Increasing Isolation Efficiency Using a Segmented Quadrupole Mass Filter Operated with Rectangular Waveforms.

The performance of a segmented quadrupole mass filter operated with rectangular waveforms and capacitively coupled rectangular waveforms applied to the prefilters was examined on a home-built quadrupole-Orbitrap platform. For peak widths of 50 m/z, 100% isolation efficiency was achieved, which fell to approximately 20% for 5 m/z peak width for a rectangular waveform of 150 V0-p. Due to a small exit aperture following the mass filter, peak structure was observed in both experimental peak shapes and those simulated using SIMION. A larger radius quadrupole was examined and achieved similar performance. While the segmented quadrupole does remove the defocusing effects of the fringing fields, the ion beam is only slightly refocused due to the low RF voltage which limits achievable gains in isolation efficiency.

Cyclable Variable Path Length Multilevel Structures for Lossless Ion Manipulations (SLIM) Platform for Enhanced Ion Mobility Separations.

Ion mobility-mass spectrometry (IMS-MS) is used to analyze complex samples and provide structural information on unknown compounds. As the complexity of samples increases, there is a need to improve the resolution of IMS-MS instruments to increase the rate of molecular identification. This work evaluated a cyclable and variable path length (and hence resolving power) multilevel Structures for Lossless Ion Manipulations (SLIM) platform to achieve a higher resolving power than what was previously possible. This new multilevel SLIM platform has eight separation levels connected by ion escalators, yielding a total path length of ∼88 m (∼11 m per level). Our new multilevel SLIM can also be operated in an "ion cycling" mode by utilizing a set of return ion escalators that transport ions from the eighth level back to the first, allowing even extendable path lengths (and higher IMS resolution). The platform has been improved to enhance ion transmission and IMS separation quality by reducing the spacing between SLIM boards. The board thickness was reduced to minimize the ions' escalator residence time. Compared to the previous generation, the new multilevel SLIM demonstrated better transmission for a set of phosphazene ions, especially for the low-mobility ions. For example, the transmission of m/z 2834 ions was improved by a factor of ∼3 in the new multilevel SLIM. The new multilevel SLIM achieved 49% better resolving powers for GRGDS1+ ions in 4 levels than our previous 4-level SLIM. The collision cross-section-based resolving power of the SLIM platform was tested using a pair of reverse sequence peptides (SDGRG1+, GRGDS1+). We achieved 1100 resolving power using 88 m of path length (i.e., 8 levels) and 1400 following an additional pass through the eight levels. Further evaluation of the multilevel SLIM demonstrated enhanced separation for positively and negatively charged brain total lipid extract samples. The new multilevel SLIM enables a tunable high resolving power for a wide range of ion mobilities and improved transmission for low-mobility ions.

Top/Middle-Down Characterization of α-Synuclein Glycoforms.

α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of ∼68 and ∼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of ∼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.

A planar quadrupole device for transmitting and trapping ions in high vacuum.

RATIONALE: Hybrid mass spectrometers combine multiple mass analyzers to achieve optimal performance in terms of tandem mass spectrometry, high mass resolving power, and mass measurement accuracy for studying highly complex samples. As a result, the need for transport, trapping, and control of ion kinetic energies is critical for the successful integration of multiple mass analyzers and hybrid instrument operation. In addition, transportation of ion populations between two physically distinct locations can result in time-of-flight (TOF) discrimination against ions with widely disparate m/z values, compromising full mass spectral performance. In this work, we demonstrated a new ion guide, referred to as a planar quadrupole (PQ) ion guide, composed of two parallel printed circuit boards (PCB) that allow radiofrequency (RF) and direct current (DC) voltages to be combined to enable both axial transport and trapping of ion populations in the ultrahigh vacuum region of the mass spectrometer. As compared with a conventional multipole ion guide, the PQ ion guide showed comparable performance in ion m/z values, signal-to-noise, and intensity and effectively reduced mass discrimination caused by TOF effects. METHODS: A PQ device was developed with two PCBs and simulated with SIMION 8.1. Electrospray ionization and Fourier transform ion cyclotron resonance mass spectrometry instrumentation were used for the testing of PQ performance. RESULTS: .In this work, we demonstrated a planar quadrupole (PQ) ion guide composed of two parallel PCB plates. The PQ enables both axial ion transport and trapping of ion populations throughout the ion transfer process from a LTQ to an ICR cell. As compared with a conventional multipole ion guide, the PQ showed comparable ion transmission efficiency and effectively reduced mass discrimination caused by TOF effects. CONCLUSIONS: The PQ is a simple design that can be implemented for ion transmission and trapping on virtually any mass spectrometer.

A Dual-Gated Structures for Lossless Ion Manipulations-Ion Mobility Orbitrap Mass Spectrometry Platform for Combined Ultra-High-Resolution Molecular Analysis.

High-resolution ion mobility spectrometry-mass spectrometry (HR-IMS-MS) instruments have enormously advanced the ability to characterize complex biological mixtures. Unfortunately, HR-IMS and HR-MS measurements are typically performed independently due to mismatches in analysis time scales. Here, we overcome this limitation by using a dual-gated ion injection approach to couple an 11 m path length structures for lossless ion manipulations (SLIM) module to a Q-Exactive Plus Orbitrap MS platform. The dual-gate setup was implemented by placing one ion gate before the SLIM module and a second ion gate after the module. The dual-gated ion injection approach allowed the new SLIM-Orbitrap platform to simultaneously perform an 11 m SLIM separation, Orbitrap mass analysis using the highest selectable mass resolution setting (up to 140 k), and high-energy collision-induced dissociation (HCD) in ∼25 min over an m/z range of ∼1500 amu. The SLIM-Orbitrap platform was initially characterized using a mixture of standard phosphazene cations and demonstrated an average SLIM CCS resolving power (RpCCS) of ∼218 and an SLIM peak capacity of ∼156, while simultaneously obtaining high mass resolutions. SLIM-Orbitrap analysis with fragmentation was then performed on mixtures of standard peptides and two reverse peptides (SDGRG1+, GRGDS1+, and RpCCS = 305) to demonstrate the utility of combined HR-IMS-MS/MS measurements for peptide identification. Our new HR-IMS-MS/MS capability was further demonstrated by analyzing a complex lipid mixture and showcasing SLIM separations on isobaric lipids. This new SLIM-Orbitrap platform demonstrates a critical new capability for proteomics and lipidomics applications, and the high-resolution multimodal data obtained using this system establish the foundation for reference-free identification of unknown ion structures.

Optimization of a Digital Mass Filter for the Isolation of Intact Protein Complexes in Stability Zone 1,1.

Digital mass filters are advantageous for the analysis of large molecules due to the ability to perform ion isolation of high-m/z ions without the generation of very high radio frequency (RF) and DC voltages. Experimentally determined Mathieu stability diagrams of stability zone 1,1 for capacitively coupled digital waveforms show a voltage offset between the quadrupole rod pairs is introduced by the capacitors which is dependent on the voltage magnitude of the waveform and the duty cycle. This changes the ion's a value from a = 0 to a < 0. These effects are illustrated for isolation for single-charge states for various protein complexes up to 800 kDa (GroEL) for stability zone 1,1. Isolation resolving power (m/Δm) of approximately 280 was achieved for an ion of m/z 12,315 (z = 65+ for 800.5 kDa GroEL D398A), which corresponds to an m/z window of 44.

Accelerating prototyping experiments for traveling wave structures for lossless ion manipulations.

Traveling wave structures for lossless ion manipulation (TW-SLIM) has proven a valuable tool for the separation and study of gas-phase ions. Unfortunately, many of the traditional components of TW-SLIM experiments manifest practical and financial barriers to the technique's broad implementation. To this end, a series of technological innovations and methodologies are presented which enable for simplified SLIM experimentation and more rapid TW-SLIM prototyping. In addition to the use of multiple independent board sets that comprise the present SLIM system, we introduce a low-cost, multifunctional traveling wave generator to produce TW within the TW-SLIM. This square-wave producing unit proved effective in realizing TW-SLIM separations compared to traditional approaches. Maintaining a focus on lowering barriers to implementation, the present set of experiments explores the use of on-board injection (OBI) methods, which offer potential alternatives to ion funnel traps. These OBI techniques proved feasible and the ability of this simplified TW-SLIM platform to enhance ion accumulation was established. Further experimentation regarding ion accumulation revealed a complexity to ion accumulation within TW-SLIM that has yet to be expounded upon. Lastly, the ability of the presented TW-SLIM platform to store ions for extended periods (1 s) without significant loss (<10%) was demonstrated. The aforementioned experiments clearly establish the efficacy of a simplified TW-SLIM platform which promises to expand adoption and experimentation of the technique.

Implementing Digital-Waveform Technology for Extended m/z Range Operation on a Native Dual-Quadrupole FT-IM-Orbitrap Mass Spectrometer.

Here, we describe a digital-waveform dual-quadrupole mass spectrometer that enhances the performance of our drift tube FT-IMS high-resolution Orbitrap mass spectrometer (MS). The dual-quadrupole analyzer enhances the instrument capabilities for studies of large protein and protein complexes. The first quadrupole (q) provides a means for performing low-energy collisional activation of ions to reduce or eliminate noncovalent adducts, viz., salts, buffers, detergents, and/or endogenous ligands. The second quadrupole (Q) is used to mass-select ions of interest for further interrogation by ion mobility spectrometry and/or collision-induced dissociation (CID). Q is operated using digital-waveform technology (DWT) to improve the mass selection compared to that achieved using traditional sinusoidal waveforms at floated DC potentials (>500 V DC). DWT allows for increased precision of the waveform for a fraction of the cost of conventional RF drivers and with readily programmable operation and precision (Hoffman, N. M. . A comparison-based digital-waveform generator for high-resolution duty cycle. Review of Scientific Instruments 2018, 89, 084101).

Application of frequency multiple FT-ICR-MS signal acquisition for improved proteome research.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with liquid chromatography (LC) is a powerful combination useful in many research areas due to the utility of high mass resolving power and mass measurement accuracy for studying highly complex samples. Ideally, every analyte in a complex sample can be subjected to accurate mass MS/MS analysis to aid in identification. FT-ICR MS can provide high mass resolving power and mass accuracy at the cost of long data acquisition periods, reducing the number of spectra that can be acquired per unit time. Frequency multiple signal acquisition has long been realized as an attractive method to obtain high mass resolving power and mass accuracy with shorter data acquisition periods. However, one of the limitations associated with frequency multiple signal acquisition is reduced signal intensity as compared to a traditional dipole detector. In this study, we demonstrated the use of a novel ICR cell to improve frequency multiple signal intensity and investigated the potential use of frequency multiple acquisition for proteome measurements. This novel ICR cell containing both dipole and frequency multiple detection electrodes was installed on a 7T FT-ICR MS coupled to an LC system. Tryptic digests of HeLa cell lysates were analyzed using dipole and frequency multiple detectors by holding either the mass resolving power or signal acquisition time constant. Compared to dipole detection, second frequency multiple detection yielded 36% or 45% more unique identified peptides from HeLa cell lysates at twice the scan rate or twice the mass resolving power, respectively. These results indicate that frequency multiple signal acquisition with either the same resolving power or the same signal acquisition duration as used with dipole signals can produce a significant increase in the number of peptides identified in complex proteome samples.

Ultra-High-Resolution Ion Mobility Separations Over Extended Path Lengths and Mobility Ranges Achieved using a Multilevel Structures for Lossless Ion Manipulations Module.

Over the past few years, structures for lossless ion manipulations (SLIM) have used traveling waves (TWs) to move ions over long serpentine paths that can be further lengthened by routing the ions through multiple passages of the same path. Such SLIM "multipass" separations provide unprecedentedly high ion mobility resolving powers but are ultimately limited in their ion mobility range because of the range of mobilities spanned in a single pass; that is, higher mobility ions ultimately "overtake" and "lap" lower mobility ions that have experienced fewer passes, convoluting their arrival time distribution at the detector. To achieve ultrahigh resolution separations over broader mobility ranges, we have developed a new multilevel SLIM possessing multiple stacked serpentine paths. Ions are transferred between SLIM levels through apertures (or ion escalators) in the SLIM surfaces. The initial multilevel SLIM module incorporates four levels and three interlevel ion escalator passages, providing a total path length of 43.2 m. Using the full path length and helium buffer gas, high resolution separations were achieved for Agilent tuning mixture phosphazene ions over a broad mobility range (K0 ≈ 3.0 to 1.2 cm2/(V*s)). High sensitivity was achieved using "in-SLIM" ion accumulation over an extended trapping region of the first SLIM level. High transmission efficiency of ions over a broad mobility range (e.g., K0 ≈ 3.0 to 1.67 cm2/(V*s)) was achieved, with transmission efficiency rolling off for the lower mobility ions (e.g., K0 ≈ 1.2 cm2/(V*s)). Resolving powers of up to ∼560 were achieved using all four ion levels to separate reverse peptides (SDGRG1+ and GRGDS1+). A complex mixture of phosphopeptides showed similar coverage could be achieved using one or all four SLIM levels, and doubly charged phosphosite isomers not significantly separated using one SLIM level were well resolved when four levels were used. The new multilevel SLIM technology thus enables wider mobility range ultrahigh-resolution ion mobility separations and expands on the ability of SLIM to obtain improved separations of complex mixtures with high sensitivity.

Parallel Detection of Fundamental and Sixth Harmonic Signals Using an ICR Cell with Dipole and Sixth Harmonic Detectors.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is a powerful instrument for high-resolution analysis of biomolecules. However, relatively long signal acquisition periods are needed to achieve mass spectra with high resolution. The use of multiple detector electrodes for detection of harmonic frequencies has been introduced as one approach to increase scan rate for a given resolving power or to obtain increased resolving power for a given detection period. The achieved resolving power and scan rate increase linearly with the order of detected harmonic signals. In recent years, ICR cell geometries have been investigated to increase the order of the harmonic frequencies and enhance harmonic signal intensities. In this study, we demonstrated PCB-based ICR cell designs with dipole and sixth harmonic detectors for parallel detection of fundamental and harmonic (6f) signals. The sixth harmonic signals from the sixth harmonic detector showed an expected 6 times higher resolving power with (M + 3H)3+ charge state insulin ions as compared with that from fundamental signals from the dipole detector. Moreover, the insulin isotopic peaks with sixth harmonic frequency signals acquired with the sixth harmonic detector were resolved for a 40 ms data acquisition period but unresolved with the same duration dipole detector signals, corresponding to a 6-fold improvement in achievable spectral acquisition rates for a given resolving power.

Traveling-Wave-Based Electrodynamic Switch for Concurrent Dual-Polarity Ion Manipulations in Structures for Lossless Ion Manipulations.

We describe the development of a dual-polarity traveling-wave (TW) structures for lossless ion manipulations (SLIM) ion mobility spectrometry (IMS) device capable of switching both positive and negative ions that are traveling simultaneously along the same path to different regions of the SLIM. Through simulations, the routing efficiency of the SLIM TW switch was compared to a SLIM direct-current-based (DC) switch developed previously for IMS-MS. We also report on the initial experimental evaluation of a dual-polarity SLIM platform, which uses the TW-based ion switch to achieve higher resolution multipass serpentine ultralong path with extended routing (SUPER) IMS separations. Overall, these results show that the dual-polarity TW switch is not only as effective as DC switching in terms of routing efficiency but also is agnostic to the polarity of the ions being routed.

High-Resolution Differential Ion Mobility Separations/Orbitrap Mass Spectrometry without Buffer Gas Limitations.

Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H2 that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultrahigh vacuum (UHV) needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the high vacuum (HV) region. Either greatly ameliorates the pressure increases upon He or H2 aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms.

Design and Performance of a Dual-Polarity Instrument for Ion Soft Landing.

A new apparatus for ion soft landing research was developed and is reported in this contribution. The instrument includes a dual polarity high-flux electrospray ionization (ESI) interface, a tandem electrodynamic ion funnel system, a collisional flatapole, a quadrupole mass filter, and a focusing lens. The instrument enables production of ionic layers by soft landing of mass-selected ions onto surfaces with balanced or imbalanced charge conditions using either layer-by-layer (LBL) or fast polarity switching modes. We present the first evidence of using weakly coordinating stable anions to protect the ionizing protons of soft-landed cations on the surface. The observed proton retention is particularly efficient when fast polarity switching of anions and cations is employed to deposit small quantities of ions in short deposition segments. Furthermore, we observe more efficient charge retention and better ionic complexation in a charge-balanced layer prepared by fast polarity switching deposition. These findings open up new opportunities for the fabrication of novel ionic assemblies using well-defined gaseous ions as building blocks.

Differential Ion Mobility Separations/Mass Spectrometry with High Resolution in Both Dimensions.

Strong orthogonality to mass spectrometry makes differential ion mobility spectrometry (FAIMS) a powerful tool for isomer separations. However, high FAIMS resolution has been achieved overall only with buffers rich in He or H2. That obstructed coupling to Fourier transform mass spectrometers operating under ultrahigh vacuum, but exceptional m/ z resolution and accuracy of FTMS are indispensable for frontline biological and environmental applications. By raising the waveform amplitude to 6 kV, we enabled high FAIMS resolution using solely N2 and thus straightforward integration with any MS platform: here Orbitrap XL with the electron transfer dissociation (ETD) option. The initial evaluation for complete histone tails (50 residues) with diverse post-translational modifications on alternative sites demonstrates a broad capability to separate and confidently identify the PTM localization variants in the middle-down range.

Parallel detection in a single ICR cell: Spectral averaging and improved S/N without increased acquisition time.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is well-renowned for its ultrahigh resolving power and mass measurement accuracy. As with other types of analytical instrumentation, achievable signal-to-noise ratio (S/N) is an important analytical figure of merit with FTICR-MS. S/N can be improved with higher magnetic fields and longer time-domain signal acquisition periods. However, serial signal averaging of spectra or time-domain signals acquired with multiple ion populations is most commonly used to improve S/N. On the other hand, serial acquisition and averaging of multiple scans significantly increases required data acquisition time and is often incompatible with on-line chromatographic separations. In this study, we investigated the potential for increased S/N by averaging 4 spectra that were acquired in parallel with a single ICR cell with 4 pairs of dipole detection electrodes, each with an independent pre-amplifier. This spectral averaging was achieved with no need for multiple ion accumulation events nor multiple, serial excitation and detection events. These efforts demonstrated that parallel signal acquisition with 4 detector electrode pairs produces S/N 1.76-fold higher than that from a single detection electrode pair. With parallel detection, improved S/N was achieved with no observable loss in resolving power (100,000) as compared with that from a single detection electrode pair. These results demonstrate that parallel detection of multiple induced image current signals with multiple preamplifiers exists as a viable option for future instrumentation to increase achievable S/N and sensitivity. This approach may have general utility especially where conventional serial signal averaging is impractical.

Characterization of Harmonic Signal Acquisition with Parallel Dipole and Multipole Detectors.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is a powerful instrument for the study of complex biological samples due to its high resolution and mass measurement accuracy. However, the relatively long signal acquisition periods needed to achieve high resolution can serve to limit applications of FTICR-MS. The use of multiple pairs of detector electrodes enables detection of harmonic frequencies present at integer multiples of the fundamental cyclotron frequency, and the obtained resolving power for a given acquisition period increases linearly with the order of harmonic signal. However, harmonic signal detection also increases spectral complexity and presents challenges for interpretation. In the present work, ICR cells with independent dipole and harmonic detection electrodes and preamplifiers are demonstrated. A benefit of this approach is the ability to independently acquire fundamental and multiple harmonic signals in parallel using the same ions under identical conditions, enabling direct comparison of achieved performance as parameters are varied. Spectra from harmonic signals showed generally higher resolving power than spectra acquired with fundamental signals and equal signal duration. In addition, the maximum observed signal to noise (S/N) ratio from harmonic signals exceeded that of fundamental signals by 50 to 100%. Finally, parallel detection of fundamental and harmonic signals enables deconvolution of overlapping harmonic signals since observed fundamental frequencies can be used to unambiguously calculate all possible harmonic frequencies. Thus, the present application of parallel fundamental and harmonic signal acquisition offers a general approach to improve utilization of harmonic signals to yield high-resolution spectra with decreased acquisition time. Graphical Abstract ᅟ.

Design and Performance of a Novel Interface for Combined Matrix-Assisted Laser Desorption Ionization at Elevated Pressure and Electrospray Ionization with Orbitrap Mass Spectrometry.

Matrix-Assisted Laser Desorption Ionization, MALDI, has been increasingly used in a variety of biomedical applications, including tissue imaging of clinical tissue samples, and in drug discovery and development. These studies strongly depend on the performance of the analytical instrumentation and would drastically benefit from improved sensitivity, reproducibility, and mass/spatial resolution. In this work, we report on a novel combined MALDI/ESI interface, which was coupled to different Orbitrap mass spectrometers (Elite and Q Exactive Plus) and extensively characterized with peptide and protein standards, and in tissue imaging experiments. In our approach, MALDI is performed in the elevated pressure regime (5-8 Torr) at a spatial resolution of 15-30 µm, while ESI-generated ions are injected orthogonally to the interface axis. We have found that introduction of the MALDI-generated ions into an electrodynamic dual-funnel interface results in increased sensitivity characterized by a limit of detection of ∼400 zmol, while providing a mass measurement accuracy of 1 ppm and a mass resolving power of 120 000 in analysis of protein digests. In tissue imaging experiments, the MALDI/ESI interface has been employed in experiments with rat brain sections and was shown to be capable of visualizing and spatially characterizing very low abundance analytes separated only by 20 mDa. Comparison of imaging data has revealed excellent agreement between the MALDI and histological images.

Serpentine Ultralong Path with Extended Routing (SUPER) High Resolution Traveling Wave Ion Mobility-MS using Structures for Lossless Ion Manipulations.

Ion mobility (IM) separations have a broad range of analytical applications, but insufficient resolution often limits their utility. Here, we report on ion mobility separations in a structures for lossless ion manipulations (SLIM) serpentine ultralong path with extended routing (SUPER) traveling wave (TW) ion mobility (IM) module in conjunction with mass spectrometry (MS). Ions were confined in the SLIM by rf fields in conjunction with a DC guard bias, enabling essentially lossless TW transmission over greatly extended paths. The extended routing utilized multiple passes (e.g., ∼1094 m over 81 passes through the 13.5 m serpentine path) and was facilitated by the introduction of a lossless ion switch that allowed ions to be directed to either the MS detector or for another pass through the serpentine separation region, allowing theoretically unlimited IM path lengths. The multipass SUPER IM-MS provided resolution approximately proportional to the square root of the number of passes (or total path length). More than 30-fold higher IM resolution (∼340 vs ∼10) for Agilent tuning mix m/z 622 and 922 ions was achieved for 40 passes compared to commercially available drift tube IM and other TWIM-based platforms. An initial evaluation of the isomeric sugars lacto-N-hexaose and lacto-N-neohexaose showed the isomeric structures to be baseline resolved, and a new conformational feature for lacto-N-neohexaose was revealed after 9 passes. The new SLIM SUPER high resolution TWIM platform has broad utility in conjunction with MS and is expected to enable a broad range of previously challenging or intractable separations.

Parallel Spectral Acquisition with Orthogonal ICR Cells.

FT-based high performance mass analyzers yield increased resolving power and mass measurement accuracy, yet require increased duration of signal acquisition that can limit many applications. The implementation of stronger magnetic fields, multiple detection electrodes for harmonic signal detection, and an array of multiple mass analyzers arranged along the magnetic field axis have been used to decrease required acquisition time. The results presented here show that multiple ion cyclotron resonance (ICR) mass analyzers can also be implemented orthogonal to the central magnetic field axis. The orthogonal ICR cell system presented here consisting of two cells (master and slave cells) was constructed with printed circuit boards and installed within a single superconducting magnet and vacuum system. A master cell was positioned, as is normally done with ICR cells, on the central magnetic field axis and a slave cell was located off this central axis, but directly adjacent and alongside the master cell. To achieve ion transfer between cells, ions that were initially trapped in the master cell were drifted across the magnetic field into the slave cell with application of a small DC field applied perpendicularly to the magnetic field axis. A subsequent population of ions was injected and accumulated in the master cell. Simultaneous excitation of cyclotron motion of ions in both cells was carried out; ICR signals from each cell were independently amplified and recorded in parallel. Presented here are the initial results of successful parallel spectral acquisition with this orthogonal dual ICR cell array. Graphical Abstract ᅟ.