Dynamically multiplexed ion mobility time-of-flight mass spectrometry

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma. To better address variable sample complexity, we have developed and rigorously evaluated a novel dynamic MP approach that ensures correlation of the analyzer performance with an ion source function and provides the improved dynamic range and sensitivity throughout the experiment. The MP IMS-TOFMS instrument has been shown to reliably detect peptides at a concentration of 1 nM in the presence of a highly complex matrix, as well as to provide a 3 orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features were found to yield approximately 700 unique peptide identifications at a false discovery rate (FDR) of approximately 7.5%. Accounting for IMS information gave rise to a projected FDR of approximately 4%. Signal reproducibility was found to be greater than 80%, while the variations in the number of unique peptide identifications were <15%. A single sample analysis was completed in 15 min that constitutes almost 1 order of magnitude improvement compared to a more conventional LC-MS approach.     

Pseudorandom sequence modifications for ion mobility orthogonal time-of-flight mass spectrometry

Due to the inherently low duty cycle of ion mobility spectrometry (IMS) experiments that sample from continuous ion sources, a range of experimental advances have been developed to maximize ion utilization efficiency. The use of ion trapping and accumulation approaches prior to the ion mobility drift tube has demonstrated significant gains over discrete sampling from continuous sources but have traditionally relied upon a signal averaging (SA) to attain analytically useful signal-to-noise ratios (SNR). Multiplexed (MP) techniques based upon the Hadamard transform offer an alternative experimental approach by which ion utilization efficiency can be elevated from approximately 1 to approximately 50%. Recently, our research group demonstrated a unique multiplexed ion mobility time-of-flight (MP-IMS-TOF) approach that incorporates ion trapping and can extend ion utilization efficiency beyond 50%. However, the spectral reconstruction of the multiplexed signal using this experiment approach requires the use of sample-specific weighting designs. Such general weighting designs have been shown to significantly enhance ion utilization efficiency using this MP technique, but cannot be universally applied. By modifying both the ion trapping and the pseudorandom sequence (PRS) used for the MP experiment, we have eliminated the need for complex weighting matrices. For both simple and complex mixtures, SNR enhancements of up to 13 were routinely observed as compared to the SA-IMS-TOF approach. In addition, this new class of PRS provides a 2-fold enhancement in the number of ion gate pulses per unit time compared to the traditional HT-IMS experiment.     

Coupling high-pressure MALDI with ion mobility/orthogonal time-of-flight mass spectrometry

A new ion mobility/time-of-flight mass spectrometer employing a high-pressure MALDI source has been designed and tested. The prototype instrument operates at a source/drift cell pressure of 1-10 Torr helium, resulting in a mobility resolution of approximately 25. A small time-of-flight mass spectrometer (20 cm) with a mass resolution of up to 200 has been attached to the drift cell to identify (in terms of mass-to-charge ratio) the separated ions. A simple tripeptide mixture has been separated in the drift tube and mass identified as singly protonated species. The ability to separate peptide mixtures, e.g., tryptic digest of a protein, is illustrated and compared to results obtained on a high-vacuum time-of-flight instrument.     

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.     

Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.     

Secondary ionization of chemical warfare agent simulants: atmospheric pressure ion mobility time-of-flight mass spectrometry

For the first time, the use of a traditional ionization source for ion mobility spectrometry (radioactive nickel ((63)Ni) beta emission ionization) and three alternative ionization sources (electrospray ionization (ESI), secondary electrospray ionization (SESI), and electrical discharge (corona) ionization (CI)) were employed with an atmospheric pressure ion mobility orthogonal reflector time-of-flight mass spectrometer (IM(tof)MS) to detect chemical warfare agent (CWA) simulants from both aqueous- and gas-phase samples. For liquid-phase samples, ESI was used as the sample introduction and ionization method. For the secondary ionization (SESI, CI, and traditional (63)Ni ionization) of vapor-phase samples, two modes of sample volatilization (heated capillary and thermal desorption chamber) were investigated. Simulant reference materials, which closely mimic the characteristic chemical structures of CWA as defined and described by Schedule 1, 2, or 3 of the Chemical Warfare Convention treaty verification, were used in this study. A mixture of four G/V-type nerve simulants (dimethyl methylphosphonate, pinacolyl methylphosphonate, diethyl phosphoramidate, and 2-(butylamino)ethanethiol) and one S-type vesicant simulant (2-chloroethyl ethyl sulfide) were found in each case (sample ionization and introduction methods) to be clearly resolved using the IM(tof)MS method. In many cases, reduced mobility constants (K(o)) were determined for the first time. Ion mobility drift times, flight times, relative signal intensities, and fragmentation product signatures for each of the CWA simulants are reported for each of the methods investigated.     

Postacquisition mass resolution improvement in time-of-flight secondary ion mass spectrometry

Good mass resolution can be difficult to achieve in time-of-flight secondary ion mass spectrometry (TOF-SIMS) when the analysis area is large or when the surface being analyzed is rough. In most cases, a significant improvement in mass resolution can be achieved by postacquisition processing of raw data. Methods are presented in which spectra are extracted from smaller regions within the original analysis area, recalibrated, and selectively summed to produce spectra with higher mass resolution than the original. No hardware modifications or specialized instrument tuning are required. The methods can be extended to convert the original raw file into a new raw file containing high mass resolution data. To our knowledge, this is the first report of conversion of a low mass resolution raw file into a high mass resolution raw file using only the data contained within the low mass resolution raw file. These methods are applicable to any material but are expected to be particularly useful in analysis of difficult samples such as fibers, powders, and freeze-dried biological specimens.     

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.     

Detection of a chemical warfare agent simulant in various aerosol matrixes by ion mobility time-of-flight mass spectrometry

For the first time, a traditional radioactive nickel (63Ni) beta emission ionization source for ion mobility spectrometry was employed with an atmospheric pressure ion mobility orthogonal reflector time-of-flight mass spectrometer (IM(tof)MS) to detect a chemical warfare agent (CWA) simulant from aerosol samples. Aerosol-phase sampling employed a quartz cyclonic chamber for sample introduction. The simulant reference material, which closely mimicked the characteristic chemical structure of CWAs as defined and described by Schedule 1, 2, or 3 of the Chemical Warfare Convention treaty verification, was used in this study. An overall elevation in arbitrary signal intensity of approximately 1.0 orders of magnitude was obtained by the progressive increase of the thermal AP-IMS temperature from 75 to 275 degrees C. A mixture of one G-type nerve simulant (dimethyl methylphosphonate (DMMP)) in four (water, kerosene, gasoline, diesel) matrixes was found in each case (AP-IMS temperature 75-275 degrees C) to be clearly resolved in less than 2.20 x 10(4) micros using the IM(tof)MS instrument. Corresponding ions, masses, drift times, K(o) values, and arbitrary signal intensities for each of the sample matrixes are reported for the CWA simulant DMMP.     

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer

A hybrid quadrupole orthogonal time-of-flight mass spectrometer optimized for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization has been equipped with a C 60 cluster ion source. This configuration is shown to exhibit a number of characteristics that improve the performance of traditional time-of-flight secondary ion mass spectrometry (TOF-SIMS) experiments for the analysis of complex organic materials and, potentially, for chemical imaging. Specifically, the primary ion beam is operated as a continuous rather than a pulsed beam, resulting in up to 4 orders of magnitude greater ion fluence on the target. The secondary ions are extracted at very low voltage into 8 mTorr of N 2 gas introduced for collisional focusing and cooling purposes. This extraction configuration is shown to yield secondary ions that rapidly lose memory of the mechanism of their birth, yielding tandem mass spectra that are identical for SIMS and MALDI. With implementation of ion trapping, the extraction efficiency is shown to be equivalent to that found in traditional TOF-SIMS machines. Examples are given, for a variety of substrates that illustrate mass resolution of 12,000-15,600 with a mass range for inorganic compounds to m/ z 40,000. Preliminary chemical mapping experiments show that with added sensitivity, imaging in the MS/MS mode of operation is straightforward. In general, the combination of MALDI and SIMS is shown to add capabilities to each technique, providing a robust platform for TOF-SIMS experiments that already exists in a large number of laboratories.     

Automated gain control ion funnel trap for orthogonal time-of-flight mass spectrometry

Time-of-flight mass spectrometry (TOF MS) is increasingly used in proteomics research. Herein, we report on the development and characterization of a TOF MS instrument with improved sensitivity equipped with an electrodynamic ion funnel trap (IFT) that employs an automated gain control (AGC) capability. The IFT-TOF MS was coupled to a reversed-phase capillary liquid chromatography (RPLC) separation and evaluated in experiments with complex proteolytic digests. When applied to a global tryptic digest of Shewanella oneidensis proteins, an order-of-magnitude increase in sensitivity compared to that of the conventional continuous mode of operation was achieved due to efficient ion accumulation prior to TOF MS analysis. As a result of this sensitivity improvement and related improvement in mass measurement accuracy, the number of unique peptides identified in the AGC-IFT mode was 5-fold greater than that obtained in the continuous mode.     

Fourier transform time-of-flight mass spectrometry in an electrostatic ion beam trap

We report on the application of an electrostatic ion beam trap as a mass spectrometer. The instrument is analogous to an optical resonator; ions are trapped between focusing mirrors. The storage time is limited by the residual gas pressure and reaches up to several seconds, resulting in long ion flight paths. The oscillation of ion bunches between the mirrors is monitored by nondestructive image charge detection in a field-free region and mass spectra are obtained via Fourier transform. The principle of operation is demonstrated by measuring the mass spectrum of trapped Ar+ and Xe+ particles, produced by a standard electron impact ion source. Also, mass spectra of heavier PEGnNa+ and bradykinin ions from a pulsed MALDI ion source were obtained. The long ion flight path, combined with mass-independent charge detection, makes this system particularly interesting for the investigation of large molecules.     

Depth profiling of fullerene-containing structures by time-of-flight secondary ion mass spectrometry

A new variant of depth profiling for thin-film fullerene-containing organic structures by the method of time-of-flight (TOF) secondary ion mass spectrometry (SIMS) on a TOF.SIMS-5 setup is described. The dependence of the yield of C₆₀ molecular ions on the energy of sputtering ions has been revealed and studied. At an energy of sputtering Cs⁺ ions below 1 keV, the intensity of C₆₀ molecular ions is sufficiently high to make possible both elemental and molecular depth profiling of multicomponent (multilayer) thin-film structures. Promising applications of TOF-SIMS depth profiling for obtaining more detailed information on the real molecular composition of functional organic materials are shown.     

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.     

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.     

Enhanced analyte detection using in-source fragmentation of field asymmetric waveform ion mobility spectrometry-selected ions in combination with time-of-flight mass spectrometry

Miniaturized ultra high field asymmetric waveform ion mobility spectrometry (FAIMS) is used for the selective transmission of differential mobility-selected ions prior to in-source collision-induced dissociation (CID) and time-of-flight mass spectrometry (TOFMS) analysis. The FAIMS-in-source collision induced dissociation-TOFMS (FISCID-MS) method requires only minor modification of the ion source region of the mass spectrometer and is shown to significantly enhance analyte detection in complex mixtures. Improved mass measurement accuracy and simplified product ion mass spectra were observed following FAIMS preselection and subsequent in-source CID of ions derived from pharmaceutical excipients, sufficiently close in m/z (17.7 ppm mass difference) that they could not be resolved by TOFMS alone. The FISCID-MS approach is also demonstrated for the qualitative and quantitative analysis of mixtures of peptides with FAIMS used to filter out unrelated precursor ions thereby simplifying the resulting product ion mass spectra. Liquid chromatography combined with FISCID-MS was applied to the analysis of coeluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching. The potential of FISCID-MS for the quantitative determination of a model peptide spiked into human plasma in the range of 0.45-9.0 μg/mL is demonstrated, showing good reproducibility (%RSD < 14.6%) and linearity (R(2) > 0.99).     

Proton transfer in time-of-flight secondary ion mass spectrometry studies of frozen-hydrated dipalmitoylphosphatidylcholine

A frozen water matrix, as found in freeze-fractured frozen-hydrated cellular samples, enhances the ionization of phosphatidylcholine lipids with static time-of-flight secondary ion mass spectrometry (TOF-SIMS). Isotopic profiles of the phosphocholine ion from deuterated forms of dipalmitoylphosphatidylcholine (DPPC) have been examined under various sample preparation conditions to show that ionization occurs through protonation from the matrix and is enhanced by the water present in freeze-fractured samples. The ionization of DPPC results in positively charged fragment ions, primarily phosphocholine, with a m/z of 184. Other ions include the M + H ion (m/z 735) and an ion representing the abstraction of the two palmitoyl fatty acid groups (m/z 224). Freeze-fracture techniques have been used to prepare frozen aqueous samples such as liposomes and cells to expose their membranes for static TOF-SIMS imaging. Due to the importance of surface water during SIMS analyses, sources of gas-phase water resulting from freeze-fracture were examined. Under proper fracturing conditions, water vapor, resulting from water in the sample and water condensed onto the outside of the sample, is released into the vacuum but does not condense back onto the surface. Combining the demonstrated enhancement of phosphatidylcholine lipid signal from water with the freeze-fracture preparation techniques described herein demonstrates potential advantages of studying biological samples in a frozen-hydrated state.     

Separation and characterization of metallosupramolecular libraries by ion mobility mass spectrometry

The self-assembly of Zn(II) ions and bis(terpyridine) (tpy) ligands carrying 120° or 180° angles between their metal binding sites was utilized to prepare metallosupramolecular libraries with the connectivity. These combinatorial libraries were separated and characterized by ion mobility mass spectrometry (IM MS) and tandem mass spectrometry (MS(2)). The 180°-angle building blocks generate exclusively linear complexes, which were used as standards to determine the architectures of the assemblies resulting from the 120°-angle ligands. The latter ligand geometry promotes the formation of macrocyclic hexamers, but other n-mers with smaller (n = 5) or larger ring sizes (n = 7-9) were identified as minor products, indicating that the angles in the bis(terpyridine) ligand and within the coordinative tpy-Zn(II)-tpy bonds are not as rigid, as previously believed. Macrocyclic and linear isomers were detected in penta- and heptameric assemblies; in the larger octa- and nonameric assemblies, ring-opened conformers with compact and folded geometries were observed in addition to linear extended and cyclic architectures. IM MS(2) experiments provided strong evidence that the macrocycles present in the libraries were already formed in solution, during the self-assembly process, not by dissociation of larger complexes in the gas phase. The IM MS/MS(2) methods provide a means to analyze, based on size and shape (architecture), supramolecular libraries that are not amenable to liquid chromatography, LC-MS, NMR, and/or X-ray techniques.