Metabolomic and Lipidomic Profiling of Bacillus Using Two-Dimensional Tandem Mass Spectrometry.

Lipidomic and metabolomic profiles of sporulated and vegetative Bacillus subtilis and Bacillus thuringiensis from irradiated lysates were recorded using a quadrupole ion trap mass spectrometer modified to perform two-dimensional tandem mass spectrometry (2D MS/MS). The 2D MS/MS data domains, acquired using a 1.2 s scan of negative ions generated by nanoelectrospray ionization of microwave irradiated spores, showed the presence of dipicolinic acid (DPA) as well as various lipids. Aside from microwave radiation to extract DPA and lipids from spores, sample preparation was minimal. Characteristic lipid and metabolic profiles were observed using 107─108 cells of the two Bacillus species. Major features of the lipid profiles observed for the vegetative states included sets of phosphatidylglycerol (PG) lipids. Product ion spectra were extracted from the 2D MS/MS data, and they provided structural information on the fatty acid components of the PG lipids. The study demonstrates the flexibility, speed, and informative power of metabolomic and lipidomic fingerprinting for identifying the presence of spore-forming biological agents using 2D MS/MS as a rapid profiling screening method.

Bacterial growth monitored by two-dimensional tandem mass spectrometry.

The growth of the bacterium E. coli was monitored by targeting the phospholipid constituents through desorption electrospray ionization and characterizing individual sets of isomers by recording the full 2D MS/MS data domain in a single scan of a modified quadrupole ion trap mass spectrometer. The experiments tested the applicability of the new instrumental capabilities which include sample interrogation at the molecular level for multiple components at speeds of <10 seconds/sample. The major lipids observed were phosphatidylethanolamines and phosphatidylglycerols and the growth experiment showed fatty acid chain modification from alkene to cyclopropyl groups over time. Notably, these novel MS scans were also performed using desorption electrospray ionization (DESI) to quickly sample complex mixtures without pre-separation. This demonstration experiment has implications for the value of ambient ionization mass spectrometry for monitoring biological systems on physiologically relevant timescales.

Novel Ion Trap Scan Modes to Develop Criteria for On-Site Detection of Sulfonamide Antibiotics.

Advances in ambient ionization techniques have facilitated the direct analysis of complex mixtures without sample preparation. Significant attention has been given to innovating ionization methods so that multiple options are now available, allowing for ready selection of the best methods for particular analyte classes. These ambient techniques are commonly implemented on benchtop systems, but their potential application with miniature mass spectrometers for in situ measurements is even more powerful. These applications require that attention be paid to tailoring the mass spectrometric methodology for the on-site operation. In this study, combinations of scan modes are employed to efficiently determine what tandem mass spectrometry (MS/MS) operations are most useful for detecting sulfonamides using a miniature ion trap after ionization. First, mixtures of representative sulfonamide antibiotics were interrogated using a 2D MS/MS scan on a benchtop ion trap in order to determine which class-specific fragments (ionic or neutral) are shared between the sulfonamides and thus have diagnostic value. Then, three less-used combination scans were recorded: (i) a simultaneous precursor ion scan was used to detect both analytes and an internal standard in a single ion injection event to optimize quantitative performance; (ii) a simultaneous precursor/neutral loss scan was used to improve detection limits; and finally, (iii) the simultaneous precursor/neutral loss scan was implemented in a miniature mass spectrometer and representative sulfonamides were detected at concentrations as low as 100 ng/mL by nano-electrospray and 0.5 ng absolute by paper spray ionization, although improvements in the stability of the home-built instrumentation are needed to further optimize performance.

Immediate and sensitive detection of sporulated Bacillus subtilis by microwave release and tandem mass spectrometry of dipicolinic acid.

Spore lysis of Bacillus species is achieved by brief (1 min) microwave irradiation while tandem mass spectrometry (MS/MS) allows identification of the characteristic spore marker, dipicolinic acid. This rapid measurement, made on 105-108 spores, has significant implications for biothreat recognition.

2D MS/MS Spectra Recorded in the Time Domain Using Repetitive Frequency Sweeps in Linear Quadrupole Ion Traps.

Ion trap mass spectrometers have emerged as powerful on-site analytical platforms, in spite of limited mass resolution, due to their compatibility with ambient ionization methods and ready implementation of tandem mass spectrometry (MS/MS). When operated at constant trapping voltage, ions can be activated at their secular frequencies and all MS/MS experiments can be performed, including the two-dimensional tandem mass scan (2D MS/MS scan) in which all precursor ions and their subsequent product ions are both identified and correlated. In the new method of performing this 2D MS/MS experiment presented here, the precursor ions are excited by a nonlinear (inverse Mathieu q) frequency sweep while the resulting product ions are identified by their ejection time within a repeating orthogonally applied nonlinear (inverse Mathieu q) frequency sweep. This resulting compact representation contains the total fragmentation behavior of a collection of ionized compounds and captures detailed chemical information efficiently (typically in 1 s). The approach is implemented using a simple single mass analyzer instrument. This methodology was tested on three different multicomponent mixtures: drugs of abuse, peptides, and fentanyl analogs. The data are compared with those obtained by more common MS/MS scan methods.

Quinoline Triradicals: A Reactivity Study.

The gas-phase reactivities of several protonated quinoline-based σ-type (carbon-centered) mono-, bi-, and triradicals toward dimethyl disulfide (DMDS) were studied by using a linear quadrupole ion trap mass spectrometer. The mono- and biradicals produce abundant thiomethyl abstraction products and small amounts of DMDS radical cation, as expected. Surprisingly, all triradicals produce very abundant DMDS radical cations. A single-step mechanism involving electron transfer from DMDS to the triradicals is highly unlikely because the (experimental) adiabatic ionization energy of DMDS is almost 3 eV greater than the (calculated) adiabatic electron affinities of the triradicals. The unexpected reactivity can be explained based on an unprecedented two-step mechanism wherein the protonated triradical first transfers a proton to DMDS, which is then followed by hydrogen atom abstraction from the protonated sulfur atom in DMDS by the radical site in the benzene ring of the deprotonated triradical to generate the conventional DMDS radical cation and a neutral biradical. Quantum chemical calculations as well as examination of deuterated and methylated triradicals provide support for this mechanism. The proton affinities of the neutral triradicals (and DMDS) influence the first step of the reaction while the vertical electron affinities and spin-spin coupling of the neutral triradicals influence the second step. The calculated total reaction exothermicities for the triradicals studied range from 27.6 up to 29.9 kcal mol-1.

Two-Dimensional Tandem Mass Spectrometry in a Single Scan on a Linear Quadrupole Ion Trap.

A two-dimensional tandem mass spectrometry (2D MS/MS) scan has been developed for the linear quadrupole ion trap. Precursor ions are mass-selectively excited using a nonlinear ac frequency sweep at constant rf voltage, while simultaneously, all product ions of the excited precursor ions are ejected from the ion trap using a broad-band waveform. The fragmentation time of the precursor ions correlates with the precursor m/z value (the first mass dimension) and also with the ejection time of the product ions, allowing the correlation between precursor and product ions. Additionally, the second mass dimension (product ions' m/z values) is recovered through fast Fourier transform of each mass spectral peak, revealing either intentionally introduced "frequency tags" or the product ion micropacket frequencies, both of which can be converted to product ion m/z through the classical Mathieu parameters, thereby revealing a product ion mass spectrum for every precursor ion without prior isolation. We demonstrate the utility of this method for analyzing a broad range of structurally related precursor ions, including chemical warfare agent simulants, fentanyls and other opioids, amphetamines, cathinones, antihistamines, and tetracyclic antidepressants.

Spin-Spin Coupling Between Two meta-Benzyne Moieties In a Quinolinium Tetraradical Cation Increases Their Reactivities.

The reactivity of a carbon-centered σ,σ,σ,σ-type singlet-ground-state tetraradical containing two meta-benzyne moieties was examined in the gas phase. Surprisingly, the tetraradical showed higher reactivity than its individual meta-benzyne counterparts. The reactivity of meta-benzynes is controlled by their (calculated) distortion energy ΔE2.3 , singlet-triplet spitting ΔES-T , and electron affinity (EA2.3 ) of the meta-benzyne moiety at the transition state geometry for hydrogen-atom abstraction reactions. The addition of a second meta-benzyne moiety to a meta-benzyne does not significantly change EA2.3 . However, ΔE2.3 is substantially decreased for both meta-benzyne moieties in the tetraradical, and this explains their higher reactivities. The decrease in ΔE2.3 for each meta-benzyne moiety in the tetraradical is rationalized by stabilizing spin-spin coupling between one radical site in each meta-benzyne moiety. Therefore, spin-spin coupling between the meta-benzyne moieties in this tetraradical increases its reactivity, whereas spin-spin coupling within each meta-benzyne moiety decreases its reactivity.

Logical MS/MS scans: a new set of operations for tandem mass spectrometry.

A new set of operations for tandem mass spectrometry in a linear ion trap is described. Logical MS/MS operations categorize compounds in mixtures based on characteristic structural features as revealed by MS/MS behavior recorded in multiple fragmentation pathways. This approach is a conceptual extension of tandem mass spectrometry in which interrogation of the full data domain is performed by simultaneous implementation of precursor and neutral loss scans. This process can be thought of as moving through the 2D MS/MS data domain along multiple scan lines simultaneously, which allows experiments that explore the 2D data domain of MS/MS to be couched in terms of logical operations, AND, NAND (not and), OR (inclusive or), XOR (exclusive or), NOT, etc. Examples of particular logical conditions include all precursor ions that fragment to both of two selected product ions (logical AND), or all precursor ions that do not produce a specified fragment ion (logical NOT). These and other operational modes (TRUE/FALSE, XOR, OR, etc.) complement and extend the existing set of conventional MS/MS scans, namely product scans, precursor scans, and neutral loss scans. We describe the implementation of logical MS/MS scans on a commercial linear ion trap mass spectrometer using simple mixtures of amphetamines and fentanyl analogues and argue their utility for complex mixture analysis.

Simultaneous and Sequential MS/MS Scan Combinations and Permutations in a Linear Quadrupole Ion Trap.

Methods of performing precursor ion scans as well as neutral loss scans in a single linear quadrupole ion trap have recently been described. In this paper we report methodology for performing permutations of MS/MS scan modes, that is, ordered combinations of precursor, product, and neutral loss scans following a single ion injection event. Only particular permutations are allowed; the sequences demonstrated here are (1) multiple precursor ion scans, (2) precursor ion scans followed by a single neutral loss scan, (3) precursor ion scans followed by product ion scans, and (4) segmented neutral loss scans. (5) The common product ion scan can be performed earlier in these sequences, under certain conditions. Simultaneous scans can also be performed. These include multiple precursor ion scans, precursor ion scans with an accompanying neutral loss scan, and multiple neutral loss scans. We argue that the new capability to perform complex simultaneous and sequential MSn operations on single ion populations represents a significant step in increasing the selectivity of mass spectrometry.

Triple Resonance Methods to Improve Performance of Ion Trap Precursor and Neutral Loss Scans.

Two experiments are described that extend the capabilities of quadrupole ion trap mass spectrometers operated in the precursor and neutral loss scan mode. The first experiment, a triple resonance precursor ion scan, is used to enhance sensitivity, selectivity, and molecular coverage. This method augments the ion trap precursor ion scan with the application of a second excitation frequency to selectively activate first-generation (MS2) product ions as they are formed and produce second-generation (MS3) product ions, which are then mass-selectively ejected with a third auxiliary signal and detected. This single mass analyzer experiment can be equated to performing the sequential precursor ion scan in a multiple analyzer system (Anal. Chem. 1990, 62 (17), 1809-1818). The second capability demonstrated is "frequency tagging", a method used to differentiate between ions ejected due to inherent instability under given trapping conditions, which causes artifacts during these scans, and ions that are resonantly ejected by the product ion ejection frequency. Beat frequencies are used to modulate resonance ejection peaks but conveniently do not modulate boundary ejection peaks. Frequency tagging provides a mechanism to identify the artifact peaks that are a consequence of operating at a high trapping voltage (i.e., low mass cutoff) for optimal precursor/product ion selectivity. The experiment is demonstrated for precursor and for neutral loss scans.

Selective Gas-Phase Mass Tagging via Ion/Molecule Reactions Combined with Single Analyzer Neutral Loss Scans to Probe Pharmaceutical Mixtures.

We have demonstrated the use of a simple single ion trap mass spectrometer to identify classes of compounds as well as individual components in complex mixtures. First, a neutral reagent was used to mass tag oxygen-containing analytes using a gas-phase ion/molecule reaction. Then, a neutral loss scan was used to indicate the carboxylic acids. The lack of unit mass selectivity in the neutral loss scan required subsequent product ion scans to confirm the presence and identity of the individual carboxylic acids. The neutral loss scan technique reduced the number of data-dependent MS/MS scans required to confirm identification of signals as protonated carboxylic acids. The method was demonstrated on neat mixtures of standard carboxylic acids as well as on solutions of relevant pharmaceutical tablets and may be generalizable to other ion/molecule reactions.

Implementation of Precursor and Neutral Loss Scans on a Miniature Ion Trap Mass Spectrometer and Performance Comparison to a Benchtop Linear Ion Trap.

Implementation of orthogonal double resonance precursor and neutral loss scans on the Mini 12 miniature rectilinear ion trap mass spectrometer is described, and performance is compared to that of a commercial Thermo linear trap quadropole (LTQ) linear ion trap. The ac frequency scan version of the technique at constant rf voltage is used here because it is operationally much simpler to implement. Remarkably, the Mini 12 shows up to two orders of magnitude higher sensitivity compared to that of the LTQ. Resolution on the LTQ is better than unit at scan speeds of ~ 400 Th/s, whereas peak widths on the Mini 12, on average, range from 0.5 to 2.0 Th full width at half maximum and depend heavily on the precursor ion Mathieu q parameter as well as the pump down time that precedes the mass scan. Both sensitivity and resolution are maximized under higher pressure conditions (short pump down time) on the Mini 12. The effective mass range of the product ion ejection waveform was found to be 5.8 Th on the Mini 12 in the precursor ion scan mode vs. that of 3.9 Th on the LTQ. In the neutral loss scan mode, the product ion selectivity was between 8 and 11 Th on the Mini 12 and between 7 and 8 Th on the LTQ. The effects of nonlinear resonance lines on the Mini 12 were also explored. Graphical Abstract ᅟ.

Precursor and Neutral Loss Scans in an RF Scanning Linear Quadrupole Ion Trap.

Methodology for performing precursor and neutral loss scans in an RF scanning linear quadrupole ion trap is described and compared to the unconventional ac frequency scan technique. In the RF scanning variant, precursor ions are mass selectively excited by a fixed frequency resonance excitation signal at low Mathieu q while the RF amplitude is ramped linearly to pass ions through the point of excitation such that the excited ion's m/z varies linearly with time. Ironically, a nonlinear ac frequency scan is still required for ejection of the product ions since their frequencies vary nonlinearly with the linearly varying RF amplitude. In the case of the precursor scan, the ejection frequency must be scanned so that it is fixed on a product ion m/z throughout the RF scan, whereas in the neutral loss scan, it must be scanned to maintain a constant mass offset from the excited precursor ions. Both simultaneous and sequential permutation scans are possible; only the former are demonstrated here. The scans described are performed on a variety of samples using different ionization sources: protonated amphetamine ions generated by nanoelectrospray ionization (nESI), explosives ionized by low-temperature plasma (LTP), and chemical warfare agent simulants sampled from a surface and analyzed with swab touch spray (TS). We lastly conclude that the ac frequency scan variant of these MS/MS scans is preferred due to electronic simplicity. In an accompanying manuscript, we thus describe the implementation of orthogonal double resonance precursor and neutral loss scans on the Mini 12 using constant RF voltage. Graphical Abstract ᅟ.