvent Assisted Ionization Inlet (SAII) produces ions in a heated inlet to a mass spectrometer from aqueous and aqueous/organic solutions with high sensitivity. However, the use of acid modifiers, which typically aids electrospray ionization, generally results in ion suppression in SAII. Here we demonstrate that the use of carbonation and other super-saturated gases as solution modifiers increases analyte ion abundance and reduces metal cation adduction in SAII. Carbonation is also found to enhance electrospray ionization. The mechanistic and practical utility of carbonation in mass spectrometry is addressed.      

Coaxial Electrospray Ionization for the Study of Rapid In-source Chemistry

Coaxial electrospray has been used effectively for several dual-emitter applications, but has not been utilized for the study of rapid in-source chemistry. In this paper, we report the fabrication of a coaxial, micro-volume dual-emitter through the modification of a manufacturer’s standard electrospray probe. This modification creates rapid mixing inside the Taylor cone and the ability to manipulate fast reactions using a variety of solvents and analytes. We demonstrate its potential as a low-cost, dual-emitter assembly for diverse applications through three examples: relative ionization in a biphasic electrospray, hydrogen-deuterium exchange, and protein supercharging.

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The Effect of Organic Modifiers on Electrospray Ionization Charge-State Distribution and Desorption Efficiency for Oligonucleotides

The chemical composition of the solution has a critical impact on the electrospray desorption efficiency of oligonucleotides. Several physiochemical properties of various organic modifiers were investigated with respect to their role in the desorption process of oligonucleotides. The Henry’s Law Constant, which reflects the volatility of alkylamines, was found to have a prominent effect on both the electrospray charge state distribution and desorption efficiency of oligonucleotides. Alkylamines with higher $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values such as hexylamine, piperidine, and imidazole reduced the charge state distribution by forming complexes with the oligonucleotide and dissociating from it in the gas phase, while alkylamines with extremely low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values reduced the electrospray charge state distribution by facilitating ion emission at an earlier stage of the electrospray desorption process. Ion-pairing agents with moderate $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values do not alter the electrospray charge state distribution of oligonucleotides and their ability to enhance oligonucleotide ionization followed the order of decreasing $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ values. The Henry’s Law Constant also correlated to the impact of the acidic modifiers on oligonucleotide ionization efficiency. Ionization enhancement effects were observed with hexafluoroisopropanol, and this effect was attributed to its low $ \mathrm{k}_{\mathrm{H,cc}}\left( {\mathrm{aq}/\mathrm{gas}} \right) $ and moderate acidity. The comprehensive effects of both alkylamine and hexafluoroisoproapnol on the electrospray ionization desorption of oligonucleotides were also evaluated, and acid-base equilibrium was found to play a critical role in determining these effects.      

OmniSpect: An Open MATLAB-Based Tool for Visualization and Analysis of Matrix-Assisted Laser Desorption/Ionization and Desorption Electrospray Ionization Mass Spectrometry Images

We present omniSpect, an open source web- and MATLAB-based software tool for both desorption electrospray ionization (DESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) that performs computationally intensive functions on a remote server. These functions include converting data from a variety of file formats into a common format easily manipulated in MATLAB, transforming time-series mass spectra into mass spectrometry images based on a probe spatial raster path, and multivariate analysis. OmniSpect provides an extensible suite of tools to meet the computational requirements needed for visualizing open and proprietary format MSI data.      

Method for Continuous Monitoring of Electrospray Ion Formation

A method for continuously monitoring the performance of electrospray ionization without the addition of hardware or chemistry to the system is demonstrated. In the method, which we refer to as SprayDx, cluster ions with solvent vapor natively formed by electrospray are followed throughout the collection of liquid chromatography–selected reaction monitoring data. The cluster ion extracted ion chromatograms report on the consistency of the ion formation and detection system. The data collected by the SprayDx method resemble the data collected for postcolumn infusion of analyte. The response of the cluster ions monitored reports on changes in the physical parameters of the ion source such as voltage and gas flow. SprayDx is also observed to report on ion suppression in a fashion very similar to a postcolumn infusion of analyte. We anticipate the method finding utility as a continuous readout on the performance of electrospray and other atmospheric pressure ionization processes.

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Gas-Phase Fragmentation of Oligoproline Peptide Ions Lacking Easily Mobilizable Protons

The fragmentation of peptides containing quaternary ammonium group, but lacking easily mobilizable protons, was examined with the aid of deuterium-labeled analogs and quantum-chemical modeling. The fragmentation of oligoproline containing quaternary ammonium group involves the mobilization of hydrogens localized at α- and γ- or δ-carbon atoms in the pyrrolidine ring of proline. The study of the dissociation pattern highlights the unusual proline residue behavior during MS/MS experiments of peptides.      

A High Voltage Power Supply That Mitigates Current Reversals in Capillary Zone Electrophoresis-Electrospray Mass Spectrometry

Capillary electrophoresis coupled with electrospray ionization typically employs two power supplies, one at each end of the capillary. One power supply is located at the proximal (injection) end of the capillary. The power supply located at the distal (detector) end of the capillary drives the electrospray. Electrophoresis is driven by the difference in potential between these power supplies. Separations that employ large capillary inner diameter, high conductivity background electrolyte, and high separation potentials generate higher current than that produced by the electrospray. Excess current flows through the electrospray power supply. Most power supplies are not designed to sink current, and the excess current will cause the electrospray voltage to deviate from its set point. We report a simple circuit to handle this excess current, allowing separations under a wide range of electrophoretic conditions.

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In-Line Desalting of Proteins from Buffer and Synthetic Urine Solution Prior to ESI-MS Analysis via a Capillary-Channeled Polymer Fiber Microcolumn

Presented here is a novel in-line solid phase extraction (SPE) method utilizing a capillary-channeled polymer (C-CP) fiber microcolumn prior to introduction to an electrospray ionization (ESI) source. The high permeability of the microcolumn allows for operation under syringe pump or HPLC driven flow, ultimately providing greater mass spectral clarity and accurate molecular weight determinations for different protein/buffer combinations. Studies presented here focus on the desalting of several target proteins from a standard phosphate buffered saline (PBS) matrix and a synthetic urine solution prior to ESI-MS determinations. In every case, responses for μM-level proteins in PBS improve from the situation of not permitting molecular weight determinations to values that are precise to better than ±10 Da, without internal standards, with relative improvements in the signal-to-background ratios (S/B) on the order of 3,000×. De-salting of a myoglobin-spiked (12 μM) synthetic urine results in equally-improved spectral quality.      

Use of an Open Port Sampling Interface Coupled to Electrospray Ionization for the On-Line Analysis of Organic Aerosol Particles

A simple design for an open port sampling interface coupled to electrospray ionization (OPSI-ESI) is presented for the analysis of organic aerosols. The design uses minimal modifications to a Bruker electrospray (ESI) emitter to create a continuous flow, self-aspirating open port sampling interface. Considerations are presented for introducing aerosol to the open port sampling interface including aerosol gas flow and solvent flow rates. The device has been demonstrated for use with an aerosol of nicotine as well as aerosol formed in the pyrolysis of biomass. Upon comparison with extractive electrospray ionization (EESI), this device has similar sensitivity with increased reproducibility by nearly a factor of three. The device has the form factor of a standard Bruker/Agilent ESI emitter and can be used without any further instrument modifications.

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Biomimetic Antireflective Silicon Nanocones Array for Small Molecules Analysis

Biomimetic antireflective silicon nanocones array is used for analysis of small molecules by mass spectrometry. The role of the absorbed laser energy and its distribution in the laser desorption/ionization process has been investigated by varying the antireflective features precisely. By optimizing the antireflective silicon array, the absorbed laser energy can be channeled completely into the desorption/ionization of analytes. The optimized silicon array exhibits excellent performance to detect peptide, amino acid, drug molecule, and carbohydrate without any interference in the low-mass region.      

Overtone Mobility Spectrometry: Part 5. Simulations and Analytical Expressions Describing Overtone Limits

Mathematical expressions for the analytical duty cycle associated with different overtones in overtone mobility spectrometry are derived from the widths of the transmitted packets of ions under different instrumental operating conditions. Support for these derivations is provided through ion trajectory simulations. The outcome of the theory and simulations indicates that under all operating conditions there exists a limit or maximum observable overtone that will result in ion transmission. Implications of these findings on experimental design are discussed.      

Structural Analysis of Triacylglycerols by Using a MALDI-TOF/TOF System with Monoisotopic Precursor Selection

A new MALDI-TOF/TOF system with monoisotopic precursor selection was applied to the analysis of triacylglycerols in an olive oil sample. Monoisotopic precursor selection made it possible to obtain product-ion mass spectra without interference from species that differed by a single double bond. Complete structure determination of all triacylglycerols, including structural isomers, was made possible by interpreting the charge-remote fragmentation resulting from high-energy collision-induced dissociation (CID) of the sodiated triacylglycerols.      

Electrospray Generated from the Tip-Sealed Fine Glass Capillary Inserted with an Acupuncture Needle Electrode

In electrospray, excess charges are supplied to a sample solution by the occurrence of electrochemical reactions. Recently, different versions of electrospray, e.g., dielectric barrier electrospray ionization, inductive desorption electrospray ionization, and electrostatic-ionization driven by dielectric polarization, have been reported in which the sample solution was not in direct contact with the metal electrode but separated by dielectric materials. The objective of the current work is to elucidate the mechanism of dielectric barrier electrospray. A sealed borosilicate glass capillary inserted with a fine acupuncture needle was used as a probe. A sample solution (~ 400 nL) was captured on the glass capillary tip and a positive high voltage (HV) pulse (+?4.5 kV) was applied to the internal metal electrode. Mass spectra were measured as a function of the HV pulse width from μs to 10 s. Ions started to be detected with the pulse width of ~ 5 ms. The ion intensities increased slowly with time and reached a plateau in a few seconds. The charge distribution of cytochrome c [M?+?nH]ⁿ⁺ shifted to higher n values from a few ms to seconds. In addition to cone-jet mode normal electrospray that lasted until all the liquid sample was depleted from the glass tip, the polarization-induced electrospray ionization was observed at the early stage of the HV application.

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Direct Analysis of Triterpenes from High-Salt Fermented Cucumbers Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

High-salt samples present a challenge to mass spectrometry (MS) analysis, particularly when electrospray ionization (ESI) is used, requiring extensive sample preparation steps such as desalting, extraction, and purification. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used to directly analyze 50-μm thick slices of cucumber fermented and stored in 1 M sodium chloride brine. From the several hundred unique substances observed, three triterpenoid lipids produced by cucumbers, β-sitosterol, stigmasterol, and lupeol, were putatively identified based on exact mass and selected for structural analysis. The spatial distribution of the lipids were imaged, and the putative assignments were confirmed by tandem mass spectrometry performed directly on the same cucumber, demonstrating the capacity of the technique to deliver confident identifications from highly complex samples in molar concentrations of salt without the need for sample preparation.

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Fischer Indole Synthesis in the Gas Phase,the Solution Phase,and at the Electrospray Droplet Interface

Previous reports have shown that reactions occurring in the microdroplets formed during electrospray ionization can, under the right conditions, exhibit significantly greater rates than the corresponding bulk solution-phase reactions. The observed acceleration under electrospray ionization could result from a solution-phase, a gas-phase, or an interfacial reaction. This study shows that a gas-phase ion/molecule (or ion/ion) reaction is not responsible for the observed rate enhancement in the particular case of the Fischer indole synthesis. The results show that the accelerated reaction proceeds in the microdroplets, and evidence is provided that an interfacial process is involved.

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Rapid Characterization of Microorganisms by Mass Spectrometry—What Can Be Learned and How?

Strategies for the rapid and reliable analysis of microorganisms have been sought to meet national needs in defense, homeland security, space exploration, food and water safety, and clinical diagnosis. Mass spectrometry has long been a candidate technique because it is extremely rapid and can provide highly specific information. It has excellent sensitivity. Molecular and fragment ion masses provide detailed fingerprints, which can also be interpreted. Mass spectrometry is also a broad band method—everything has a mass—and it is automatable. Mass spectrometry is a physiochemical method that is orthogonal and complementary to biochemical and morphological methods used to characterize microorganisms.      

Unexpected Reduction of Iminoquinone and Quinone Derivatives in Positive Electrospray Ionization Mass Spectrometry and Possible Mechanism Exploration

Unexpected reduction of iminoquinone (IQ) and quinone derivatives was first reported during positive electrospray ionization mass spectrometry. Upon increasing spray voltage, the intensities of IQ and quinone derivatives decreased drastically, accompanying the increase of the intensities of the reduction products, amodiaquine (AQ) and phenol derivatives. To gain more insight into the mechanism of such reduction, we explored the experimental factors that are influential to corona discharge (CD). The results show that experimental parameters that favor severe CD, including metal spray emitter, using water as spray solvent, sheath gas with low dielectric strength (e.g., nitrogen), and shorter spray tip-to-mass spectrometer inlet distance, facilitated the reduction of IQ and quinone derivatives, implying that the reduction should be closely related to CD in the gas phase.

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Decarboxylative Coupling Reaction in ESI(−)-MS/MS of 4-Nitrobenzyl 4-Hydroxybenzoates: Triplet Ion–Neutral Complex-Mediated 4-Nitrobenzyl Transfer

In negative electrospray ionization mass spectrometry of 4-nitrobenzyl 4-hydroxybenzoates, a decarboxylation reaction, which was significantly promoted by the presence of a nitro group on the benzyl group, competed with radical elimination reactions. Density functional theory calculations indicated that decarboxylation of deprotonated 4-nitrobenzyl vanillate occurred via a radical route involving homolytic cleavage of the C_benzyl–O bond to give a triplet ion–neutral complex, followed by decarboxylative coupling.

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Action-FRET of a Gaseous Protein

Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question—and thus further develop mass spectrometry as a tool for structural biology—structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state—where a systematic decrease is observed—and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization.

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Discrimination of Isobaric Leu/Ile Residues by MALDI In-Source Decay Mass Spectrometry

MALDI in-source decay (ISD) has been used for top-down sequencing of proteins. The use of the matrix 1,5-diaminonapthalene (1,5-DAN) gave abundant w ions, which are formed from the unimolecular dissociation of z? radical fragments via α cleavage reaction and thus help identify which of the isobaric amino acids, Leu or Ile, is present. The high abundance of w ions in MALDI-ISD with 1,5-DAN results from the low collision rate in the MALDI plume. MALDI-ISD with 1,5-DAN appears to be an useful method for the top-down sequencing of proteins, including discrimination of Leu and Ile near the C-terminal end.