Hyperspectral Visualization-Based Mass Spectrometry Imaging by LMJ-SSP: A Novel Strategy for Rapid Natural Product Profiling in Bacteria.

Mass spectrometry imaging (MSI) has been widely used to discover natural products (NPs) from underexplored microbiological sources. However, the technique is limited by incompatibility with complicated/uneven surface topography and labor-intensive sample preparation, as well as lengthy compound profiling procedures. Here, liquid micro-junction surface sampling probe (LMJ-SSP)-based MSI is used for rapid profiling of natural products from Gram-negative marine bacteria Pseudoalteromonas on nutrient agar media without any sample preparation. A conductance-based autosampling platform with 1 mm spatial resolution and an innovative multivariant analysis-driven method was used to create one hyperspectral image for the sampling area. NP discovery requires general spatial correlation between m/z and colony location but not highly precise spatial resolution. The hyperspectral image was used to annotate different m/z by straightforward color differences without the need to directly interrogate the spectra. To demonstrate the utility of our approach, the rapid analysis of Pseudoalteromonas rubra DSM6842, Pseudoalteromonas tunicata DSM14096, Pseudoalteromonas piscicida JCM20779, and Pseudoalteromonas elyakovii ATCC700519 cultures was directly performed on Agar. Various natural products, including prodiginine and tambjamine analogues, were quickly identified from the hyperspectral image, and the dynamic extracellular environment was shown with compound heatmaps. Hyperspectral visualization-based MSI is an efficient and sensitive strategy for direct and rapid natural product profiling from different Pseudoalteromonas strains.

Structural Identification of Eicosanoids with Ring Structures Using Differential Mobility Spectrometry-Electron Impact Excitation of Ions from Organics Mass Spectrometry.

We developed a structural identification method for eicosanoids with various ring structures using mass spectrometry. We discovered that an electron beam with a kinetic energy of 10 eV, which is in the Electron Impact Excitation of Ions from Organics (EIEIO) regime, cleaved the fatty acids enough to distinguish constitutional and cis/trans isomers. In addition to EIEIO, a comparison to authentic standards using differential mobility spectrometry (DMS) can identify diastereomers, which was difficult by EIEIO. The combination of EIEIO and DMS can provide a high-throughput method to identify complete structures of eicosanoids in mixed samples, which is not allowed with conventional analytical methods though eicosanoids are important signaling molecules in biosystems.

Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes.

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant (Kd) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of Kd value for a protein-aptamer complex. We conclude that "accurate constant via transient incomplete separation" (ACTIS) can serve as a reference method for affinity characterization of protein-aptamer binding in solution.

Localization of Multiple O-Linked Glycans Exhibited in Isomeric Glycopeptides by Hot Electron Capture Dissociation.

We describe a method to obtain a comprehensive profile of multiple glycosylations in glycopeptide isoforms. We detected a wide range of abundances of various O-glycoforms in isomeric glycopeptides using hot electron capture dissociation (hot ECD) in liquid chromatography-tandem mass spectrometry. To capture low abundant glycosylated species, a prototype of a ZenoTOF 7600 system incorporating an efficient electron-activated dissociation device to perform hot ECD was operated in targeted or scheduled high-resolution multiple reaction monitoring workflows. In addition, Zeno trap pulsing was activated to enhance the sensitivity of the time-of-flight mass spectrometer. Sixty-nine O-glycopeptides of the long O-glycopeptides in tryptic bovine fetuin digest were obtained with a relative abundance range from 100 to 0.2%, which included sialylated glycans with Neu5Ac and Neu5Gc.

Electron Impact Excitation of Ions from Organics on Singly Protonated Peptides with and without Post-Translational Modifications.

We report on the dissociation of singly protonated peptides by electrons using electron-activated dissociation (EAD), which comprises electron impact excitation of ions from organics (EIEIO), electronic-excitation dissociation (EED), and electron ionization dissociation (EIoD). Various singly protonated peptides were dissociated using a recently reported fast EAD device. The dissociation can be induced through two pathways: (i) vibrational dissociation similar to collision-activated dissociation (CAD, or collision-induced dissociation, CID) by relaxation from a molecular electronic excited state to high vibrational states; and (ii) radical-induced dissociation where molecular electronic excitation is followed by homolytic cleavage. EAD is complementary to CAD as additional molecular information can be obtained; e.g., fragile PTM moieties, such as glycosylation and sulfation, can be localized. Simultaneously, the energetic production of radical z• fragments enables Leu and Ile discrimination, like in a hot ECD process. Using the fast EAD device, LC-EIEIO-time-of-flight mass spectrometry was applied to a tryptic monoclonal antibody digest containing short singly protonated peptides.

Circular Geometry in Molecular Stream Separation to Facilitate Nonorthogonal Field-to-Flow Orientation.

Molecular stream separation (MSS) is a promising complement for continuous-flow synthesis. MSS is driven by forces exerted on molecules by a field applied at an angle to the stream-carrying flow. MSS has only been performed with a 90° field-to-flow angle because of a rectangular geometry of canonic MSS; the second-order rotational symmetry of a rectangle prevents any other angle. Here, we propose a noncanonic circular geometry for MSS, which better aligns with the polar nature of MSS and allows changing the field-to-flow. We conducted in silico and experimental studies of circular geometry for continuous-flow electrophoresis (CFE, an MSS method). We proved two advantages of circular CFE over its rectangular counterpart. First, circular CFE can support better flow and electric-field uniformity than rectangular CFE. Second, the nonorthogonal field-to-flow orientation, achievable in circular CFE, can result in a higher stream resolution than the orthogonal one. Considering that circular CFE devices are not more complex in fabrication than rectangular ones, we foresee that circular CFE will serve as a new standard and a testbed for the investigation and creation of new CFE modalities.

Discontinuously Dewetting Solvent Arrays: Droplet Formation and Poly-Synchronous Surface Extraction for Mass Spectrometry Imaging Applications.

We describe a new liquid tissue stamping method called poly-synchronous surface extraction (PSSE) that utilizes an omniphobic substrate patterned with hydrophilic surface energy traps (SETs), which when wet with a solvent form a dense microdroplet array. When contacted with a tissue sample, each droplet locally extracts analytes from the tissue surface, which subsequentially can be analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-IMS) or ambient ionization-MS techniques. Optimization of the patterned surface with six different solvents was carried out to increase the droplet density, height, and reproducibility of volume deposition. Once optimized, sister slices of a strawberry (Fragaria × ananassa) were spatially extracted using the PSSE technique and the chemical distribution of selected compounds was analyzed with both MALDI-IMS and a lower resolution but faster ambient liquid microjunction surface sampling probe (LMJ-SSP) approach. Heat maps for target analytes for the PSSE approach are compared to those produced using traditional MALDI-IMS analysis. The PSSE method aligned well with direct analysis and demonstrated the potential to increase the speed of ambient MS tissue imaging techniques by decreasing the number of steps required for sample preparation.

Electronic spectroscopy of differential mobility-selected prototropic isomers of protonated para-aminobenzoic acid.

para-Aminobenzoic acid (PABA) was electrosprayed from mixtures of protic and aprotic solvents, leading to formation of two prototropic isomers in the gas phase whose relative populations depended on the composition of the electrospray solvent. The two ion populations were separated in the gas phase using differential mobility spectrometry (DMS) within a nitrogen-only environment at atmospheric pressure. Under high-field conditions, the two prototropic isomers eluted with baseline signal separation with the N-protonated isomer having a more negative CV shift than the O-protonated isomer, in accord with previous DMS studies. The conditions most favorable for formation and separation of each tautomer were used to trap each prototropic isomer in a quadrupole ion trap for photodissociation action spectroscopy experiments. Spectral interrogation of each prototropic isomer in the UV region (3-6 eV) showed good agreement with previously recorded spectra, although a previously reported band (4.8-5.4 eV) was less intense for the O-protonated isomer in our measured spectrum. Without DMS selection, the measured spectra contained features corresponding to both protonated isomers even when solvent conditions were optimised for formation of a single isomer. Interconversion between protonated isomers within the ion trap was observed when protic ESI solvents were employed, leading to spectral cross contamination even with mobility selection. CCSD vertical excitation energies and vertical gradient (VG) Franck-Condon simulations are presented and reproduce the measured spectral features with near-quantitative agreement, providing supporting evidence for spectral assignments.

UVPD spectroscopy of differential mobility-selected prototropic isomers of protonated adenine.

Two prototropic isomers of adenine are formed in an electrospray ion source and are resolved spatially in a differential mobility spectrometer before detection in a triple quadrupole mass spectrometer. Each isomer is gated in CV space before being trapped in the linear ion trap of the modified mass spectrometer, where they are irradiated by the tuneable output of an optical parametric oscillator and undergo photodissociation to form charged fragments with m/z 119, 109, and 94. The photon-normalised intensity of each fragmentation channel is measured and the action spectra for each DMS-gated tautomer are obtained. Our analysis of the action spectra, aided by calculated vibronic spectra and thermochemical data, allow us to assign the two signals in our measured ionograms to specific tautomers of protonated adenine.

Template Instrumentation for "Accurate Constant via Transient Incomplete Separation".

Accurate Constant via Transient Incomplete Separation (ACTIS) is a new method for finding the equilibrium dissociation constant Kd of a protein-small molecule complex based on transient incomplete separation of the complex from the unbound small molecule in a capillary. This separation is caused by differential transverse diffusion of the complex and the small molecule in a pressure-driven flow. The advection-diffusion processes underlying ACTIS can be described by a system of partial differential equations allowing for a virtual ACTIS instrument to be built and ACTIS to be studied in silico. The previous in silico studies show that large variations in the fluidic system geometry do not affect the accuracy of Kd determination, thus, proving that ACTIS is conceptually accurate. The conceptual accuracy does not preclude, however, instrumental inaccuracy caused by run-to-run signal drifts. Here we report on assembling a physical ACTIS instrument with a fluidic system that mimics the virtual one and proving the absence of signal drifts. Furthermore, we confirmed method ruggedness by assembling a second ACTIS instrument and comparing the results of experiments performed with both instruments in parallel. Despite some unintentional differences between the instruments (caused by tolerances in sizes, positions, etc.) and noticeable differences in their respective separagrams, we found that the Kd values determined for identical samples with these instruments were equal. Conclusively, the fluidic system presented here can serve as a template for reliable ACTIS instrumentation.

UVPD Spectroscopy of Differential Mobility-Selected Prototropic Isomers of Rivaroxaban.

Two ion populations of protonated Rivaroxaban, [C19H18ClN3O5S + H]+, are separated under pure N2 conditions using differential mobility spectrometry prior to characterization in a hybrid triple quadrupole linear ion trap mass spectrometer. These populations are attributed to bare protonated Rivaroxaban and to a proton-bound Rivaroxaban-ammonia complex, which dissociates prior to mass-selecting the parent ion. Ultraviolet photodissociation (UVPD) and collision-induced dissociation (CID) studies indicate that both protonated Rivaroxaban ion populations are comprised of the computed global minimum prototropic isomer. Two ion populations are also observed when the collision environment is modified with 1.5% (v/v) acetonitrile. In this case, the protonated Rivaroxaban ion populations are produced by the dissociation of the ammonium complex and by the dissociation of a proton-bound Rivaroxaban-acetonitrile complex prior to mass selection. Again, both populations exhibit a similar CID behavior; however, UVPD spectra indicate that the two ion populations are associated with different prototropic isomers. The experimentally acquired spectra are compared with computed spectra and are assigned to two prototropic isomers that exhibit proton sharing between distal oxygen centers.

Assessing Accuracy of an Analytical Method In Silico: Application to "Accurate Constant via Transient Incomplete Separation" (ACTIS).

Analytical methods may not have reference standards required for testing their accuracy. We postulate that the accuracy of an analytical method can be assessed in the absence of reference standards in silico if the method is built upon deterministic processes. A deterministic process can be precisely computer-simulated, thus allowing virtual experiments with virtual reference standards. Here, we apply this in silico approach to study "Accurate Constant via Transient Incomplete Separation" (ACTIS), a method for finding the equilibrium dissociation constant (Kd) of protein-small-molecule complexes. ACTIS is based on a deterministic process: molecular diffusion of the interacting protein-small-molecule pair in a laminar pipe flow. We used COMSOL software to construct a virtual ACTIS setup with a fluidic system mimicking that of a physical ACTIS instrument. Virtual ACTIS experiments performed with virtual samples-mixtures of a protein and a small molecule with defined rate constants and, thus, Kd of their interaction-allowed us to assess ACTIS accuracy by comparing the determined Kd value to the input Kd value. Further, the influence of multiple system parameters on ACTIS accuracy was investigated. Within multifold ranges of parameter values, the values of Kd did not deviate from the input Kd values by more than a factor of 1.25, strongly suggesting that ACTIS is intrinsically accurate and that its accuracy is robust. Accordingly, further development of ACTIS can focus on achieving high reproducibility and precision. We foresee that in silico accuracy assessment, demonstrated here with ACTIS, will be applicable to other analytical methods built upon deterministic processes.

Separating Isomers, Conformers, and Analogues of Cyclosporin using Differential Mobility Spectroscopy, Mass Spectrometry, and Hydrogen-Deuterium Exchange.

Cyclosporins are an invaluable class of drug used to prevent the rejection of transplanted tissue. While the most popular drug in this group is cyclosporin A, several other analogues are available, including some enantiomeric and structurally isomeric forms. Unfortunately, the presence of such isomers can make the detection and identification of these drugs by mass spectrometry (MS) alone quite challenging. Here, we demonstrate the separation and analysis of six cyclosporin analogues using liquid chromatography (LC) and differential mobility spectroscopy (DMS) coupled to MS. Using DMS, we demonstrate the separation of three isomers: CycA and CycH (cyclosporin H), which are enantiomers, and isocyclosporin A (a structural isomer of CycA and CycH). For several of the cyclosporins, we can separate different conformers for each isomeric form. After DMS separation, tandem mass spectrometry (MS/MS) analyses of the separated isomers also distinguish these isomeric forms of cyclosporin. In addition, we have probed differences between each isomer by using gas-phase hydrogen-deuterium exchange (HDX) immediately after DMS separation, which reveals differences in the levels of intramolecular hydrogen bonding between each of the cyclosporins.

rPTMDetermine: A Fully Automated Methodology for Endogenous Tyrosine Nitration Validation, Site-Localization, and Beyond.

We present herein rPTMDetermine, an adaptive and fully automated methodology for validation of the identification of rarely occurring post-translational modifications (PTMs), using a semisupervised approach with a linear discriminant analysis (LDA) algorithm. With this strategy, verification is enhanced through similarity scoring of tandem mass spectrometry (MS/MS) comparisons between modified peptides and their unmodified analogues. We applied rPTMDetermine to (1) perform fully automated validation steps for modified peptides identified from an in silico database and (2) retrieve potential yet-to-be-identified modified peptides from raw data (that had been missed through conventional database searches). In part (1), 99 of 125 3-nitrotyrosyl-containing (nitrated) peptides obtained from a ProteinPilot search were validated and localized. Twenty nitrated peptides were falsely assigned because of incorrect monoisotopic peak assignments, leading to erroneous identification of deamidation and nitration. Five additional nitrated peptides were, however, validated after performing nonmonoisotopic peak correction. In part (2), an additional 236 unique nitrated peptides were retrieved and localized, containing 113 previously unreported nitration sites; 25 endogenous nitrated peptides with novel sites were selected and verified by comparison with synthetic analogues. In summary, we identified and confidently validated 296 unique nitrated peptides-collectively representing the largest number of endogenously identified 3-nitrotyrosyl-containing peptides from the cerebral cortex proteome of a Macaca fascicularis model of stroke. Furthermore, we harnessed the rPTMDetermine strategy to complement conventional database searching and enhance the confidence of assigning rarely occurring PTMs, while recovering many missed peptides. In a final demonstration, we successfully extended the application of rPTMDetermine to peptides featuring tryptophan oxidation.

Enhancing signal and mitigating up-front peptide fragmentation using controlled clustering by gas-phase modifiers.

Up-front CID fragmentation is a phenomenon where molecular ions are activated and fragment as they enter the atmosphere-to-vacuum region of the mass spectrometer, and consequently can complicate the mass spectra and their analysis. This phenomenon can be minimized by controlling the voltages on lens/optic elements where ions are sampled from the atmospheric region, but this approach can also have a negative effect on overall ion sensitivity. In this study, we introduce gas-phase modifiers (acetonitrile, acetone, cyclohexane, water, and methanol) to the curtain gas to mitigate up-front CID fragmentation. These modifiers cluster with incoming ions, increasing the energy barrier to fragmentation and consequently reducing the complexity of mass spectra. The clustering is monitored by differential mobility spectrometry-mass spectrometry (DMS-MS) and precursor mass spectrum-scanning. Unlike typical singly charged species, peptide ion-modifier clusters were found to survive through the atmosphere-to-vacuum interface of the mass spectrometer, showing that highly charged peptides cluster most strongly with acetonitrile and acetone. In addition, when peptides cluster with acetonitrile, they produce a large increase in signal intensity for the most highly charged and fragile ions. This results in a significant reduction, up to 90% with some modifiers, in up-front CID fragmentation for these fragile highly charged peptides, increasing the overall analytical sensitivity and decreasing the limits of detection by up to 82% depending on the analyte. The proposed technique has no significant detrimental effect on the peptide mass fingerprinting of a BSA or mAb protein digest, but it does reduce the amount of redundant and data-deficient spectra needed to produce adequate sequence coverage using information-dependent acquisition methods by ~ 40%. We propose that this technique could have a benefit in the fields of proteomics and peptidomics where up-front CID fragmentation and chemical noise routinely mask targets of biological importance. Graphical abstract.

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein-Small Molecule Complex.

Current practical methods for finding the equilibrium dissociation constant, Kd , of protein-small molecule complexes have inherent sources of inaccuracy. Introduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy. Conceptually, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing fast transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit and used to calculate a fraction of unbound small molecule, which, in turn, is used to calculate Kd . Herein the validity of ACTIS is proven theoretically, its accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become a reference-standard method for determining Kd  values of protein-small molecule complexes.

Determining molecular properties with differential mobility spectrometry and machine learning.

The fast and accurate determination of molecular properties is highly desirable for many facets of chemical research, particularly in drug discovery where pre-clinical assays play an important role in paring down large sets of drug candidates. Here, we present the use of supervised machine learning to treat differential mobility spectrometry - mass spectrometry data for ten topological classes of drug candidates. We demonstrate that the gas-phase clustering behavior probed in our experiments can be used to predict the candidates' condensed phase molecular properties, such as cell permeability, solubility, polar surface area, and water/octanol distribution coefficient. All of these measurements are performed in minutes and require mere nanograms of each drug examined. Moreover, by tuning gas temperature within the differential mobility spectrometer, one can fine tune the extent of ion-solvent clustering to separate subtly different molecular geometries and to discriminate molecules of very similar physicochemical properties.

Quantitative structural multiclass lipidomics using differential mobility: electron impact excitation of ions from organics (EIEIO) mass spectrometry.

We report a method for comprehensive structural characterization of lipids in animal tissues using a combination of differential ion mobility spectrometry (DMS) with electron-impact excitation of ions from organics (EIEIO) mass spectrometry. Singly charged lipid ions in protonated or sodiated forms were dissociated by an electron beam having a kinetic energy of 10 eV in a branched radio-frequency ion trap. We established a comprehensive set of diagnostics to characterize the structures of glycerophospholipids, sphingolipids, and acylglycerols, including glycosylated, plasmalogen, and ester forms. This EIEIO mass spectrometer was combined with DMS as a separation tool to analyze complex lipid extracts. Deuterated quantitative standards, which were added during extraction, allowed for the quantitative analysis of the lipid molecular species in various lipid classes. We applied this technique to the total lipids extracted from porcine brain, and we structurally characterized over 300 lipids (with the exception of cis/trans double-bond isomerism in the acyl chains). The structural dataset of the lipidomes, whose regioisomers were distinguished, exhibit a uniquely defined distribution of acyl chains within each lipid class; that is, sn-1 and sn-2 in the cases of glycerophospholipids or sn-2 and (sn-1, sn-3) in the cases of triacylglycerols.

Fast quantitation of opioid isomers in human plasma by differential mobility spectrometry/mass spectrometry via SPME/open-port probe sampling interface.

Mass spectrometry (MS) based quantitative approaches typically require a thorough sample clean-up and a decent chromatographic step in order to achieve needed figures of merit. However, in most cases, such processes are not optimal for urgent assessments and high-throughput determinations. The direct coupling of solid phase microextraction (SPME) to MS has shown great potential to shorten the total sample analysis time of complex matrices, as well as to diminish potential matrix effects and instrument contamination. In this study, we demonstrate the use of the open-port probe (OPP) as a direct and robust sampling interface to couple biocompatible-SPME (Bio-SPME) fibres to MS for the rapid quantitation of opioid isomers (i.e. codeine and hydrocodone) in human plasma. In place of chromatography, a differential mobility spectrometry (DMS) device was implemented to provide the essential selectivity required to quantify these constitutional isomers. Taking advantage of the simplified sample preparation process based on Bio-SPME and the fast separation with DMS-MS coupling via OPP, a high-throughput assay (10-15 s per sample) with limits of detection in the sub-ng/mL range was developed. Succinctly, we demonstrated that by tuning adequate ion mobility separation conditions, SPME-OPP-MS can be employed to quantify non-resolved compounds or those otherwise hindered by co-extracted isobaric interferences without further need of coupling to other separation platforms.

Distinguishing Cis and Trans Isomers in Intact Complex Lipids Using Electron Impact Excitation of Ions from Organics Mass Spectrometry.

We present a mass spectrometry-based method for the identification of cis and trans double-bond isomers within intact complex lipid mixtures using electron impact excitation of ions from organics (EIEIO) mass spectrometry. EIEIO involves irradiating singly charged lipid ions with electrons having kinetic energies of 5-16 eV. The resulting EIEIO spectra can be used to discern cis and trans double-bond isomers by virtue of the differences in the fragmentation patterns at the carbon-carbon single bonds neighboring the double bonds. For trans double bonds, these characteristic fragments include unique closed-shell and open-shell (radical) products. To explain this fragmentation pattern in trans double bonds, we have proposed a reaction mechanism involving excitation of the double bond's π electrons followed by hydrogen atom rearrangement. Several lipid standards were analyzed using the EIEIO method, including mixtures of these standards. Prior to EIEIO, some of the lipid species in these mixtures were separated from their isomeric forms by using differential mobility spectrometry (DMS). For example, mixed cis and trans forms of triacylglycerols and phosphatidylcholines were identified by this DMS-EIEIO workflow. With this combined gas-phase separation and subsequent fragmentation, we could eliminate the need for authentic standards for identification. When DMS could not separate cis and trans isomers completely, as was the case with sphingomyelins, we relied upon the aforementioned diagnostic EIEIO fragment peaks to determine the relative contribution of the trans double-bond isomer in the mixed samples. We also applied the DMS-EIEIO methodology to natural samples extracted from a ruminant (bovine), which serve as common origins of trans fatty acids in a typical Western diet that includes dairy products.