Rapid resolution of carbohydrate isomers by electrospray ionization ambient pressure ion mobility spectrometry-time-of-flight mass spectrometry (ESI-APIMS-TOFMS)

Carbohydrates are an extremely complex group of isomeric molecules that have been difficult to analyze in the gas phase by mass spectrometry because (1) precursor ions and product ions to successive stages of MS(n) are frequently mixtures of isomers, and (2) detailed information about the anomeric configuration and location of specific stereochemical variants of monosaccharides within larger molecules has not been possible to obtain in a general way. Herein, it is demonstrated that gas-phase analyses by direct combination of electrospray ionization, ambient pressure ion mobility spectrometry, and time-of-flight mass spectrometry (ESI-APIMS-TOFMS) provides sufficient resolution to separate different anomeric methyl glycosides and to separate different stereoisomeric methyl glycosides having the same anomeric configuration. Reducing sugars were typically resolved into more than one peak, which might represent separation of cyclic species having different anomeric configurations and/or ring forms. The extent of separation, both with methyl glycosides and reducing sugars, was significantly affected by the nature of the drift gas and by the nature of an adducting metal ion or ion complex. The study demonstrated that ESI-APIMS-TOFMS is a rapid and effective analytical technique for the separation of isomeric methyl glycosides and simple sugars, and can be used to differentiate glycosides having different anomeric configurations.     

A novel surface ionization source for ion mobility spectrometer

A surface ionization (SI) source is designed and prepared for ion mobility spectrometer (IMS). The source acts not only as an emitter but also an ion injector which can inject ions periodically into the drift region of drift tube. Using the dual-role source, the dimension of the drift tube can be decreased and the circuit for high voltage can be simplified efficiently. The IMS with the SI source has a response range of ∼4 orders of magnitude and a good reproducibility to tri-ethylamine. Compared with radioactive ionization (RI), the ultra-short time for ion injection and the zero level base line of ion mobility spectrum are characteristics of the surface ionization.     

Monte Carlo simulation of ion transport in non-linear ion mobility spectrometry

A program for Monte Carlo simulation of ion transport in non-linear ion mobility spectrometry, also known as field asymmetric ion mobility spectrometry (FAIMS) or differential mobility spectrometry (DMS), has been developed. Simulations are based on elastic collisions between the ions and the gas particles, and take into account the effects of flow dynamics and asymmetric electric fields. Using this program, the separation and diffusion of the ions moving in a planar DMS filtration gap are demonstrated. Ion focusing in a cylindrical filtration gap is also confirmed. A characteristic compensation voltage is found to provide insight for understanding separation in non-linear ion mobility spectrometry. The simulation program is used to study the characteristics of non-linear ion mobility spectrometry, the effect of the carrier gas flow, and the dependence of the compensation voltage and nonlinear mobility coefficient (α) on the applied asymmetric electric field.     

An alternative field switching ion gate for ESI-ion mobility spectrometry

In electrospray ionization (ESI)-ion mobility spectrometry, continuously generated ions must be desolvated in a first tube before short ion pulses are introduced into a second (drift) tube. Both tubes are separated by an ion-gate. The resolving power of the resulting drift time spectrum is strongly influenced by the design of the ion gate. In the case of the Bradbury-Nielsen gates typically used, an orthogonal field between oppositely charged, parallel wires blocks ions from entering the drift tube. However, the blocking field also distorts the entering ion cloud. One alternative, which eliminates these effects and therefore enables a potentially higher resolving power, is already known for spectrometers with small ionization volumes, where ions are formed between two electrodes and subsequently transferred into the drift tube by a high voltage pulse. Based on this setup, we introduce an alternative ion gate design for liquid samples, named field switching ion gate (FSIG). The continuous flow of ions generated by ESI is desolvated in the first tube and introduced into the space between two electrodes (repeller and transfer electrodes). A third (blocking) electrode prevents the movement of ions into the drift tube in the closed state. Ions are transferred during the open state by pulsing the voltages of the repeller and blocking electrodes. First results demonstrate an increase of the resolving power by 100% without intensity losses and further changes in the spectrometer setup. The parameters of the FSIG, such as electrode voltages and pulse width, are characterized allowing the optimization of the spectrometer’s resolving power.     

Applying a dynamic method to the measurement of ion mobility

A dynamic method is applied to measure the mobility of gas-phase ions in the dual ion funnel interface of the electrospray source of a quadrupole orthogonal time-of-flight mass spectrometer. In a new operational mode, a potential barrier was formed in the second ion funnel of the mass spectrometer and then progressively increased. In this region, a flow of gas drags the ions into the mass spectrometer while the electric force applied by the potential barrier decelerates them. Ions with lower mobility can be carried by the gas flow more easily than those with high mobility. Thus, electrical forces can block the more mobile ions more easily. Hence, the electric barrier formed in the ion funnel permits only ions below a certain mobility threshold to enter the mass spectrometer. When the barrier voltage is increased, this threshold moves from high to low mobilities. Ions with mobilities above the threshold cannot enter the mass spectrometer, and their signal decreases to zero. Thus, in a barrier voltage scan, mass spectrometric signals of ions sequentially disappear. Differentiation of these decreasing ion signal curves produces peaks from which an ion mobility spectrum can be reconstructed. Blocking voltages, i.e., the positions of the peaks on the barrier voltage scale are directly related to the mobility of these ions. An internal calibration using ions with known mobility values helps determine the unknown ion mobilities and allows calculation of ionic cross sections.     

A novel non-radioactive electron source for ion mobility spectrometry

Typical ion mobility spectrometers work by employing a radioactive source to provide electrons with high energy to ionize the analytes in a series of chemical reactions. General security as well as regulatory concerns related to the use of radioactivity resulted in a need for a different ionization source which on the other hand produces ions in a similar manner as a radioactive source because the mechanisms are well known. Here we introduce a novel non-radioactive electron source which is capable of providing high energy electrons in a way that is similar to beta radiating substances yielding correspondingly similar peak spectra.     

Novel ion mobility setup combined with collision cell and time-of-flight mass spectrometer

An ion mobility cell of a novel type was coupled to an orthogonal injection time-of-flight (TOF) mass spectrometer. The mobility cell operates at low-pressure and contains a segmented RF ion guide providing an axial electric field that drives the ions towards the exit. A flow of gas is arranged inside the ion guide in such a way that the gas drag counteracts the force exerted by the axial field. Ions with different mobility coefficients can be scanned out of the ion guide by ramping the axial field strength. The ions can be analyzed intact or fragmented in a collision cell before introduction into an orthogonal TOF mass spectrometer. An ion source with matrix assisted laser desorption/ionization (MALDI) was attached to the instrument. The setup was evaluated for the analysis of peptide and protein mixture, with sequential fragmentation of multiple precursor ions from a protein digest and with mobility separation of fragment ions formed by in-source fragmentation of pure peptides. The mobility resolution for peptides was observed to be three times higher than the theoretical resolution predicted for a classical mobility setup with similar operating conditions (pressure, field strength, and length).     

Optimum collision energies for proteomics: The impact of ion mobility separation

Ion mobility spectrometry (IMS) is a widespread separation technique used in various research fields. It can be coupled to liquid chromatography–mass spectrometry (LC–MS/MS) methods providing an additional separation dimension. During IMS, ions are subjected to multiple collisions with buffer gas, which may cause significant ion heating. The present project addresses this phenomenon from the bottom-up proteomics point of view. We performed LC–MS/MS measurements on a cyclic ion mobility mass spectrometer with varied collision energy (CE) settings both with and without IMS. We investigated the CE dependence of identification score, using Byonic search engine, for more than 1000 tryptic peptides from HeLa digest standard. We determined the optimal CE values—giving the highest identification score—for both setups (i.e., with and without IMS). Results show that lower CE is advantageous when IMS separation is applied, by 6.3 V on average. This value belongs to the one-cycle separation configuration, and multiple cycles may supposedly have even larger impact. The effect of IMS is also reflected in the trends of optimal CE values versus m/z functions. The parameters suggested by the manufacturer were found to be almost optimal for the setup without IMS; on the other hand, they are obviously too high with IMS. Practical consideration on setting up a mass spectrometric platform hyphenated to IMS is also presented. Furthermore, the two CID (collision induced dissociation) fragmentation cells of the instrument—located before and after the IMS cell—were also compared, and we found that CE adjustment is needed when the trap cell is used for activation instead of the transfer cell. Data have been deposited in the MassIVE repository (MSV000090944).     

Comparative measurements of toxic industrial compounds with a differential mobility spectrometer and a time of flight ion mobility spectrometer

Small concentrations of toxic compounds in atmospheric air have often to be measured selectively by portable equipment. Ion mobility spectrometers are instruments used to monitor explosives, drugs and chemical warfare agents. First responders also need to detect hazardous gases released in accidents while transporting them or in their production in chemical plants. Not all toxic gases can be measured with the time of flight ion mobility spectrometer at concentrations required by safety standards applied in workplace areas. The time of flight ion mobility spectrometer is based on an inlet membrane, an ionization region, a shutter grid and the drift region with a detector in the drift tube. The separation of ions is due to the different mobility of the ions when they are exposed to a weak electric field (E = 200…300 V/cm). High field asymmetric waveform spectrometry or differential mobility spectrometry is a relative new ion mobility spectrometer technology. The separation is due to the different mobilities of the ions in the high (E = 15000...30000 V/cm) and the weak electric fields. About 30 different toxic industrial chemical compounds were analyzed with both systems under comparable conditions. For selected examples the detection limits, the selectivity and the identification capabilities of the two systems for some of the main compounds will be discussed.     

Selective ion suppression as a pre-separation method in ion mobility spectrometry using a pulsed electron gun

Ion mobility spectrometry is a well-known method for fast trace gas detection. Detection limits in the very low ppb- and even ppt-range, fast response times down to a second and good separation power combined with a reasonable instrumental effort make ion mobility spectrometry more and more attractive. Aiming for higher separation power we investigate the ion specific lifetime of different ion species in a field free reaction region of a drift tube ion mobility spectrometer equipped with a pulsed non-radioactive electron gun. When turning off the electron gun ionization stops and the total ion concentration in the reaction region starts to decrease, while different ion species have different decay times. By varying the time delay between the end of the ionization and the injection pulse transferring all remaining ions of one polarity from the reaction region into the drift region the individual decay times can be measured. Our experimental data show that the lifetime of ion species in a field free reaction region mainly depends on ion-ion-recombination and charge transfer reactions leading to significant lifetime differences. Therefore, short-lived ions can be effectively suppressed in the reaction region by introducing a sufficient time delay between the end of the ionization and the injection pulse. This allows detecting even smallest concentrations of long-lived ions in a complex short-lived background. From our experimental data it can be also concluded that wall losses and the ion transport within the sample gas stream out of the reaction region just play a minor role in the ion loss.     

Bradbury-Nielsen vs. Field switching shutters for high resolution drift tube ion mobility spectrometers

A key component in the design of every drift tube ion mobility spectrometer (IMS) is the ion shutter which controls the injection of ions into the drift tube. Especially, compact drift tube IMS require very short injection pulses to achieve high resolution and therefore require fast ion shutters. Thus, it is important to find an ion shutter principle that can be readily scaled towards these short injection widths without causing major non-idealities in the injection process, such as drift field inhomogeneities, ion loss and ion discrimination by mobility. In this paper, we compare different ion shutter principles, foremost the Bradbury-Nielsen gate and a field switching design. It is shown through theoretical considerations and field simulations that the Bradbury-Nielsen shutter is more universally applicable and typically less complex for long injections widths but field inhomogeneities associated with its operating principle impede the scaling process. Thus, the currently less used field switching shutters will become the superior principle when very short injection widths are required, as this shutter principle allows for single digit microsecond widths.     

Traveling-wave ion mobility mass spectrometry analysis of isomeric modified peptides arising from chemical cross-linking

Traveling-wave ion mobility (TWIM) coupled to mass spectrometry (MS) has emerged as a powerful tool for structural and conformational analysis of proteins and peptides, allowing the analysis of isomeric peptides (or proteins) with the same sequence but modified at different residues. This work demonstrates the use of the novel TWIM-MS technique to separate isomeric peptide ions derived from chemical cross-linking experiments, which enables the acquisition of distinct product ion spectra for each isomer, clearly indicating modification on different sites. Experiments were performed with four synthetic peptides, for which variable degrees of mobility separation were achieved. In cases of partially overlapping mobility arrival time distributions (ATDs), extracting the ATDs of fragment ions belonging to each individual isomer allowed their separation into two distinct ATDs. Accumulation over regions from the specific ATDs generates the product ion spectrum of each isomer, or a spectrum highly enriched in their fragments. The population of both modified peptide isomers was correlated with the intrinsic reactivities of different Lys residues from reactions conducted at different pH conditions.     

Temperature effects on resolution in ion mobility spectrometry

The separation efficiency of ion mobility spectrometry (IMS) may be measured in terms of either resolving power, based on a single-peak definition, or peak-to-peak resolution, based on the separation of pairs of adjacent peaks. Usually resolving power decreases with temperature. However, the experimental results show that the peak-to-peak resolution may be increased in some cases. Negative ion mobility spectra of halide ions are better resolved at elevated temperatures. In addition, the peaks corresponding to protonated monomer of amylacetate and the proton-bound dimer of ethylacetate are well separated at 100 °C while they fully overlap at 18 °C. This paper focuses on the effect of temperature on peak-to-peak resolution. It was also observed that in some cases peak-to-peak resolution decreases with temperature. Examples are the spectra of cyclohexanone and methyl-iso-butyl ketone (MIBK) as well as dimethyl methyl phosphonate (DMMP) and MIBK. The increase or decrease in resolution at elevated temperatures has been attributed to the changes in separation factor (α) which is governed by the different hydration and clustering tendency of ions.     

Cross sections and ion kinetic energy analysis for the electron impact ionization of acetylene

Using a Nier-type electron impact ion source in combination with a double focusing two sector field mass spectrometer, partial cross sections for electron impact ionization of acetylene are measured for electron energies up to 1000 eV. Discrimination factors for ions are determined using the deflection field method in combination with a three-dimensional ion trajectory simulation of ions produced in the ion source. Analysis of the ion yield curves obtained by scanning the deflectors allows the assignment of ions with the same mass-to-charge ratio to specific production channels on the basis of their different kinetic energy distributions. This analysis also allows to determine, besides kinetic energy distributions of fragment ions, partial cross sections differential in kinetic energy. Moreover a charge separation reaction, the Coulomb explosion of the doubly charged parent ions C2H2++ into the fragment ions C2H+ and H+, is investigated and its mean kinetic energy release (KER=3.88 eV) is deduced.     

Optimum waveforms for differential ion mobility spectrometry (FAIMS)

Differential mobility spectrometry or field asymmetric waveform ion mobility spectrometry (FAIMS) is a new tool for separation and identification of gas-phase ions, particularly in conjunction with mass spectrometry. In FAIMS, ions are filtered by the difference between mobilities in gases (K) at high and low electric field intensity (E) using asymmetric waveforms. An infinite number of possible waveform profiles make maximizing the performance within engineering constraints a major issue for FAIMS technology refinement. Earlier optimizations assumed the non-constant component of mobility to scale as E(2), producing the same result for all ions. Here we show that the optimum profiles are defined by the full series expansion of K(E) that includes terms beyond the first that is proportional to E(2). For many ion/gas pairs, the first two terms have different signs, and the optimum profiles at sufficiently high E in FAIMS may differ substantially from those previously reported, improving the resolving power by up to 2.2 times. This situation arises for some ions in all FAIMS systems, but becomes more common in recent miniaturized devices that employ higher E. With realistic K(E) dependences, the maximum waveform amplitude is not necessarily optimum, and reducing it by up to approximately 20% to 30% is beneficial in some cases. The present findings are particularly relevant to targeted analyses where separation depends on the difference between K(E) functions for specific ions.     

Determination of gas phase protein ion densities via ion mobility analysis with charge reduction

We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1?:?49.5?:?49.5, formic acid?:?methanol?:?water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase measurements to be correlated to biomolecular structures in solution, low charge state ions should be analyzed. Further, to determine if different solution conditions give rise to ions of different structure, ions of similar charge state should be compared. Non-denatured protein ion densities are found to be in excellent agreement with non-denatured protein ion densities inferred from prior DMA and drift tube measurements made without charge reduction (all ions with densities in the 0.85-1.10 g cm(-3) range), showing that these ions are not strongly influenced by Coulombic stretching nor by analysis method.     

An open source ion gate pulser for ion mobility spectrometry

Excluding the ion source, an ion mobility spectrometer is fundamentally comprised of drift chamber, ion gate, pulsing electronics, and a mechanism for amplifying and recording ion signals. Historically, the solutions to each of these challenges have been custom and rarely replicated exactly. For the IMS research community few detailed resources exist that explicitly detail the construction and operation of ion mobility systems. In an effort to address this knowledge gap we outline a solution to one of the key aspects of a drift tube ion mobility system, the ion gate pulser. Bradbury-Nielsen or Tyndall ion gates are found in nearly every research-grade and commercial IMS system. While conceptually simple, these gate structures often require custom, high-voltage, floating electronics. In this report we detail the operation and performance characteristics of a wifi-enabled, MOSFET-based pulser design that uses a lithium-polymer battery and does not require high voltage isolation transformers. Currently, each output of this circuit follows a TTL signal with ~20 ns rise and fall times, pulses up to +/? 200 V, and is entirely isolated using fiber optics. Detailed schematics and source code are provided to enable continued use of robust pulsing electronics that ease experimental efforts for future comparison.     

Ion characterisation by comparison of ion mobility spectrometry and mass spectrometry data

The major uncertainty related to ion mobility spectrometry is the lack of knowledge about the characteristics of the ions detected. When using a radioactive atmospheric pressure ionisation source (e.g. ⁶³Ni), from theory proton bound water clusters are expected as reactant ions. When analyte ions occur, proton transfer should lead to proton-bound monomer and dimer ions. To increase the knowledge about those ionisation processes in an ion mobility spectrometer (IMS), a ß-radiation ionisation source was coupled to a mass spectrometer (MS) and an identical one to an IMS. Exemplarily, acetone, limonene and 2- and 5-nonanone were introduced into both instruments in varying concentrations. By correlating the MS and IMS spectra, conclusions about the identities of the ions detected by IMS could be drawn. Proton-bound monomer, dimer and even trimer ions (MH⁺, 2MH⁺, 3MH⁺) could be observed in the MS spectra for acetone and 5-nonanone and could be assigned to the related signals detected by IMS. The oligomers could be expected from theory for increasing concentration. Limonene and 2-nonanone yielded in a variety of different ions and fragments indicating complex gas phase ion chemistry. Those findings on the obviously different behaviour of different analytes require further research focussed on the ion chemistry in IMS including the comparison of different ionisation sources.     

Liquid phase ion mobility spectrometry

A novel analytical method, called Liquid Phase Ion Mobility Spectrometry (LiPIMS) was demonstrated, where aqueous phase analytes were ionized and introduced into non-aqueous liquids, transported by an external electric field from the point of generation to a collection electrode. Ions were produced from a unique liquid phase ionization process, called Electrodispersion Ionization. Spectra of analyte ions illustrated the potential of LiPIMS as a new separation technique. Experimental data showed that electrodispersion ionization was effective in generating nanoampere level of ion current in hexane and benzene from aqueous samples. By controlling the ionization voltage in relation to the sample flow rate, it was possible to operate the electrodispersion ionization source in both continuous and pulsed ionization modes. Unique LiPIMS spectra of aqueous samples of tetramethylammonium bromide, tetrabutylammonium bromide and bradykinin were presented and their respected liquid phase ion mobility values were determined.     

Characterizing the resolution and accuracy of a second-generation traveling-wave ion mobility separator for biomolecular ions

High-accuracy, high-resolution ion mobility measurements enable a vast array of important contemporary applications in biological chemistry. With the recent advent of both new, widely available commercial instrumentation and also new calibration datasets tailored for the aforementioned commercial instrumentation, the possibilities for extending such high performance measurements to a diverse set of applications have never been greater. Here, we assess the performance characteristics of a second-generation traveling-wave ion mobility separator, focusing on those figures of merit that lead to making measurements of collision cross-section having both high precision and high accuracy. Through performing a comprehensive survey of instrument parameters and settings, we find instrument conditions for optimized drift time resolution, cross-section resolution, and cross-section accuracy for a range of peptide, protein and multi-protein complex ions. Moreover, the conditions for high accuracy IM results are significantly different from those optimized for separation resolution, indicating that a balance between these two metrics must be attained for traveling wave IM separations of biomolecules. We also assess the effect of ion heating during IM separation on instrument performance.