Ultrasensitive identification of localization variants of modified peptides using ion mobility spectrometry

Localization of the modification sites on peptides is challenging, particularly when multiple modifications or mixtures of localization isomers (variants) are involved. Such variants commonly coelute in liquid chromatography and may be undistinguishable in tandem mass spectrometry (MS/MS) for lack of unique fragments. Here, we have resolved the variants of singly and doubly phosphorylated peptides employing drift tube ion mobility spectrometry (IMS) coupled to time-of-flight mass spectrometry. Even with a moderate IMS resolving power of ～80-100, substantial separation was achieved for both 2+ and 3+ ions normally generated by electrospray ionization, including for the variants indistinguishable by MS/MS. Variants often exhibit a distribution of 3-D conformers, which can be adjusted for optimum IMS separation by prior field heating of ions in a funnel trap. The peak assignments were confirmed using MS/MS after IMS separation, but known species could be identified using just the ion mobility "tag". Avoiding the MS/MS step lowers the detection limit of localization variants to <100 amol, an order of magnitude better than that provided by electron transfer dissociation in an Orbitrap MS.     

Enhanced direct ambient analysis by differential mobility-filtered desorption electrospray ionization-mass spectrometry

Desorption electrospray ionization (DESI) is rapidly becoming established as one of the most powerful ionization techniques allowing direct surface analysis by mass spectrometry (MS) in the ambient environment. DESI provides a significant number of unique analytical capabilities for a broad range of applications, both quantitative and qualitative in nature including biological tissue imaging, pharmaceutical quality control, in vivo analysis, proteomics, metabolomics, forensics, and explosives detection. Despite its growing adoption as a powerful high throughput analysis tool, DESI-MS analysis at trace levels often suffers from background chemical interferences generated during the electrospray ionization processes. In order to improve sensitivity and selectivity, a differential mobility (DM) ion separation cell was successfully interfaced to a custom-built DESI ion source. This new hybrid platform can be operated in two modes: the "DM-off" mode for standard DESI analysis and "DM-on mode" where DESI-generated ions are detected after discrimination by the differential mobility cell. The performance of the DESI-DM-MS platform was tested with several samples typically amenable to DESI analysis, including counterfeit pharmaceuticals and binary mixtures of isobaric chemicals of importance in the pharmaceutical and food industries. In the DM-on mode, DESI-MS signal-to-noise ratios were improved by 70-190% when compared to the DM-off mode. Also, the addition of the DM cell enabled selective in-source ion activation of specific DESI-generated precursor ions, providing tandem MS-like spectra in a single stage mass spectrometer.     

Polarity switching accurate mass measurement of pharmaceutical samples using desorption electrospray ionization and a dual ion source interfaced to an orthogonal acceleration time-of-flight mass spectrometer

A novel approach for the rapid, accurate mass analysis of pharmaceutical solid, liquid, and cream formulations using desorption electrospray ionization (DESI) is described. The method is based on polarity switching and real-time accurate mass measurement in an orthogonal acceleration time-of-flight mass spectrometer fitted with a dual-inlet electrospray ion source. Infusion of a reference compound into one inlet provides a single-point "lock mass" for accurate mass measurement. The other inlet sprays solvent at the sample being investigated using DESI. Minimal sample preparation was required. Results demonstrate the ability to acquire simultaneously positive and negative accurate mass DESI data within the same acquisition, thus negating the need for repeat analysis in each ion mode. In this paper, drugs that preferentially ionize in a particular mode depending on their physiochemical properties are presented. Mass accuracy to within 2 mTh was obtained for all drugs sampled.     

Non-proximate detection of small and large molecules by desorption electrospray ionization and desorption atmospheric pressure chemical ionization mass spectrometry: instrumentation and applications in forensics, chemistry, and biology

Ambient surfaces are examined by mass spectrometry at distances of up to 3 m from the instrument without any prior sample preparation. Non-proximate versions of the desorption electrospray ionization (DESI) and desorption atmospheric pressure chemical ionization experiments are shown to allow rapid, sensitive, and selective detection of trace amounts of active ingredients in pharmaceutical drug formulations, illicit drugs (methamphetamine, cocaine, and diacetylmorphine), organic salts, peptides, chemical warfare agent simulants, and other small organic compounds. Utilizing an ion transport tube to transport analyte ions to the mass spectrometer, nonproximate DESI allows one to collect high-quality, largely interference-free spectra with signal-to-noise (S/N) ratios of more than 100. High selectivity is achieved by tandem mass spectrometry and by reactive DESI, a variant experiment in which reagents added into the solvent spray allow bond-forming reactions with the analyte. Ion/molecule reactions were found to selectively suppress the response of mixture components other than the analyte of interest in nonproximate-DESI. Flexible ion transport tubing is also investigated, allowing performance similar to stainless steel tubing in the transport of ions from the sample to the mass spectrometer. Transfer tube temperature effects are examined. A multiple sprayer DESI source capable of analyzing a larger sample area was evaluated to decrease the sampling time and increase sample throughput. Low nanogram detection limits were obtained for the compounds studied from a wide variety of surfaces, even those present in complex matrixes.     

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.     

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%.     

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry combined with mass spectrometry for peptide analysis

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (ultra-FAIMS) combined with mass spectrometry (MS) has been applied to the analysis of standard and tryptic peptides, derived from α-1-acid glycoprotein, using electrospray and nanoelectrospray ion sources. Singly and multiply charged peptide ions were separated in the gas phase using ultra-FAIMS and detected by ion trap and time-of-flight MS. The small compensation voltage (CV) window for the transmission of singly charged ions demonstrates the ability of ultra-FAIMS-MS to generate pseudo-peptide mass fingerprints that may be used to simplify spectra and identify proteins by database searching. Multiply charged ions required a higher CV for transmission, and ions with different amino acid sequences may be separated on the basis of their differential ion mobility. A partial separation of conformers was also observed for the doubly charged ion of bradykinin. Selection on the basis of charge state and differential mobility prior to tandem mass spectrometry facilitates peptide and protein identification by allowing precursor ions to be identified with greater selectivity, thus reducing spectral complexity and enhancing MS detection.     

Electrospray ionization high-resolution ion mobility spectrometry for the detection of organic compounds, 1. Amino acids

Our aim in this investigation was to demonstrate the potential of the high-resolution electrospray ionization ion mobility spectrometry (ESI-IMS) technique as an analytical separation tool in analyzing biomolecular mixtures to pursue astrobiological objectives of searching for the chemical signatures of life during an in-situ exploration of solar system bodies. Because amino acids represent the basic building blocks of life, we used common amino acids to conduct the first part of our investigation, which is being reported here, to demonstrate the feasibility of using the ESI-IMS technique for detection of the chemical signatures of life. The ion mobilities of common amino acids were determined by electrospray ionization ion mobility spectrometry using three different drift gases (N2, Ar, and CO2). We demonstrated that the selectivity can be vastly improved in ion mobility spectroscopy (IMS) in detecting organic molecules by using different drift gases. When a judicial choice of drift gas is made, a vastly improved separation of two different amino acid ions resulted. It was found that each of the studied amino acids could be uniquely identified from the others, with the exception of alanine and glycine, which were never separable by more then 0.1 ms. This unique identification is a result of the different polarizabilities of the various drift gases. In addition, a better separation was achieved by changing the drift voltage in successive experimental runs without significantly degrading the resolution. We also report the result of our analysis of liquid samples containing mixtures of amino acids.     

Resolving structural isomers of monosaccharide methyl glycosides using drift tube and traveling wave ion mobility mass spectrometry

Monosaccharide structural isomers including sixteen methyl-D-glycopyranosides and four methyl-N-acetylhexosamines were subjected to ion mobility measurements by electrospray ion mobility mass spectrometry. Two ion mobility-MS systems were employed: atmospheric pressure drift tube ion mobility time-of-flight mass spectrometry and a Synapt G2 HDMS system which incorporates a low pressure traveling wave ion mobility separator. All the compounds were investigated as [M + Na](+) ions in the positive mode. A majority of the monosaccharide structural isomers exhibited different mobility drift times in either system, depending on differences in their anomeric and stereochemical configurations. In general, drift time patterns (relative drift times of isomers) matched between the two instruments. Higher resolving power was observed using the atmospheric pressure drift tube. Collision cross section values of monosaccharide structural isomers were directly calculated from the atmospheric pressure ion mobility experiments, and a collision cross section calibration curve was made for the traveling wave ion mobility instrument. Overall, it was demonstrated that ion mobility-mass spectrometry using either drift tube or traveling wave ion mobility is a valuable technique for resolving subtle variations in stereochemistry among the sodium adducts of monosaccharide methyl glycosides.     

Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs

A secondary electrospray ionization (SESI) method was developed as a nonradioactive ionization source for ion mobility spectrometry (IMS). This SESI method relied on the gas-phase interaction between charged particles created by electrospray ionization (ESI) and neutral gaseous sample molecules. Mass spectrometry (MS) was used as the detection method after ion mobility separation for ion identification. Preliminary investigations focussed on understanding the ionization process of SESI. The performance of ESI-IMS and SESI-IMS for illicit drug detection was evaluated by determining the analytical figures of merit. In general, SESI had a higher ionization efficiency for small volatile molecules compared with the electrospray method. The potential of developing a universal interface for both GC- and LC-MS with an addition stage of mobility separation was demonstrated.     

Enhanced ion utilization efficiency using an electrodynamic ion funnel trap as an injection mechanism for ion mobility spectrometry

Conventional ion mobility spectrometers that sample ion packets from continuous sources have traditionally been constrained by an inherently low duty cycle. As such, ion utilization efficiencies have been limited to <1% in order to maintain instrumental resolving power. Using a modified electrodynamic ion funnel, we demonstrated the ability to accumulate, store, and eject ions in conjunction with ion mobility spectrometry (IMS), which elevated the charge density of the ion packets ejected from the ion funnel trap (IFT) and provided a considerable increase in the overall ion utilization efficiency of the IMS instrument. A 7-fold increase in signal intensity was revealed by comparing continuous ion beam current with the amplitude of the pulsed ion current in IFT-IMS experiments using a Faraday plate. Additionally, we describe the IFT operating characteristics using a time-of-flight mass spectrometer attached to the IMS drift tube.     

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.     

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.     

Desorption electrospray ionization mass spectrometry for high-throughput analysis of pharmaceutical samples in the ambient environment

Desorption electrospray ionization (DESI) allows mass spectrometry to be used for on-line high-throughput monitoring of pharmaceutical samples in the ambient environment, without prior sample preparation. Positive and negative ion DESI are used to characterize the active ingredients in pharmaceutical samples formulated as tablets, ointments, and liquids. Compounds of a wide variety of chemical types are detected in these complex matrices. The effects on analytical performance of operating parameters, including the electrospray high voltage, heated capillary temperature, solvent infusion rate, and solvent composition, are evaluated and optimized. In addition to experiments in which a simple solvent is sprayed onto the solid analyte samples, reactive desorption is performed by adding reagents to the solvent spray to generate particularly stable or characteristic ions with the analytes of interest. A variable-speed moving belt was built for high-throughput sampling and used to provide rapid qualitative and semiquantitative information on drug constituents in tablets. Sampling rates as high as 3 samples/s are achieved in the ambient environment. Relative standard deviations of the relative ion abundances for major components in the mass spectra are in the range of 2-8%. Impurities and components present at levels as low as approximately 0.1% are identified and carryover effects are minimized in high-throughput on-line analysis of pharmaceutical samples.     

Ion mobility spectrometry: a comprehensive and versatile tool for occupational pharmaceutical exposure assessment

The qualitative and quantitative capabilities of ion mobility spectrometry (IMS) as a comprehensive and powerful tool in workplace air monitoring have been demonstrated on the example of a Spanish pharmaceutical company. The developed IMS based procedure is capable of detecting and determining in air samples the active pharmaceutical ingredients (APIs) manipulated and/or produced in this pharmaceutical industry. Sensitivity, in the ng-pg range, selectivity, possibly to provide results in near real time, and reduction of analysis costs are the most important properties that ratify IMS as a serious alternative in occupational exposure assessment. The possibility of false positives by drift time interferences and false negatives by competitive ionization and also desorption process interferences has been deeply evaluated. Moreover, chemometric strategies based on self-modeling curve resolution (SMCR) have been applied to obtain qualitative and quantitative individual component information from overlapped peaks. The IMS procedure has been successfully applied to evaluate the concentration of APIs (nimesulide, dexketoprofen, deflazacort) handled by the pharmaceutical company employees in the making of tablets and granulates, and control measures have been suggested in accordance.     

Multiplexed ion mobility spectrometry-orthogonal time-of-flight mass spectrometry

Ion mobility spectrometry (IMS) coupled to orthogonal time-of-flight mass spectrometry (TOF) has shown significant promise for the characterization of complex biological mixtures. The enormous complexity of biological samples (e.g., from proteomics) and the need for both biological and technical analysis replicates imposes major challenges for multidimensional separation platforms with regard to both sensitivity and sample throughput. A major potential attraction of the IMS-TOF MS platform is separation speeds exceeding that of conventional condensed-phase separations by orders of magnitude. Known limitations of the IMS-TOF MS platforms that presently mitigate this attraction include the need for extensive signal averaging due to factors that include significant ion losses in the IMS-TOF interface and an ion utilization efficiency of less than approximately 1% with continuous ion sources (e.g., ESI). We have developed a new multiplexed ESI-IMS-TOF mass spectrometer that enables lossless ion transmission through the IMS-TOF as well as a utilization efficiency of >50% for ions from the ESI source. Initial results with a mixture of peptides show a approximately 10-fold increase in signal-to-noise ratio with the multiplexed approach compared to a signal averaging approach, with no reduction in either IMS or TOF MS resolution.     

IMS-IMS and IMS-IMS-IMS/MS for separating peptide and protein fragment ions

Multidimensional ion mobility spectrometry (IMS-IMS and IMS-IMS-IMS) techniques have been combined with mass spectrometry (MS) and investigated as a means of generating and separating peptide and protein fragment ions. When fragments are generated inside a drift tube and then dispersed by IMS prior to MS analysis, it is possible to observe many features that are not apparent from MS analysis alone. The approach is demonstrated by examining fragmentation patterns arising from electrospray ion distributions of insulin chain B and ubiquitin. The multidimensional IMS approach makes it possible to select individual components for collisional activation and to disperse fragments based on differences in mobility prior to MS analysis. Such an approach makes it possible to observe many features not apparent by MS analysis alone.     

Ion mobility spectrometry of halothane, enflurane, and isoflurane anesthetics in air and respired gases

Three common gaseous anesthetics, halothane, enflurane, and isoflurane, were characterized by using ion mobility spectrometry (IMS)/mass spectrometry, and the dependence of product ion distributions on temperature and concentration was evaluated. At 40 degrees C and 500 ppb, negative ion mobility spectra in air largely consisted of monomer or dimer adducts with Br- or Cl- formed through dissociative electron capture of molecular neutrals. With increased temperature or decreased vapor concentrations, declustering and dissociation of product ions became pronounced. Ion-molecule reactions in the drift region of the IMS were evident as distortions in peak shape in the mass-resolved mobility spectra and in variable reduced mobilities for the same ions. A portable hand-held IMS was used for convenient, real-time detection of enflurane in respired gases following a controlled inhalation episode.     

Two-dimensional gas-phase separations coupled to mass spectrometry for analysis of complex mixtures

Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-flight (TOF) MS. Implementation of FAIMS/IMS and IMS/MS interfaces using electrodynamic ion funnels greatly improves sensitivity. Evaluation of FAIMS/IMS/TOF performance for a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions and, hence, the benefit of FAIMS filtering prior to IMS/MS. The effective peak capacities in analyses of tryptic peptides are approximately 500 for FAIMS/IMS separations and approximately 10(6) for 3-D FAIMS/IMS/MS, providing a potential platform for ultrahigh-throughput analyses of complex mixtures.