MS Inlet Research Articles

Detection of radiation-exposure biomarkers by differential mobility prefiltered mass spectrometry (DMS–MS)

Technology to enable rapid screening for radiation exposure has been identified as an important need, and, as a part of a NIH/NIAD effort in this direction, metabolomic biomarkers for radiation exposure have been identified in a recent series of papers. To reduce the time necessary to detect and measure these biomarkers, differential mobility spectrometry–mass spectrometry (DMS–MS) systems have been developed and tested. Differential mobility ion filters preselect specific ions and also suppress chemical noise created in typical atmospheric-pressure ionization sources (ESI, MALDI, and others). Differential-mobility-based ion selection is based on the field dependence of ion mobility, which, in turn, depends on ion characteristics that include conformation, charge distribution, molecular polarizability, and other properties, and on the transport gas composition which can be modified to enhance resolution. DMS–MS is able to resolve small-molecule biomarkers from isobaric interferences, and suppresses chemical noise generated in the ion source and in the mass spectrometer, improving selectivity and quantitative accuracy. Our planar DMS design is rapid, operating in a few milliseconds, and analyzes ions before fragmentation. Depending on MS inlet conditions, DMS-selected ions can be dissociated in the MS inlet expansion, before mass analysis, providing a capability similar to MS/MS with simpler instrumentation. This report presents selected DMS–MS experimental results, including resolution of complex test mixtures of isobaric compounds, separation of charge states, separation of isobaric biomarkers (citrate and isocitrate), and separation of nearly isobaric biomarker anions in direct analysis of a bio-fluid sample from the radiation-treated group of a mouse-model study. These uses of DMS combined with moderate resolution MS instrumentation indicate the feasibility of field-deployable instrumentation for biomarker evaluation.

Imaging of Surface Charge and the Mechanism of Desorption Electrospray Ionization Mass Spectrometry

Distributions of charge deposited on surfaces in desorption electrospray ionization mass spectrometry (DESI-MS) were investigated using a static charge measurement apparatus, which gives an output voltage proportional to the local surface charge density. By scanning the probe along the surface and taking measurements at fixed intervals, a contour image of relative charge density reflecting the charge distribution on the surface can be plotted. Through the measured charge distribution and the derived charge density gradient, the motion of charged droplets in the DESI experiment can be inferred. Measurements taken under various DESI operating conditions, including spray pressure, angle, flow rate, and sprayer tip-to-surface distance, show that charge is spread over an area of a few square centimeters under typical conditions; effective desorption occurs from a much smaller area (∼1 mm2) of highest charge density. Higher sheath gas pressures and smaller sprayer tip-to-surface distances lead to concentration of charge distribution into a smaller area, whereas smaller spray angles favor charge distribution over a larger area. The appearance of the highest charge density in front of the DESI sprayer tip and near the MS inlet suggests that charged droplets are moved toward the MS inlet by pneumatic forces and by the vacuum suction, in agreement with results of earlier simulations. The present observations are consistent with previous studies using other techniques and support the accepted droplet splashing DESI mechanism.

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e −, 1H + processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na + and K + adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.

Mechanistic Aspects of Soot Formation from the Combustion of Pine Wood

Carbonaceous soot has been generated from pine in a range of appliances to simulate different combustion conditions. The fuel as well as biomass cell wall components have been studied by pyrolysis-GC−MS and pyrolysis-GC−TCD. In addition, the soots have been probed using both pyrolysis-GC−MS and direct inlet mass spectrometry (DI−MS). The material collected from the pine combustion is smoke, and the major component is a carbonaceous soot. The soots contain both organic carbon (adsorbed species) and black (solid) soot, and the organic carbon consists of primary pyrolysis products from the cell wall components, as well as decomposition products, PAH and oxidized PAH. The black carbon contains oxygen functionality (of the order of 5−10 wt % O), and there are indications that this is incorporated during soot growth, although surface oxidation on reactive sites could also be important. The decomposition products suggest an important additional PAH route is via cyclopentadiene, which is derived after cracking of lignin monomer fragments. Kinetic modeling also highlights the lignin monomers as important contributions to the soot production pathways. A model is proposed which, in addition to the hydrogen abstraction carbon addition (HACA) mechanism, incorporates the cyclopentadiene and the O-PAH addition routes to soot.

Development of a Sonic Spray Ionization Source for the Mass Spectrometric Analysis of Proteins

Sonic spray ionization source (SSI), developed in our lab, was coupled to a commercially available linear ion trap mass spectrometer for the analysis of proteins. Mass spectra of proteins such as ribonuclease A (RNase A) and lysozyme were obtained with substantially abundant peaks of low charge states. The experimental data show that SSI source is apt to form multiply charged ions of proteins, but with charge states lower than those obtained with a conventional ESI source. The effects of the operation parameters of the SSI source, including nebulizing gas pressure, desolvation temperature, sample infusion rate, spray solution constitution, etc., on the charge state distribution were systematically investigated. The signal of RNase A was optimized to the best level under the following operation conditions: the nebulizing gas pressure was 3.4–3.6 MPa, the distance between the spray tip and the MS inlet was 4–6 mm, the desolvation temperature was 250–300 °C, and the sample infusion rate (2%–5% formic acid solution) was 50 μl min –1 . The ions of low charge state were favored by elevated desolvation temperature and high nebulizing gas pressure or high content of volatile constituents (e.g. methanol) in the spray solution. A mechanism that the desolvation process contributes most to the formation of the low charge states was proposed.

Internal Energy Distributions in Desorption Electrospray Ionization (DESI)

The internal energy distributions of typical ions generated by desorption electrospray ionization (DESI) were measured using the "survival yield" method, and compared with corresponding data for electrospray ionization (ESI) and electrosonic spray ionization (ESSI). The results show that the three ionization methods produce populations of ions having internal energy distributions of similar shapes and mean values (1.7-1.9 eV) suggesting similar phenomena, at least in the later stages of the process leading from solvated droplets to gas-phase ions. These data on energetics are consistent with the view that DESI involves "droplet pick-up" (liquid-liquid extraction) followed by ESI-like desolvation and gas-phase ion formation. The effects of various experimental parameters on the degree of fragmentation of p-methoxy-benzylpyridinium ions were compared between DESI and ESSI. The results show similar trends in the survival yields as a function of the nebulizing gas pressure, solvent flow rate, and distance from the sprayer tip to the MS inlet. These observations are consistent with the mechanism noted above and they also enable the user to exercise control over the energetics of the DESI ionization process, through manipulation of external and internal ion source parameters.

Fully Automated Four-Column Capillary LC−MS System for Maximizing Throughput in Proteomic Analyses

We describe a four-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput liquid chromatography-mass spectrometry (LC-MS(/MS)) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of postcolumn switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.

Probing the Mechanisms of an Air Amplifier Using a LTQ-FT-ICR-MS and Fluorescence Spectroscopy

We report the first quantitative assessment of electrosprayed droplet/ion focusing enabled by the use of a voltage-assisted air amplifier between an electrospray ionization emitter and a hybrid linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (ESI-LTQ-FT-ICR-MS). A solution of fluorescent dye was electrosprayed with a stainless steel mesh screen placed in front of the MS inlet capillary acting as a gas-permeable imaging plate for fluorescence spectroscopy. Without use of the air amplifier, no detectable FT-ICR signal was observed, as well as no detectable fluorescence on the screen upon imaging using a fluorescence scanner. When the air amplifier was turned ON while electrospraying the fluorescent dye, FT-ICR mass spectra with high signal to noise ratio were obtained with an average ion injection time of 21 ms for an AGC target value of 5 x 10(5). Imaging of the screen using a fluorescence scanner produced a distinct spot of cross-sectional area approximately 33.5 mm(2) in front of the MS inlet capillary. These experimental results provide direct evidence of aerodynamic focusing of electrosprayed droplets/ions enabled by an air amplifier, resulting in improved electrospray droplet/ion capture efficiency and reduced ion injection time. A second set of experiments was carried out to explore whether the air amplifier assists in desolvation. By electrospraying a mix of quaternary amines, ratios of increasingly hydrophobic molecules were obtained. Observation of the solvophobic effect associated with electrospray ionization resulted in a higher abundance of the hydrophobic molecule. This bias was eliminated when the air amplifier was turned ON and a response indicative of the respective component concentrations of the molecules in the bulk solution was observed.

Future directions for electrospray ionization for biological analysis using mass spectrometry

Future directions for electrospray ionization for biological analysis using mass spectrometry

Control of electrochemical reactions at the capillary electrophoresis outlet/electrospray emitter electrode under CE/ESI-MS through the application of redox buffers.

It was found that combining capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS) overlays two controlled current techniques to form a three-electrode system (CE inlet, CE outlet/ES emitter, and MS inlet electrodes) in which the CE outlet electrode and the ES emitter electrode were shared between the CE and the ESI-MS circuits. Depending on the polarities and magnitudes of the voltages at the CE inlet, CE outlet/ES emitter, and MS inlet electrodes, the nature of the two redox reactions at the shared electrode was the same or different (both reduction, both oxidation, or one oxidation and the other reduction). Several redox buffers were introduced for controlling electrochemical reactions at the shared electrode. By reacting at this electrode, redox buffers were able to maintain electrode potentials below the onset of water electrolysis, thereby eliminating gas bubble formation and/or pH drift. The volume of the gas generated due to water electrolysis was used to quantitate water oxidation or reduction at this electrode. Two types of redox buffers were used. A reactive electrode with an oxidation potential below that of water was used as the electrode under anodic conditions. Also, a reactive compound with a redox potential below that of water was added to the CE and/or ESI running buffer. When the shared electrode was the anode of both CE and ESI-MS circuits, the use of iron or etched and sanded stainless steel (ss) wire, instead of platinum wire, suppressed bubble formation at the shared electrode. Under these conditions, corrosion of the Fe wire and formation of Fe2+ replaced oxidation of water, eliminating O2 gas bubble and H+ formation. When mixtures of peptides were analyzed, iron adducts of peptides were observed. For a fresh wire, however, the intensities of adduct ions were less than 3% of the protonated molecules. After a few days of operation, the intensities of the adduct ions increased to approximately 50%, due to rust formation on the Fe wire. On-column rinsing with a 40% solution of citric acid rejuvenated the Fe wire and reduced the adduct peak intensities to less than 3%. Unmodified ss wire did not quench bubble formation, which was attributed to its passivated surface. When Fe, ss, and Pt wires were used as the shared electrode under forward polarity CE and positive ESI mode, where the shared electrode acted as a cathode with respect to CE inlet and as an anode with respect to MS inlet, reduction of water at the cathodic end of the electrode and, in the case of ss and Pt wires, oxidation of water at the anodic end of the shared electrode produced a significant amount of bubbles. Under these conditions, however, a buffer containing 50 mM p-benzoquinone completely suppressed both cathodic reduction and anodic oxidation of water for CE currents up to 4 microA. Reduction of p-benzoquinone at the cathodic end of the shared electrode to hydroquinone, and oxidation of this hydroquinone at the anodic end of the electrode, replaced reduction and oxidation of water, eliminating bubble formation. A 0.1% acetic acid solution saturated with I2 was also found to suppress bubble formation at the cathode for CE currents up to 3 microA; however, strong iodine adduct ions were observed under CE/ESI-MS when a mixture of peptides was analyzed. The application of iron as an in-capillary electrode for the analysis of a peptide mixture and a protein digest demonstrated a high separation efficiency similar to when hydroquinone was used as a redox buffer.

Experimental characterization of a short retention time gas-solid separator

An inertial gas-solid separator has been specifically designed to collect the recycling products from solids in CFB reactors operating at very high gas velocities. The main feature of this device is an integrated layout of the riser separator set that allows gas retention times as short as 3 ms and maximum inlet solid/gas mass ratios up to 30. The separation efficiency and the pressure drop for different particle sizes, gas velocities and solid mass flow rates has been determined and their dependence on solid loading and design features has been discussed.