Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.

Installation protocol for charge transfer dissociation mass spectrometry on ion trapping mass spectrometers.

RATIONALE: Charge transfer dissociation (CTD) is a novel fragmentation technique that demonstrates enhanced structural characterization for a wide variety of molecules compared to standard fragmentation techniques like collision-induced dissociation (CID). Alternative fragmentation techniques, such as electron transfer dissociation, electron capture dissociation, and ultraviolet photodissociation, also overcome many of the shortfalls of CID, but none of them are a silver bullet that can adequately characterize a wide variety of structures and charge states of target compounds. Given the diversity of structural classes and their occasional obstinance towards certain activation techniques, alternative fragmentation techniques are required that rely on novel or alternative modes of activation. METHODS: Herein, we present a step-by-step protocol for the installation of CTD on a quadrupole ion trap mass spectrometer and best practices for optimizing the signal-to-noise ratio and acquisition times for CTD mass spectra. RESULTS: In addition to two CTD installations in the Jackson laboratory, CTD has also been installed, and is currently in operation, on two 3D ion trap mass spectrometers in France: one in the laboratory of Dr. David Ropartz and Dr. Hélène Rogneaux at INRAE in Nantes, and the other in the laboratory of Dr. Jean-Yves Salpin at Université d'Évry Val-d'Essonne, part of the Paris-Saclay University system. CONCLUSIONS: Here, we provide a visual protocol to help others accomplish the instrument modification.

Conotoxin Prediction: New Features to Increase Prediction Accuracy.

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

-Omics potential of in vitro skin models for radiation exposure.

There is a growing need to uncover biomarkers of ionizing radiation exposure that leads to a better understanding of how exposures take place, including dose type, rate, and time since exposure. As one of the first organs to be exposed to external sources of ionizing radiation, skin is uniquely positioned in terms of model systems for radiation exposure study. The simultaneous evolution of both MS-based -omics studies, as well as in vitro 3D skin models, has created the ability to develop a far more holistic understanding of how ionizing radiation affects the many interconnected biomolecular processes that occur in human skin. However, there are a limited number of studies describing the biomolecular consequences of low-dose ionizing radiation to the skin. This review will seek to explore the current state-of-the-art technology in terms of in vitro 3D skin models, as well as track the trajectory of MS-based -omics techniques and their application to ionizing radiation research, specifically, the search for biomarkers within the low-dose range.

Structural Characterization of Natural and Synthetic Macrocycles Using Charge-Transfer Dissociation Mass Spectrometry.

Research in natural products (NPs) has gained interest as drug developers turn to nature to combat problems with drug resistance, drug delivery, and emerging diseases. Whereas NPs offer a tantalizing source of new pharmacologically active compounds, their structural complexity presents a challenge for analytical characterization and organic synthesis. Of particular concern is the characterization of cyclic-, polycyclic-, or macrocyclic compounds. One example of endogenous compounds as inspiration for NP development are cobalamins, like vitamin B12. An example of exogenous NPs is the class of macrolides that includes erythromycin. Both classes of macrocycles feature analogues with a range of modifications on their macrocyclic cores, but because of their cyclic nature, they are generally resistant to fragmentation by collision-induced dissociation (CID). In the present work, charge-transfer dissociation (CTD) was employed, with or without supplemental collisional activation, to produce radical-driven, high-energy fragmentation products of different macrocyclic precursors. With the assistance of collisional activation of CTnoD products, CTD frequently cleaved two covalent bonds within the macrocycle cores to reveal rich, informative spectra that helped identify sites of modification and resolve structural analogues. In a third example of macrocycle fragmentation, CTD enabled an impurity in a biological sample to be characterized as a cyclic polymer of nylon-6,6. In each example, CTD spectra are starkly different from CID and are highly reminiscent of other high-energy fragmentation techniques like extreme ultraviolet dissociative photoionization (XUV-DPI) and electron ionization-induced dissociation (EID). The results indicate that CTD-MS is a useful tool for the characterization of natural and synthetic macrocycles.

Ultra-high-performance liquid chromatography charge transfer dissociation mass spectrometry (UHPLC-CTD-MS) as a tool for analyzing the structural heterogeneity in carrageenan oligosaccharides.

Ultra-high-performance liquid chromatography (UHPLC) with charge transfer dissociation mass spectrometry (CTD-MS) is presented for the analysis of a mixture of complex sulfated oligosaccharides. The mixture contained kappa (κ), iota (ι), and lambda (λ) carrageenans that contain anhydro bridges, different degrees of sulfation ranging from one to three per dimer, different positioning of the sulfate groups along the backbone, and varying degrees of polymerization (DP) between 4 and 12. Optimization studies using standard mixtures of carrageenans helped establish the optimal conditions for online UHPLC-CTD-MS/MS analysis. Optimization included (1) UHPLC conditions; (2) ion source conditions, such as the capillary voltage, drying gas and nebulizing gas temperature, and flow rate; and (3) CTD-MS conditions, including data-dependent CTD-MS. The UHPLC-CTD results were contrasted with UHPLC-CID results of the same mixture on the same instrument. Whereas CID tends to produce B/Y and C/Z ions with many neutral losses, CTD produced more abundant A/X ions and less abundant neutral losses, which enabled more confident structural detail. The results demonstrate that He-CTD is compatible with the timescale of UHPLC and provides more structural information about carrageenans compared to state-of-the-art methods like UHPLC-CID analysis.

Charge transfer dissociation of a branched glycan with alkali and alkaline earth metal adducts.

Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He-CTD) and low-energy collision-induced dissociation (LE-CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H+ , Na+ , K+ , Ca2+ , and Mg2+ . Regardless of the metal adduct, He-CTD generated abundant and numerous glycosidic and cross-ring cleavages that were structurally informative and able to identify the 1,4-linkage and 1,6-branching patterns. In contrast, the LE-CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE-CID of [M + K + H]2+ and [M + Na]+ precursors generated a few cross-ring cleavages, but they were not sufficient to identify the 1,4-linkage and 1,6-branching pattern of the XXXG xyloglucan. He-CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE-CID and He-CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H+ from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports for magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He-CTD, the [M + Mg]2+ precursor generated more singly charged product ions than [M + Ca]2+ , either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He-CTD of the [M + 2Na]2+ and the [M + 2 K]2+ precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross-ring cleavages observed.

Quantitative Assessment of Six Different Reagent Gases for Charge Transfer Dissociation (CTD) of Biological Ions.

Charge transfer dissociation mass spectrometry (CTD-MS) has been shown to induce high energy fragmentation of biological ions in the gas phase and provide fragmentation spectra similar to extreme ultraviolet photodissociation (XUVPD). To date, CTD has typically employed helium cations with kinetic energies between 4-10 keV to initiate radical-directed fragmentation of analytes. However, as a reagent, helium has recently been listed as a critical mineral that is becoming scarcer and more expensive, so this study explored the potential for using cheaper and more readily available reagent gases. A model peptide, bradykinin, and a model oligosaccharide, κ-carrageenan with a degree of polymerization of 4, were fragmented using a variety of CTD reagent gases, which included helium, hydrogen, oxygen, nitrogen, argon and lab air. The CTD results were also contrasted with low-energy collision-induced dissociation (LE-CID), which were collected on the same 3D ion trap. Using constant reagent ion fluxes and kinetic energies, all five alterative reagent gases generated remarkably consistent sequence coverage and fragmentation efficiencies relative to He-CTD, which suggests that the ionization energy of the reagent gas has a negligible effect on the activation of the biological ions. The CTD efficiencies of all the gases ranged from 11-13% for bradykinin and 7-8% for κ-carrageenan. Within these tight ranges, the abundance of the CTnoD peak of bradykinin and the efficiency of CTD fragmentation of bradykinin both correlated with the ionization energy of the CTD reagent gas, which suggests that resonant charge transfer plays a small role in the activation of this peptide. The majority of the excitation energy for bradykinin and for κ-carrageenan comes from an electron stopping mechanism, which is described by long-range interactions between the reagent cations and electrons in the highest occupied molecular orbitals (HOMOs) of the biological ions. The CTD spectra do not provide any evidence for covalently bound products between the biological ions and the more-reactive gases like hydrogen, oxygen and nitrogen, which implies that the high kinetic energies of the reagent ions make them unavailable for covalent reactions. This work demonstrates that any of the substitute reagent gases tested are viable options for future CTD-MS experiments.

Structural Characterization of Isomeric Oligogalacturonan Mixtures Using Ultrahigh-Performance Liquid Chromatography-Charge Transfer Dissociation Mass Spectrometry.

Pectins are natural polysaccharides made from galacturonic acid residues, and they are widely used as an excipient in food and pharmaceutical industries. The degree of methyl-esterification, the monomeric composition, and the linkage pattern are all important factors that influence the physical and chemical properties of pectins, such as the solubility. This work focuses on the successful online coupling of charge transfer dissociation-mass spectrometry (CTD-MS) with ultrahigh-performance liquid chromatography (UHPLC) to differentiate isomers of oligogalacturonans derived from citrus pectins. This work employed CTD fragmentation of the pectin mixtures in data-dependent acquisition mode. Compared to the UHPLC with collision-induced dissociation mass spectrometry (UHPLC-CID-MS), UHPLC-CTD-MS yielded fewer ambiguous ions and more structurally informative results. The developed UHPLC-CTD-MS method resulted in abundant cross-ring cleavages-and especially 1,4Xn, 1,5Xn, and 2,4Xn ions-which helped to identify most of the isomers. The Gal A isomers differed only in the methyl group position along the galacturonic acid backbone. The combination of CTD in real time with UHPLC provides a new tool for the structural characterization of complex mixtures of oligogalacturonans and potentially other classes of oligosaccharides.

Comparison of in-source collision-induced dissociation and beam-type collision-induced dissociation of emerging synthetic drugs using a high-resolution quadrupole time-of-flight mass spectrometer.

In-source collision-induced dissociation (CID) is commonly used with single-stage high-resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in-source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in-source CID and beam-type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in-source CID. Electrospray ionization-quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to generate product ion spectra from in-source CID and beam-type CID for a series of well-characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in-source CID and a range of collision energies for beam-type CID. The results indicate that large fragmentor potentials for in-source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam-type CID. Although conditions can be found in which in-source CID and beam-type CID provide similar overall spectra, the in-source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam-type CID.

Structural Characterization of Sulfated Glycosaminoglycans Using Charge-Transfer Dissociation.

Glycosaminoglycans (GAGs) participate in a broad range of physiological processes, and their structures are of interest to researchers in structural biology and medicine. Although they are abundant in tissues and extracellular matrices, their structural heterogeneity makes them challenging analytes. Mass spectrometry, and more specifically, tandem mass spectrometry, is particularly well suited for their analysis. Many tandem mass spectrometry techniques have been examined for their suitability toward the structural characterization of GAGs. Threshold activation methods such as collision-induced dissociation (CID) produce mainly glycosidic cleavages and do not yield a broad range of structurally informative cross-ring fragments. Considerable research efforts have been directed at finding other means of dissociating gas-phase GAG ions to produce more comprehensive structural information. Here, we compare the structural information on GAGs obtained by charge-transfer dissociation (CTD) and electron detachment dissociation (EDD). EDD has previously been applied to GAGs and is known to produce both glycosidic and cross-ring cleavages in similar abundance. CTD has not previously been used to analyze GAGs but has been shown to produce abundant cross-ring cleavages and no sulfate loss when applied to another class of sulfated carbohydrates like algal polysaccharides. In contrast to EDD, which is restricted to FTICR mass spectrometers, CTD can be implemented on other platforms, such as ion trap mass spectrometers (ITMS). Here, we show the capability of CTD-ITMS to produce structurally significant details of the sites of modification in both heparan sulfate (HS) and chondroitin sulfate (CS) standards ranging in length from degree of polymerization (dp) 4 to dp6. EDD and CTD both yield more structural information than CID and yield similar fractional abundances to one another for glycosidic fragments, cross-ring fragments, and neutral losses.

The influence of chemical modifications on the fragmentation behavior of fentanyl and fentanyl-related compounds in electrospray ionization tandem mass spectrometry.

Fentanyl is a synthetic opioid that has been approved by the FDA as a general anesthetic because of its rapid onset and high potency. However, since 2013 an opioid epidemic involving fentanyl or fentanyl-related compounds (FRCs) has swept the United States and caused numerous deaths in every state. The identification of novel FRCs is complicated by the rapid turnover of modifications to the core fentanyl structure. In this study, a series of 16 FRCs were analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS) to gain a deeper understanding of the conserved and unique fragmentation behaviors associated with substitution to the core fentanyl structure. This work provides an approach, based on the product ions from ESI-MS/MS, to identify the modification site(s) on the core fentanyl structure for FRCs. Five common locations of substitution to the core fentanyl structure were used to assess the effect of substitution on the fragmentation behavior of FRCs. The proposed fragmentation pathways are supported through the combination of isotopic labeling, multi-stage mass spectrometry (MSn ), and accurate mass measurements with high-resolution mass spectrometry (HRMS). The identification of primary product ions specific to regions of substitution provides an additional tool for the identification of the location of substitution to the core fentanyl structure, which ultimately will assist toxicologists and seized drug analysts in the identification of emerging FRCs.

The characterization of isobaric product ions of fentanyl using multi-stage mass spectrometry, high-resolution mass spectrometry and isotopic labeling.

This study uses a combination of multi-stage mass spectrometry (MSn ), accurate mass measurements - with high-resolution mass spectrometry (HRMS) - and isotopic labeling to characterize the fragmentation behavior of fentanyl and 4-ANPP. By understanding the fragmentation behavior of fentanyl and its analogs in more detail, toxicologists and seized drug analysts will be better poised to identify new and emerging fentalogs, which are increasingly common and deadly adulterants in the growing opioid crisis. Throughout the literature the product ion at m/z 188 is often the most abundant fragment in the mass spectrometric analysis of fentanyl and fentanyl analogs, and this fragment is used for both qualitative and quantitative determinations. Our work shows there are at least three different structures for the isobaric fentanyl product ions at m/z 188, and they each form and fragment via different pathways. The development of fragmentation mechanisms to explain the observed fragmentation pathways of fentanyl and its main precursor 4-ANPP helps contribute to the advancement of knowledge about fentanyl fragmentation and could provide important information for the identification of future fentanyl analogs.

Laser ablation coupled with DAPNe-NSI-MS applied to redacted documents.

Laser ablation has been applied to redacted documents, where the text has been concealed by other ink. This technique strips the redacting ink revealing the text that was once redacted. Once removed, a nanomanipulation technique is used to extract the ink of the underlying text where mass spectrometry is then implemented to analyze its ink chemistry. In order to facilitate microscopy with direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry (DAPNe-NSI-MS), laser ablation must be executed prior to ink extraction. Laser ablation has a nondestructive approach of stripping the ink used to redact the document. Not only does this reveal the text, it clears an area for DAPNe to directly extract ink, in miniscule amounts, from the document without inducing destruction. The redacting ink was concluded to affect the aging process of the concealed handwritten ink more than the printed text. The redacted handwritten sample obtained higher relative peak area (%) values than the control samples (text that was not redacted) and the control for the printed text produced higher amounts of low molecular weight products than the sample. Implementing laser ablation on these samples could also affect the chemical properties of the underlying ink due to the additional UV radiation and plasma heating. Results indicate by using laser ablation to remove the redacting ink, the relative peak area of the underlying ink deviates by 1.25%. The thermal degradation of binding agents such as polymethylene, polyethylene glycol, and diethylene glycol was monitored by calculating the relative peak area for five days which, in turn, tracks the oxidation process. The relative peak area values were also used to determine the chemical kinetics of polyethylene glycol, where degradation and polymerization occur.

Vehicle-Mounted Portable Mass Spectrometry System for the Covert Detection via Spatial Analysis of Clandestine Methamphetamine Laboratories.

The ability to detect atmospheric effluent from clandestine methamphetamine manufacture is a useful tool for law enforcement. A membrane inlet mass spectrometer is mounted onto an all-electric drive capable hybrid vehicle that samples the atmosphere while in motion. Attributing a latitude and longitude to each spectrum collected, unique chemical fingerprints from clandestine manufacture are then mapped. This location-based mass spectrum data provides a localization to an area of interest. The synthesis of methamphetamine precursors was performed, and the impurities from such reactions were observed. A mock manufacture was setup, and the impurities were introduced into the atmosphere via heating. The detection of products and impurities using this mobile platform has shown the effectiveness of locating and localizing the manufacture of methamphetamine.