Separation of isomeric peptides using electrospray ionization/high-resolution ion mobility spectrometry

In this paper, the first examples of baseline separation of isomeric macromolecules by electrospray ionization/ion mobility spectrometry (ESI/IMS) at atmospheric pressure are presented. The behavior of a number of different isomeric peptides in the IMS was investigated using nitrogen as a drift gas. The IMS was coupled to a quadrupole mass spectrometer, which was used for identification and selective detection of the electrosprayed ions. The mobility data were used to determine their average collision cross sections. The gas-phase ions of isomeric peptides were found to have different collision cross sections. In all cases, doubly charged ions exhibited significantly (8-20%) larger collision cross sections than the respective singly charged species. The analysis of mixtures of the isomeric peptides clearly demonstrated the capability of IMS to separate gas-phase peptide ions due to small differences in their conformational structures, which cannot be determined by mass spectrometry. An actual resolving power of 80 was achieved for two doubly charged reversed sequenced pentapeptides. Baseline separation was provided for ions differing by only 2.5% in their measured collision cross sections; partial separation was shown for isomeric ions exhibiting differences as small as 1.1%.     

High-sensitivity ion mobility spectrometry/mass spectrometry using electrodynamic ion funnel interfaces

The utility of ion mobility spectrometry (IMS) for separation of mixtures and structural characterization of ions has been demonstrated extensively, including in biological and nanoscience contexts. A major attraction of IMS is its speed, several orders of magnitude greater than that of condensed-phase separations. Nonetheless, IMS combined with mass spectrometry (MS) has remained a niche technique, substantially because of limited sensitivity resulting from ion losses at the IMS-MS junction. We have developed a new electrospray ionization (ESI)-IMS-QTOF MS instrument that incorporates electrodynamic ion funnels at both front ESI-IMS and rear IMS-QTOF interfaces. The front funnel is of the novel "hourglass" design that efficiently accumulates ions and pulses them into the IMS drift tube. Even for drift tubes of 2-m length, ion transmission through IMS and on to QTOF is essentially lossless across the range of ion masses relevant to most applications. The rf ion focusing at the IMS terminus does not degrade IMS resolving power, which exceeds 100 (for singly charged ions) and is close to the theoretical limit. The overall sensitivity of the present ESI-IMS-MS system is comparable to that of commercial ESI-MS, which should make IMS-MS suitable for analyses of complex mixtures with ultrahigh sensitivity and exceptional throughput.     

Screening of solid commercial pharmaceuticals using ion mobility spectrometry

Ion mobility spectrometry (IMS) was used to analyze vapors generated directly above pharmaceutical solids warmed in air to 100-200 degrees C. Vapors were characterized by IMS/mass spectrometry to evaluate the air-based atmospheric pressure chemical ionization of components in analgesic medicines. A hand-held IMS was used to determine the suitability of membrane-based instrumentation for routine analyses of large polar molecules. Mobility spectra for seven compounds could be individually represented by single or a few intense product ion peaks. Ion source fragmentations were nonexistent with these pharmaceuticals but complex behavior in the IMS involving ion-molecule clustering was pronounced in certain spectra. Mobility spectra were distinct and recognizable for each compound and binary mixtures of individual ingredients and actual over-the-counter medicines produced mobility spectra suggestive of composite spectra. This work represents a first delineation of complex ion mobility spectra for mixtures using spectra from individual components. These findings support further development of IMS for use in quality control during manufacture of such preparations and in routine screening of powders containing analgesic pharmaceuticals.     

Characterizing electrospray ionization using atmospheric pressure ion mobility spectrometry

Reduced flow rate electrospray ionization has been proven to provide improved sensitivity, less background noise, and improved limits of detections for ESI-MS analysis. Miniaturizing the ESI source from conventional electrospray to microelectrospray and further down to nanoelectrospray has resulted in higher and higher sensitivity; however, when effects of flow rate were investigated for atmospheric pressure ESI-IMS using a nanospray emitter, a striking opposite result was observed. The general tendency we observed in ESI-IMS was that higher flow rate offered higher ion signal intensity throughout a variety of conditions investigated. Thus, further efforts were undertaken to rationalize these contradictory results. It is well accepted that decreased flow rate increases both ionization efficiency and transmission efficiency, thus improving ion signal in ESI-MS. However, our study revealed that decreased flow rate results in decreased ion signal because ion transfer is constant, no matter how flow rate changes in ESI-IMS. Since ion transfer is constant in atmospheric pressure ESI-IMS, ionization efficiency can be studied independently, which otherwise is not possible in ESI-MS in which both ionization efficiency and transmission efficiency vary as conditions alter. In this article, we present a systematic study of signal intensity and ionization efficiency at various experimental conditions using ESI-IMS and demonstrate the ionization efficiency as a function of flow rate, analyte concentration, and solvent composition.     

Ultrahigh-resolution differential ion mobility spectrometry using extended separation times

Ion mobility spectrometry (IMS), and particularly differential IMS or field asymmetric waveform IMS (FAIMS), is emerging as a versatile tool for separation and identification of gas-phase ions, especially in conjunction with mass spectrometry. For over two decades since its inception, the utility of FAIMS was constrained by resolving power (R) of less than ～20. Stronger electric fields and optimized gas mixtures have recently raised achievable R to ～200, but further progress with such approaches is impeded by electrical breakdown. However, the resolving power of planar FAIMS devices using any gas and field intensity scales as the square root of separation time (t). Here, we extended t from the previous maximum of 0.2 s up to 4-fold by reducing the carrier gas flow and increased the resolving power by up to 2-fold as predicted, to >300 for multiply charged peptides. The resulting resolution gain has enabled separation of previously "co-eluting" peptide isomers, including folding conformers and localization variants of modified peptides. More broadly, a peak capacity of ～200 has been reached in tryptic digest separations.     

Surface ionization ion mobility spectrometry

A surface ionization (SI) source was designed and constructed for ion mobility spectrometry (IMS). Compared with a conventional (63)Ni source, the surface ionization source is as simple and reliable, has an extended dynamic response range, is more selective in response, and does not have regulatory problems associated with radioactive ionization sources. The performance of this SI-IMS was evaluated with several different classes of compounds. Triethylamine was employed for studying the behavior of the ionization source under different source conditions and gaseous environments. Amines, tobacco alkaloids, and triazine herbicides were also investigated. Picogram level detection limits were achieved for target compounds with a response dynamic range of 5 orders of magnitude. Selective monitoring by IMS was also demonstrated. While the surface ionization source does not have the universality of response that is obtained with a (63)Ni ionization source, it is an excellent nonradioactive alternative for the ionization and ion mobility detection of those compounds to which it responds.     

Hadamard transform ion mobility spectrometry

Traditionally, the spectrum acquired using ion mobility spectrometry (IMS) is an average of multiple experimental cycles. Each cycle is initiated by passing a short burst of ions into a drift tube containing a homogeneous electric field. Prior to starting the subsequent cycle, all ions in the system must arrive at the detector or spectral overlap may occur. To maximize resolution, the ion pulse admitted to the drift tube is small in relation to the total scan time with the unfortunate consequence of an inherently low duty cycle (approximately 1%). Offering an improved SNR through a 50% duty cycle, the Hadamard transform (HT) applied to ion mobility spectrometry represents a fresh alternative to signal-averaged data acquisition. Initial results from measurements of amphetamine and cytochrome c samples indicate a 2-10-fold increase in SNR for the HT-IMS technique with no reduction in resolution.     

Hadamard transform ion mobility spectrometry

A detection scheme that makes use of the Hadamard transform has been employed with an atmospheric-pressure ion mobility spectrometer fitted with an electrospray ionization source. The Hadamard transform was implemented through the use of a linear-feedback shift register to produce a pseudorandom sequence of 1023 points. This pseudorandom sequence was applied to the ion gate of the spectrometer, and deconvolution of the ion signal was accomplished by the Hadamard transform to reconstruct the mobility spectrum. Ion mobility spectra were collected in both a conventional and Hadamard mode, with comparisons made between the two approaches. Initial results exhibited low spectral definition, so an oversampling technique was applied to increase the number of data points across each analyte spectral peak. The use of the Hadamard transform increases the duty cycle of the instrument to 50% and results in a roughly 5-fold enhancement of the signal-to-noise ratio with a negligible loss of instrument resolution. It is also shown that any potential multiplex disadvantage, which limits the attractiveness of some high-throughput techniques, is not a limiting factor in this new implementation.     

Voltage sweep ion mobility spectrometry

Ion mobility spectrometry (IMS) is a rapid, gas-phase separation technique that exhibits excellent separation of ions as a standalone instrument. However, IMS cannot achieve optimal separation power with both small and large ions simultaneously. Similar to the general elution problem in chromatography, fast ions are well resolved using a low electric field (50-150 V/cm), whereas slow drifting molecules are best separated using a higher electric field (250-500 V/cm). While using a low electric field, IMS systems tend to suffer from low ion transmission and low signal-to-noise ratios. Through the use a novel voltage algorithm, some of these effects can be alleviated. The electric field was swept from low to high while monitoring a specific drift time, and the resulting data were processed to create a 'voltage-sweep' spectrum. If an optimal drift time is calculated for each voltage and scanned simultaneously, a spectrum may be obtained with optimal separation throughout the mobility range. This increased the resolving power up to the theoretical maximum for every peak in the spectrum and extended the peak capacity of the IMS system, while maintaining accurate drift time measurements. These advantages may be extended to any IMS, requiring only a change in software.     

Protein analyses using differential ion mobility microchips with mass spectrometry

Differential ion mobility spectrometry (FAIMS) integrated with mass spectrometry (MS) is a powerful new tool for biological and environmental analyses. Large proteins occupy regions of FAIMS spectra distinct from peptides, lipids, or other medium-size biomolecules, likely because strong electric fields align huge dipoles common to macroions. Here we confirm this phenomenon in separations of proteins at extreme fields using FAIMS chips coupled to MS and demonstrate their use to detect even minor amounts of large proteins in complex matrixes of smaller proteins and peptides.     

Quantifying peptides in isotopically labeled protease digests by ion mobility/time-of-flight mass spectrometry

Ion mobility/time-of-flight techniques have been used to analyze mixtures of isotopically labeled peptides. The isotopic labels were generated by treatment of peptides with N-acetoxysuccinimide (or the deuterated analogue), which results in acetylation (or deuterioacetylation) of the primary amines (i.e., the N-terminus and lysine residues). The relative concentrations of a peptide in each sample are determined by comparing the peak intensities for isotopic pairs. An important consideration is that as mixtures become increasingly complex, isotopic pairs of peaks may overlap with other peaks in the mass spectrum. The influence of the acetyl and deuterioacetyl groups on the mobilities of peptides is considered. The coincidence in mobilities of isotopic pairs provides a means of distinguishing isotopic pairs from other isobaric interferences.     

Low-temperature plasma ionization ion mobility spectrometry

In this research work, the capability of low-temperature plasma (LTP) as an ionization source for ion mobility spectrometry (IMS) has been investigated for the first time. This new ionization source enhances the potential of IMS as a portable analytical tool and allows direct analysis of various chemical compounds without having to evaporate the analyte or seek a solvent or reagent whatsoever. The effects of parameters such as the flow rate of the discharge gas, plasma voltage, and positioning of the LTP on the IMS signal were investigated. The positive reactant ions generated by the LTP ionization source were similar to those created in a corona discharge ionization source, where the proton clusters ((H(2)O)(n)H(+)) are the most abundant reactant ion, and in the negative mode, in addition to a saturated electron peak, several negative reactant ions (e.g., NO(x)(-)) were observed too. These reactant ions subsequently ionized the gaseous samples directly and liquids or solids after evaporation by plasma desorption. The ion mobility spectra of a few selected compounds, including explosives, drugs, and amines, were obtained to evaluate the new ionization source in positive and negative modes, and the reduced mobility values (K(0)) of the originated ions were calculated. Furthermore, the method has also been applied to obtain the figures of merit for acetaminophen as a test compound. The results obtained are promising enough to ensure the use of LTP as a desorption/ionization source in IMS for analytical applications.     

Peak-peak repulsion in ion mobility spectrometry

The space charge effect has an important role in instruments dealing with ion packets and charged particles in gas phase such as the mass spectrometer and ion mobility spectrometer (IMS). It has been shown that the space charge is partially responsible for peak broadening in IMS depending on the ion density. Here, we explore the effect of space charge on peak shifting in IMS. We show that the field created by a large peak influences the drift time of a neighboring small peak. An experimental method was introduced to accurately measure the effect of space charge between two peaks. In this method, a double pulse was applied to the shutter grid to create two closed ion packets with a given initial spacing. The final spacing was then measured at the collector through the separation of the two peaks. This study shows that space charge repulsion must be considered for accurate measurements of ion mobilities. The experiments were performed in both normal and inverse modes. A theoretical model was also proposed to describe the repulsion between two ion packets in IMS.     

Separation and identification of isomeric glycopeptides by high field asymmetric waveform ion mobility spectrometry

The analysis of intact glycopeptides by mass spectrometry is challenging due to the numerous possibilities for isomerization, both within the attached glycan and the location of the modification on the peptide backbone. Here, we demonstrate that high field asymmetric wave ion mobility spectrometry (FAIMS), also known as differential ion mobility, is able to separate isomeric O-linked glycopeptides that have identical sequences but differing sites of glycosylation. Two glycopeptides from the glycoprotein mucin 5AC, GT(GalNAc)TPSPVPTTSTTSAP and GTTPSPVPTTST(GalNAc)TSAP (where GalNAc is O-linked N-acetylgalactosamine), were shown to coelute following reversed-phase liquid chromatography. However, FAIMS analysis of the glycopeptides revealed that the compensation voltage ranges in which the peptides were transmitted differed. Thus, it is possible at certain compensation voltages to completely separate the glycopeptides. Separation of the glycopeptides was confirmed by unique reporter ions produced by supplemental activation electron transfer dissociation mass spectrometry. These fragments also enable localization of the site of glycosylation. The results suggest that glycan position plays a key role in determining gas-phase glycopeptide structure and have implications for the application of FAIMS in glycoproteomics.     

Fundamentals of traveling wave ion mobility spectrometry

Traveling wave ion mobility spectrometry (TW IMS) is a new IMS method implemented in the Synapt IMS/mass spectrometry system (Waters). Despite its wide adoption, the foundations of TW IMS were only qualitatively understood and factors governing the ion transit time (the separation parameter) and resolution remained murky. Here we develop the theory of TW IMS using derivations and ion dynamics simulations. The key parameter is the ratio (c) of ion drift velocity at the steepest wave slope to wave speed. At low c, the ion transit velocity is proportional to the squares of mobility (K) and electric field intensity (E), as opposed to linear scaling in drift tube (DT) IMS and differential mobility analyzers. At higher c, the scaling deviates from quadratic in a way controlled by the waveform profile, becoming more gradual with the ideal triangular profile but first steeper and then more gradual for realistic profiles with variable E. At highest c, the transit velocity asymptotically approaches the wave speed. Unlike with DT IMS, the resolving power of TW IMS depends on mobility, scaling as K(1/2) in the low-c limit and less at higher c. A nonlinear dependence of the transit time on mobility means that the true resolving power of TW IMS differs from that indicated by the spectrum. A near-optimum resolution is achievable over an approximately 300-400% range of mobilities. The major predicted trends are in agreement with TW IMS measurements for peptide ions as a function of mobility, wave amplitude, and gas pressure. The issues of proper TW IMS calibration and ion distortion by field heating are also discussed. The new quantitative understanding of TW IMS separations allows rational optimization of instrument design and operation and improved spectral calibration.     

Species-specific bacteria identification using differential mobility spectrometry and bioinformatics pattern recognition

As bacteria grow and proliferate, they release a variety of volatile compounds that can be profiled and used for speciation, providing an approach amenable to disease diagnosis through quick analysis of clinical cultures as well as patient breath analysis. As a practical alternative to mass spectrometry detection and whole cell pyrolysis approaches, we have developed methodology that involves detection via a sensitive, micromachined differential mobility spectrometer (microDMx), for sampling headspace gases produced by bacteria growing in liquid culture. We have applied pattern discovery/recognition algorithms (ProteomeQuest) to analyze headspace gas spectra generated by microDMx to reliably discern multiple species of bacteria in vitro: Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and Mycobacterium smegmatis. The overall accuracy for identifying volatile profiles of a species within the 95% confidence interval for the two highest accuracy models evolved was between 70.4 and 89.3% based upon the coordinated expression of between 5 and 11 features. These encouraging in vitro results suggest that the microDMx technology, coupled with bioinformatics data analysis, has potential for diagnosis of bacterial infections.     

High-resolution field asymmetric waveform ion mobility spectrometry using new planar geometry analyzers

Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as a powerful tool of broad utility for separation and characterization of gas-phase ions, especially in conjunction with mass spectrometry (MS). In FAIMS, ions are filtered by the dependence of mobility on electric field while being carried by gas flow through the analytical gap between two electrodes of either planar (p-) or cylindrical (c-) geometry. Most FAIMS/MS systems employ c-FAIMS because of its ease of coupling to MS, yet the merits of the two geometries have not been compared in detail. Here, a priori simulations reveal that reducing the FAIMS curvature always improves resolution at equal sensitivity. In particular, the resolving power of p-FAIMS exceeds that of c-FAIMS, typically by a factor of 2-4 depending on the ion species and carrier gas. We have constructed a new planar FAIMS incorporating a curtain plate interface for effective operation with an ESI ion source and joined to an MS using an ion funnel interface with a novel slit aperture. The resolution increases up to 4-fold over existing c-FAIMS, even though the analysis is approximately 2 times faster. This allows separation of species not feasible in previous FAIMS studies, e.g., protonated leucine and isoleucine or new bradykinin isomers. The improvement for protein conformers (of ubiquitin) is less significant, possibly because of multiple unresolved geometries.     

Mass analysis of mobility-selected ion populations using dual gate, ion mobility, quadrupole ion trap mass spectrometry

An electrospray ionization, dual gate, ion mobility, quadrupole ion trap mass spectrometer (ESI-DG-IM-QIT-MS) was constructed and evaluated for its ability to select mobility-filtered ions prior to mass analysis. While modification of the common signal-averaged ion mobility experiment was required, no modifications to the QIT were necessary. The dual gate scanning mode of operation was used to acquire mobility spectra, whereas the single mobility monitoring experiment selectively filtered ions for concentration and subsequent fragmentation within the QIT. Ion mobility separation of positively charged peptides and negatively charged carbohydrates, followed by MS fragmentation, was demonstrated. For a 1-min acquisition time, it was possible to obtain complete de novo sequence information for the examined peptides. Fragmentation of the negative carbohydrate chlorine adducts yielded ions characteristic of cross-ring and glycosidic bond cleavage. Previous unions of atmospheric pressure ion mobility and mass spectrometry have been limited in their ability to reproducibly obtain MSn data for mobility separation ions. The union of high-pressure ion mobility with quadrupole ion trap mass spectrometry presents the unique opportunity to obtain more detailed information regarding the chemistries of gas-phase ions.