Abstract Collisional activation is available ubiquitously in commercial mass spectrometers, but, due to differences in instrumentation, it can be difficult to “translate” instrument settings from one instrument in a collisional activation experiment to another instrument to achieve quantitatively predictable results. With the goal of a more universal understanding and translatability of collision-induced dissociation and unfolding (CID/U) experiments, we recently introduced software (“IonSPA”) for simulating ion heating, cooling, and kinetic energy damping during collisional activation. A key parameter in IonSPA is the “pseudo-atom mass”, which controls the efficiency of energy transfer between an ion’s vibrational modes and the gas particle on a collision-by-collision basis. Here, we use molecular dynamic (MD) simulations to study energy transfer efficiency for native charge states of three different protein ions. Energy transfer efficiency is found to depend primarily on the collision gas type, relative speed of the collision, and total vibrational energy of the ion. Efficiency trends toward unity (completely inelastic and thermalizing) at zero collision speed, decreases to a minimum at a moderate collision speed, and increases again toward highly inelastic at high collision speeds. Dependence on collision geometry and the role of the centrifugal barrier and interaction potential are also explored. A pseudo-atom mass function based on gas type, ion vibrational temperature, and collision speed is derived from these MD simulations and implemented into IonSPA to calculate thermochemical barrier values in CID and CIU.
Abstract Understanding and quantifying activation parameters associated with the gas-phase isomerization kinetics of stereoisomeric ions, such as Z- and E-azobenzene ions, remains a key challenge in modern mass spectrometry. Here, we explore collisional activation as a fast and efficient method to probe isomerization kinetics on QToF instruments equipped with traveling wave-ion mobility spectrometry (TWIMS), such as the Waters Synapt platform. Using sequence-defined peptoids bearing an azobenzene chromophore, we investigate the collision-induced isomerization (CII) of Z-isomer ions into stable E-isomer ions within the Trap Cell–IMS configuration. The extent of Z → E conversion is monitored as a function of the activation voltage (Trap CV), allowing the extraction of kinetic constants based on the known residence time of ions in the Trap Cell. By integrating previously determined gas-phase activation parameters (ΔH‡ and ΔS‡) into a modified Eyring framework, we derived effective temperatures (Teff) that characterize the internal energy distribution of activated ions. The dependence of Teff on Trap CV yields two critical parameters: α, describing the kinetic-to-internal energy conversion efficiency, and T0, the pre-activation ion population temperature. The measured α values are of the same order as those previously derived for protein ions. However, T0 values are found to be system-dependent, which is detrimental for establishing a universal temperature calibration. Ongoing work focuses on expanding the switchable molecule library to map α and T0 across diverse molecular architectures and decipher at the molecular level the origin of these two parameters.
Abstract Personalized treatment strategies are rising within modern medicine approaches to dynamic and heterogeneous diseases such as cancer. Human metabolism is important for the understanding of these diseases and their mechanisms, but metabolomics remains often unused in multiomics approaches due to practical challenges in sample analysis and, more importantly, data analysis. Automated feature analysis with many false-positive annotations and a rather long analysis time is effectively hindering rapid metabolite screening, as it would be needed in medical research. Here, we report a novel and efficient workflow for rapid metabolite screening with the capability of yielding high-quality metabolomics data, including data analysis. Therefore, a HILIC (superficially porous sulfobetaine) method has been optimized to separate polar metabolites in complex samples in less than 9 min, and an open-source compatible retention time database has been implemented for this rapid screening method, including about 400 metabolites in central metabolic pathways. The method operates on a Q-Exactive Plus Orbitrap in DDA full-scan acquisition based on an inclusion list and has been shown to be highly repeatable and sensitive. Moreover, false-positive annotations could be severely reduced, enabling a simplified and slim data analysis approach. Using the supplied database and the code for data filtering, a rapid metabolite screening can be conducted, suitable for the application as a rapid metabolic profiling strategy in biological and medical research.
Abstract Mass spectrometry imaging (MSI) can complement liquid chromatography–mass spectrometry (LC-MS), the gold standard for impurity assessment, by enabling spatial visualization of active pharmaceutical ingredients (APIs), byproducts, and excipients in solid formulations. With new advances in matrix-assisted laser desorption/ionization (MALDI) MSI, critical factors such as ablated spot size, raster speed, and standardized tablet surface preparation still need to be properly addressed. This study presents an innovative sample holder and sample preparation protocol enabling reproducible up to 10-μm step size acquisition for MSI analysis of tablets. The workflow was validated by embedding several commercial tablets containing various APIs in blocks with epoxy resin, then transversally cut them to expose their surfaces. Following this, the tablet surfaces were trimmed using a cryostat and subsequently polished with lapping diamond films to achieve flat and even surfaces. The tablets were then placed in a custom metal holder for MALDI-trapped ion mobility spectrometry (TIMS)-MSI analysis using a timsTOF fleX. The optimized protocol provides high-quality sample preparation and avoids loss of laser focus, enabling high-throughput analysis with high sensitivity for tablet imaging. This robustness was essential for spatial resolution measurements using the Smartbeam 3D, where precise initial focusing is required to preserve data quality across imaging areas. Consistent high signal sensitivity was achieved for single pixels in 10 mg cetirizine or 20 mg famotidine tablets and digoxin fragments were detected in a 0.25 mg/130 mg tablet. This approach provides an important tool for pharmaceutical development by enabling reliable, high-resolution spatial analysis of active ingredients and impurities in solid dosage forms. Collision cross section (CCS) values from reference standards and tablets, as well as MS/MS data were used for confident identification. LC-MS-based workflows remain essential for quantitative analysis of impurity assessment, while infrared spectroscopy (IR) and nuclear magnetic resonance (NMR) can support formulation level characterization. MALDI-TIMS-MSI complements these approaches by adding direct spatial localization of APIs and API-related molecular features within the tablet matrix.
Abstract Niemann-Pick disease type C (NPC) is a lysosomal storage disorder characterized by the accumulation of unesterified cholesterol and glycosphingolipids in the late endosomal/lysosomal system, resulting in region-specific neurodegeneration. While the mechanism through which defective NPC1 protein and thus altered cholesterol transport engenders neurodegeneration is unknown, selective reintroduction of the NPC1 gene in different cell types in the mouse brain can alleviate disease symptoms, suggesting that neuronal and glial cells may play distinct roles in disease mechanisms. Here, in an in vitro NPC model, we evaluate cell-specific changes in the proteome upon treatment with a small-molecule cholesterol-trafficking inhibitor, U18666A, which inhibits NPC1 function across three immortalized cell lines in the central nervous system (CNS): mouse hippocampal neuronal cells (HT-22), mouse microglial cells (SIM-A9), and human hybridized oligodendrocytes (MO3.13). Using nanoliquid chromatography–mass spectrometry (LC–MS) techniques, we observe 21 common proteins upregulated in all U18666A-treated cells. We also note that markers of activated microglia were significantly altered in SIM-A9 microglial cells, whereas cytoskeletal and membrane-related proteins were elevated in MO3.13. In HT-22 hippocampal neuronal cells, we identify protein changes associated with altered glucose metabolism and postulate that ATP-citrate lyase (ACLY), a protein central to multiple biological pathways, might play a role in responding to aberrant cholesterol trafficking and metabolism in NPC.
Abstract Denatured and unstructured proteins have been observed to take up high charge densities in native ESI-MS through the chain ejection model (CEM). This behavior is hypothesized to originate from exposed interior hydrophobic residues upon denaturation with nearly complete unfolding upon ejection of the protein. In this work, we demonstrate that natively unstructured, strongly hydrophilic proline/alanine/serine (PAS) sequences and even folded proteins derivatized with PAS tails can also ionize at least partly through CEM. To determine the mechanistic origin of hydrophilic CEM, PAS tails of different lengths were analyzed and conjugated to bovine serum albumin. The solution-phase sizes of the species correlate well with the size of droplets that efficiently emit small ions through ion evaporation, thus offering a mechanism for CEM in the absence of hydrophobicity. The charge-state distributions and folding state of these structured/unstructured hybrids upon ionization were monitored by IMS-MS, showing that CEM ionization can induce partial as well as complete unfolding of folded proteins. This results in CEM-like charge acquisition and signal enhancement to an extent that is dependent on the relative size of the natively unfolded region.
Abstract Thermoembolization (TE) is a new, minimally invasive approach that could improve outcomes in select patients with hepatocellular carcinoma. However, little is known about the effects of this procedure at the molecular level. To gain greater insight into the molecular effects of TE, we performed a pilot study of transarterial TE with 2-propylpentanoyl chloride in a swine model with 3 animals. Liver tissues were harvested 7 days after the procedure, and formalin-fixed specimens were subjected to serial mass spectrometry imaging (MSI) analyses of metabolites, glycans, and tryptic peptides. We observed striking differences in spatial distributions for multiple features in the TE-treated and untreated areas. Our results show that TE is highly effective in causing coagulative necrosis in the target tissue and based on the data we propose that TE may cause localized chemical alterations in tissues. Our study further demonstrates that MSI is a powerful tool that can provide fundamentally new insights into the potential of using TE or other image-guided in vivo chemistry techniques to alter local biology.
Abstract Apolipoprotein E4 (ApoE4), one of the three common isoforms of ApoE, differs from ApoE3 and ApoE2 by just one amino acid substitution each. Yet these changes in ApoE4 profoundly impact protein structure and function to make it the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). ApoE4 exhibits a tendency to form oligomers (e.g., tetramers) that complicate high-resolution structural characterization in aqueous environments. Very little is known about the self-associated oligomeric structures. In this study, we employed ultracentrifugation, native mass spectrometry (MS), and fast photochemical oxidation of proteins (FPOP), a mass spectrometry-based protein footprinting approach, to investigate conformational differences among wild-type ApoE4 and two of its mutant variants ApoE4(V236E) (Jacksonville, JV) and ApoE4(V236E/L268E) (VL). These substitutions shift the oligomeric equilibrium toward dimeric and monomeric states, respectively, and allow the tetramer state to be compared with the dimer and monomer. Comparative footprinting reveals that conformational changes driven by these mutations result in altered solvent accessibility in key regions of the protein, suggesting disruption of interfaces responsible for oligomerization and altered access of lipid-binding regions. Given ApoE4’s central role in lipid metabolism and its altered interaction with lipid surfaces compared to other isoforms, these structural changes likely modulate lipid binding affinity and may contribute to its pathological role in AD. Our findings highlight the importance of conformational states in ApoE4 function and provide new insights into the structural basis of its disease-associated behavior.
Abstract The detection and structural analysis of hydrocarbons by mass spectrometry (MS) are challenging because of poor ionization. Here, we report a plasma-based strategy that selectively converts nonpolar hydrocarbons to derivatives that are mass spectrometry-friendly by controlled oxygen and nitrogen insertion reactions. A subatmospheric pressure plasma facilitates stable discharges under mild conditions, allowing the formation of ketones, unsaturated ketones, and imines without structural fragmentation or extensive oxidation. The reaction pathways can be tuned by adjusting the carrier gas and sample introduction mode, allowing controlled switching between simple oxidation, dehydrogenation, and nitrogen insertion. Mass spectrometric analysis reveals well-defined molecular ions that show characteristic fragmentation behavior in tandem MS, facilitating improved detection and structural interpretation across linear, branched, cyclic, and aromatic hydrocarbons. Furthermore, a scalable method has been developed to achieve preparative levels of modified samples for NMR analysis, and this confirmed ketone formation as the dominant oxygen-insertion pathway. Overall, this work demonstrates a simple and versatile plasma-based approach for enhancing the detection and structural characterization of otherwise challenging hydrocarbon species.
Glucose is a key metabolite involved in energy production, biosynthesis, and signaling. The metabolism of glucose is also dysregulated in many diseases, such as cancer, in which metabolic reprogramming of the carbohydrate is a hallmark of malignancy. In spite interest in using mass spectrometry imaging (MSI) to study the spatial temporal concentrations of glucose in normal and diseased tissues, poor ionization efficiency inhibits matrix-assisted laser desorption ionization (MALDI) analysis of the endogenous metabolite. A novel mass tag featuring a diol-targeting boronic acid warhead linked to a positively charged absorbing moiety is reported for on-tissue chemical derivatization to enhance glucose detection sensitivity and ionization efficiency. Spray deposition of the borate mass tag followed by 2,5-dihydroxyacetophenone matrix and MSI acquisition in the positive ion mode gave up to 10-fold greater sensitivity for glucose compared to standard analysis using N-(1-naphthyl)ethylenediamine dinitrate matrix and enabled imaging at 10 and 5 μm spatial resolution on brain tissue, which displayed glucose distribution without delocalization or edge effects. Empowering spatially resolved analysis of glucose metabolism with high sensitivity, boronic acid tag offers strong potential for studying disease-specific metabolic reprogramming.
Relative to liquid chromatography coupled to electrospray ionization (ESI), paper spray ionization (PSI) can more rapidly ionize analytes from complex matrices with less sample preparation and less expensive instrumentation. However, matrix effects can lead to ion suppression and poorer sensitivity. To overcome these problems, we combined on-paper faradaic ion concentration polarization (f-ICP), a form of electrokinetic stacking, with PSI to preconcentrate analytes directly on the spray substrate prior to ionization. Electrokinetic stacking also enables desalting because of the separation of analyte molecules from small ions like sodium. 3D-printed cartridges containing chemically modified PTFE papers were utilized with a high voltage isolated power supply to perform on-paper stacking from the same device as paper spray. In this study, ion suppression was compared for paper spray with and without electrokinetic stacking in the presence of five salts and artificial urine at varying concentrations. The signal enhancement achieved for f-ICP/PSI relative to normal PSI was also quantified. Finally, the effect of salt type and concentration on stacking time was assessed. Increasing salt concentrations generally results in longer stacking times and increased currents, indicative of increasing quantities of charge being desalted. In the presence of single salt-containing matrices, f-ICP/PSI frequently decreased ion suppression and gave a signal enhancement of 2 to >200× for small molecule drugs compared to unstacked paper spray (uPSI). In artificial urine, a more complex matrix with various salts and metabolites, f-ICP/PSI did not reliably decrease matrix effects due to creatinine and other matrix components costacking with the analytes. Nevertheless, f-ICP/PSI still resulted in signal enhancements of 8 to >50× relative to uPSI due to analyte preconcentration from electrokinetic stacking. Overall, this research demonstrated that f-ICP/PSI results in significant sensitivity improvements relative to uPSI.
Permethylation is a widely used derivatization technique for mass spectrometry-based glycan analysis, applicable to both research and drug product characterization. It stabilizes sialic acids, improves glycan ionization, and is commonly used to analyze PNGase F-released N-linked glycans for distribution and composition profiling, yielding semiquantitative or quantitative data. We occasionally observed peaks 32 and 54 Da below expected ion masses, anomalies which are unreported in the literature. Investigation revealed these losses stem from slower-than-expected spontaneous exchange of C-1 amines for water in nascent glycosylamines following PNGase F release. Here, we report the origin of these mass losses, propose a mechanistic explanation, and suggest mitigation strategies.
The existing open file format for mass spectrometry imaging (MSI) data, imzML is suitable for high-mass complexity images. However, when the pixel count exceeds many megapixels to a gigapixel, imzML becomes an inefficient storage option due to the required per-pixel XML component. In some targeted MSI cases, the Open Microscopy Environment tagged image file format (OME-TIFF) is preferred; however, OME-TIFF images are unable to capture the mass complexity of even a low-mass resolution image. Here we present "mspix", a file format that is suitable for high-pixel count single-stage mass spectrometry (MS1) images. We convert mass spectral images from differing file formats to mspix and benchmark their access speed with our Python implementation. We show that, in our cases, mspix is more space-efficient than both native and imzML file formats, especially for high-pixel count images. For mass images of fewer than 10 million pixels, imzML remains the recommended open, vendor-neutral format. Our implementation of mspix can process mass images of greater than 1 billion pixels, which, to our knowledge, is not supported in any other MSI software library or package.
Positional isomers of carboxylic acid-substituted tetrahydrocarbazole (Schemes 1A and 1B) were studied as model compounds for the N1O2 class of neutral nitrogen species in heavy crude oils with elemental composition CcHhNnOo (where n is 1 and o is 2). These species can contribute to the corrosion of the petroleum pipelines and storage tanks. We combined positive electrospray ionization collision-induced dissociation tandem mass spectrometry (+ESI CID MS/MS) with theoretical methods to analyze the model compounds. Each protonated precursor ion at m/z 216 exhibited distinct dissociation behavior at a laboratory collision energy, Elab, of 15 eV. The isomers with the COOH group at C-1 or C-3 in the cyclohexane ring generated the m/z 170 ion as a major fragment via consecutive losses of CO and H2O with distinct relative abundances. When the COOH group is located at C-6 in the benzene ring, the m/z 172 ion was a significant fragment formed via CO2 loss from the precursor ion. The principal fragment was the m/z 198 ion formed via H2O loss from the precursor ion when the COOH group is located at C-8 in the benzene ring. Theoretical analyses of each fragmentation mechanism revealed that multistep proton transfer was crucial for the formation of most products. For some products, formation was favored by entropy, in addition to sufficient internal energy. These results contribute to a broader foundation for investigating a wider range of structurally related carbazole-based carboxylic acids that are relevant to corrosion. Ultimately, understanding their fragmentation chemistries contributes valuable insights into developing strategies to mitigate crude oil corrosion and catalyst fouling.
Phosphatidylethanols (PEths) are direct alcohol biomarkers of clinical and forensic importance. Their accurate quantification depends critically on the regioisomeric purity of reference materials, as deviations from the natural isomer distribution can bias measurement outcomes. In this study, we systematically evaluated the relative isomer abundance of synthetic PEth 16:0/18:1 standards using nuclear magnetic resonance spectroscopy (NMR), ozone-induced dissociation (CID/OzID), collision-induced dissociation (CID), and electron-activated dissociation (EAD). Three commercial preparations were investigated: a mixed isomer standard and two "IsoPure" materials claimed to contain exclusively the PEth 16:0/18:1 or 18:1/16:0 regioisomer, respectively. Quantitative 13C NMR enabled unambiguous discrimination of the carbonyl resonances, revealing a mixture of ∼83/17% in the non-IsoPure material and <1% cross-contamination in the IsoPure standards. CID/OzID provided complementary structural information, with isomeric compositions consistent with NMR results. CID and EAD (sodiated, positive ionization) yielded characteristic product ion ratios and homologue-specific product ions, respectively, that distinguished isomers, although precise quantification remained limited. In contrast, EAD spectra in negative-ion mode were dominated by nondiagnostic product ions and did not allow isomer differentiation. Together, our results demonstrate that 13C NMR and CID/OzID are robust, quantitative approaches for assessing regioisomeric purity in synthetic PEth standards, while CID and EAD can support qualitative profiling. This multiplatform evaluation provides a framework for quality control of PEth reference materials and underlines the necessity of verifying regioisomeric composition to ensure reliable biomarker quantification.
Mass spectrometry imaging (MSI) has emerged as a powerful modality for spatially resolved molecular profiling of tumor and stromal compartments; however, computational frameworks for MSI data analysis lag significantly behind those developed for spatial transcriptomics, limiting its translational potential. Here, we introduce the Spatial Omics Toolkit (SPOT), an end-to-end, open-source analytical pipeline that operationalizes established statistical methods from single-cell and spatial transcriptomics into accessible workflows for MSI data. SPOT is implemented in both R and Python, uses vendor-neutral community data formats, and integrates classification modeling, dimensionality reduction, and trajectory inference to enable spatially resolved comparative analysis across disease states with minimal computational overhead. We demonstrate the utility of SPOT on stromal proteomic profiles derived from ductal carcinoma in situ (DCIS) lesion archetypes, identifying differentially expressed peptides across disease states by orthogonal statistical approaches, and reconstructing a pseudotime trajectory from DCIS to invasive breast cancer from the same patient genetics. Collectively, SPOT provides researchers with a framework for interrogating molecular pathology across diverse MSI data sets. SPOT can be found at https://github.com/angel-omics-lab.
Per- and polyfluoroalkyl substances (PFAS) are an evolving class of synthetic chemicals that are pervasive in the environment due to widespread manufacturing and consumer use, such that PFAS contamination is of great concern. Although thousands of PFAS structures have been reported to date, this number increases daily with the identification of new PFAS from advances in non-targeted analysis (NTA) workflows. Ion mobility spectrometry in combination with mass spectrometry (IMS-MS) has recently been incorporated into PFAS NTA studies. Although MS provides essential precursor and fragmentation data, IMS offers complementary structural information via the measurement of the ion-neutral collision cross section (CCS) values. Experimental CCS values can then be compared with those calculated in silico from candidate 3D structures to meet confidence-level criteria in analyte assignments within NTA workflows. Although this approach has been applied since the late 1990s, PFAS often exhibited poorer agreement between the calculated and experimental CCS values. To address this limitation, we propose an optimized computational workflow to calculate Boltzmann-weighted CCS values for PFAS structures generated via quantum-chemical calculations. This workflow was assessed with experimental CCS values from 56 known PFAS structures across seven classes and resulted in an average percent error of 2.0%. Moreover, 11 new PFAS structures were proposed from NTA, with average errors of 1.3%. The combination of this new computational workflow and experimental IMS-MS measurements therefore establishes a workflow for structural elucidation of emerging PFAS.
Validation of epitopes and paratopes for antibodies and antigens is crucial for understanding antibody binding and is of interest in the pharmaceutical field. Traditionally, high-resolution techniques like X-ray and NMR are used for delineating protein-protein interactions, but they are prone to sample preparation problems and size limitations. Mass spectrometry (MS)-based structural analysis, like H/D exchange (HDX), covalent labeling, and cross-linking, contributes unique information and advantages relative to traditional methods. As such, it is desirable to compare all three techniques side by side and assess how complementary their results are for defining epitopes on the targets of interest. In this study, we probed interaction sites of epidermal growth factor receptor (EGFR) with cetuximab and zalutumumab using HDX, covalent labeling, and cross-linking techniques. For covalent labeling experiments, sulfo-NHS acetate and DEPC were used as the labeling reagents. In cross-linking experiments, multiple cross-linkers, including BS3, EDC, DSSO, and ADH/DMTMM, were used for a comprehensive search of protein-protein interactions. The results from cross-linking and covalent labeling were compared to HDX as well as known epitopes. Covalent labeling, cross-linking, and HDX data showed consistent results for EGFR-Cetuximab epitopes. For the EGFR-Zalutumumab complex, no intermolecular cross-links within reasonable distances were identified, but the epitopes observed from covalent labeling agree with HDX. The results are consistent with reported crystal structures. Overall results with DEPC and sulfo-NHS labeling are comparable to HDX with a faster cycle time, making them amenable for rapid screening of many molecules. DEPC labeling has more residue coverage compared to sulfo-NHS acetate but faces unique challenges like faster hydrolysis and more complex modifications. We further extended the use of covalent labeling experiments in a cellular environment with the same model system and with results consistent with purified proteins, making covalent labeling a suitable method to screen epitopes for complex targets and receptors directly from cells.
Detecting vapor from drugs and explosives is challenging due to their extremely low vapor pressures, often in the parts-per-trillion (pptv) to parts-per-quadrillion (ppqv) range. Atmospheric flow tube-mass spectrometry (AFT-MS) overcomes this limitation by extending ion-molecule reaction times, achieving detection of ppqv levels of vapor and femtogram levels of residue. However, the prior AFT-MS system was large (∼435 lbs.), limiting field deployment. To enhance portability, the AFT was integrated with a miniaturized, ruggedized, field-portable linear ion trap mass spectrometer (∼45 lbs.). This work presents results demonstrating the feasibility of portable AFT-MS for detecting illicit drugs and explosives in the field. Initial tests of the AFT with the portable MS were conducted to detect trace vapors from both solutions and residues. Real-time detection of fentanyl vapor from residue indicates detection levels of less than 100 ppqv based on the equilibrium vapor pressure of fentanyl. Adjusting ionization parameters, sampling speeds, trapping times, pressure levels, and system timing revealed pathways for further sensitivity enhancements. Early field tests at a U.S. Customs and Border Protection port of entry confirmed the ability of the system to simultaneously detect multiple drugs in complex mixtures. This is the first demonstration of portable vapor detection with AFT-MS, achieving subpptv-level sensitivity for real-time field detection of drugs and explosives.
