Novel Hybrid Quadrupole-Multireflecting Time-of-Flight Mass Spectrometry System.

A novel mass spectrometry system is described here comprising a quadrupole-multireflecting time-of-flight design. The new multireflecting time-of-flight analyzer has an effective path length of 48 m and employs planar, gridless ion mirrors providing fourth-order energy focusing resulting in resolving power over 200 000 fwhm and sub-ppm mass accuracy. We show how these attributes are maintained with relatively fast acquisition speeds, setting the system apart from other high resolution mass spectrometers. We have integrated this new system into both liquid chromatography-mass spectrometry and mass spectrometry imaging workflows to demonstrate how the instrument characteristics are of benefit to these applications.

Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved "noise" centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion-ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and "mobility fractions", that is, extracted mass spectra for segments of the 2D mobiligram (m/z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.

Surface-induced dissociation of protein complexes on a cyclic ion mobility spectrometer.

We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20-50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after ∼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared 'native' in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.

A Cyclic Ion Mobility-Mass Spectrometry System.

Improvements in the performance and availability of commercial instrumentation have made ion mobility-mass spectrometry (IM-MS) an increasingly popular approach for the structural analysis of ionic species as well as for separation of complex mixtures. Here, a new research instrument is presented which enables complex experiments, extending the current scope of IM technology. The instrument is based on a Waters SYNAPT G2-S i IM-MS platform, with the IM separation region modified to accept a cyclic ion mobility (cIM) device. The cIM region consists of a 98 cm path length, closed-loop traveling wave (TW)-enabled IM separator positioned orthogonally to the main ion optical axis. A key part of this geometry and its flexibility is the interface between the ion optical axis and the cIM, where a planar array of electrodes provides control over the TW direction and subsequent ion motion. On either side of the array, there are ion guides used for injection, ejection, storage, and activation of ions. In addition to single and multipass separations around the cIM, providing selectable mobility resolution, the instrument design and control software enable a range of "multifunction" experiments such as mobility selection, activation, storage, IMS n, and importantly custom combinations of these functions. Here, the design and performance of the cIM-MS instrument is highlighted, with a mobility resolving power of approximately 750 demonstrated for 100 passes around the cIM device using a reverse sequence peptide pair. The multifunction capabilities are demonstrated through analysis of three isomeric pentasaccharide species and the small protein ubiquitin.

Cyclic Ion Mobility Mass Spectrometry Distinguishes Anomers and Open-Ring Forms of Pentasaccharides.

There is increasing biopharmaceutical interest in oligosaccharides and glycosylation. A key requirement for these sample types is the ability to characterize the chain length, branching, type of monomers, and importantly stereochemistry and anomeric configuration. Herein, we showcase the multi-function capability of a cyclic ion mobility (cIM) separator embedded in a quadrupole/time-of-flight mass spectrometer (Q-ToF MS). The instrument design enables selective activation of mobility-separated precursors followed by cIM separation of product ions, an approach analogous to MSn. Using high cIM resolution, we demonstrate the separation of three isomeric pentasaccharides and, moreover, that three components are present for each compound. We show that structural differences between product ions reflect the precursor differences in some cases but not others. These findings are corroborated by a heavy oxygen labelling approach. Using this methodology, the identity of fragment ions may be assigned. This enables us to postulate that the two main components observed for each pentasaccharide are anomeric forms. The remaining low abundance component is assigned as an open-ring form.

Scanning Quadrupole Data-Independent Acquisition, Part A: Qualitative and Quantitative Characterization.

A novel data-independent acquisition (DIA) method incorporating a scanning quadrupole in front of a collision cell and orthogonal acceleration time-of-flight mass analyzer is described. The method has been characterized for the qualitative and quantitative label-free proteomic analysis of complex biological samples. The principle of the scanning quadrupole DIA method is discussed, and analytical instrument characteristics, such as the quadrupole transmission width, scan/integration time, and chromatographic separation, have been optimized in relation to sample complexity for a number of different model proteomes of varying complexity and dynamic range including human plasma, cell lines, and bacteria. In addition, the technological merits over existing DIA approaches are described and contrasted. The qualitative and semiquantitative performance of the method is illustrated for the analysis of relatively simple protein digest mixtures and a well-characterized human cell line sample using untargeted and targeted search strategies. Finally, the results from a human cell line were compared against publicly available data that used similar chromatographic conditions but were acquired with DDA technology and alternative mass analyzer systems. Qualitative comparison showed excellent concordance of results with >90% overlap of the detected proteins.

Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs.

Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive "aspergillosis" caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.

Lipid profiling of complex biological mixtures by liquid chromatography/mass spectrometry using a novel scanning quadrupole data-independent acquisition strategy.

RATIONALE: A novel data-independent acquisition method is detailed that incorporates a scanning quadrupole in front of an orthogonal acceleration time-of-flight (TOF) mass analyser. This approach is described and the attributes are compared and contrasted to other DIA approaches. METHODS: Specific application of the method to both targeted and untargeted lipidomic identification strategies is discussed, with data from both shotgun and LC separated lipidomics experiments presented. RESULTS: The benefits of the fast quadrupole scanning technique are highlighted, and include improvements in speed and specificity for complex mixtures providing high quality qualitative and quantitative data. CONCLUSIONS: In particular the high specificity afforded by the scanning quadrupole improves qualitative information for lipid identification.

Ion mobility tandem mass spectrometry enhances performance of bottom-up proteomics.

One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A.

Quadrupole-time-of-flight mass spectrometer modified for higher-energy dissociation reduces protein assemblies to peptide fragments.

The gas-phase dissociation of protein assemblies is becoming crucial for the application of mass spectrometry to structural biology. However certain aspects of the dissociation mechanism remain elusive. Moreover, many protein complexes resist dissociation at the energies accessible with current instrumentation. Here we report new insights into the collision-induced dissociation mechanism of protein assemblies. By holding activation energy constant and varying the charge state of the precursor ion, we show that the total charge of the precursor ion dramatically influences the internal energy required to dissociate monomers from the protein assembly. Furthermore, we have developed a modified quadrupole-time-of-flight instrument capable of accessing activation energies higher than previously possible. Under these conditions, protein assemblies eject subunits with excess internal energy that subsequently fragment into peptides. Together, these data indicate that the non-covalent dissociation is limited by the amount of charge available and not merely the activation energy, and they project the exciting possibility of extracting sequence information directly from intact protein complexes in the gas phase.

Applications of a travelling wave-based radio-frequency-only stacked ring ion guide.

The use of radio-frequency (RF)-only ion guides for efficient transport of ions through regions of a mass spectrometer where the background gas pressure is relatively high is widespread in present instrumentation. Whilst multiple collisions between ions and the background gas can be beneficial, for example in inducing fragmentation and/or decreasing the spread in ion energies, the resultant reduction of ion axial velocity can be detrimental in modes of operation where a rapidly changing influx of ions to the gas-filled ion guide needs to be reproduced at the exit. In general, the RF-only ion guides presently in use are based on multipole rod sets. Here we report investigations into a new mode of ion propulsion within an RF ion guide based on a stack of ring electrodes. Ion propulsion is produced by superimposing a voltage pulse on the confining RF of an electrode and then moving the pulse to an adjacent electrode and so on along the guide to provide a travelling voltage wave on which the ions can surf. Through appropriate choice of the travelling wave pulse height, velocity and gas pressure it will be shown that the stacked ring ion guide with the travelling wave is effective as a collision cell in a tandem mass spectrometer where fast mass scanning or switching is required, as an ion mobility separator at pressures around 0.2 mbar, as an ion delivery device for enhancement of duty cycle on an orthogonal acceleration time-of-flight (oa-TOF) mass analyser, and as an ion fragmentation device at higher wave velocities.

Ion dispersion near parallel wire grids in orthogonal acceleration time-of-flight mass spectrometry: predicting the effect of the approach angle on resolution.

Ions experience small deflections in the vicinity of grids in accelerators and ion mirrors in time-of-flight (TOF) mass spectrometers. Recent experiments with an orthogonal acceleration (oa) TOF instrument have verified that the effect can significantly degrade resolution when ions approach grids at an angle deviating from 90 degrees. The phenomenon becomes significant only when ions have components of velocity at right angles to the wires of the grids. A model is presented in this study to predict this phenomenon for parallel wire grids. The fractional energy spread of ions (calculated in the static TOF-spectrometer frame of reference) scales directly with the approach angle of ions to the grid (as measured from normal approach). The energy spread also scales with the range of angles that is a consequence of the focusing effect in each gap between the wires of the grid. The equations imply that closely spaced parallel wire grids are best for deployment in oa-TOF systems where non-zero approach angles are unavoidable. Such grids are relatively impractical to manufacture and support but rectangular repeat cell grids with relatively few wires at right angles to the source axis are shown experimentally to introduce minimal energy spread. When these grids are rotated by 90 degrees, the resolution measured in a Q-TOF spectrometer is degraded in approximate agreement with the parallel wire model. A practical implication of this work is that grid transmissions in oa-TOF systems may be significantly increased without loss of resolution. Improvements of approximately 200% (V-mode) and approximately 400% (W-mode) in ion transmission were obtained in this study without compromising resolution. This was achieved with approximately 73% transmission grids and greater potential improvements in transmission are being realised since this study with approximately 89% transmission grids having similar geometry.