Comprehensive Lipidomic Automation Workflow using Large Language Models.

Lipidomics generates large data that makes manual annotation and interpretation challenging. Lipid chemical and structural diversity with structural isomers further complicates annotation. Although, several commercial and open-source software for targeted lipid identification exists, it lacks automated method generation workflows and integration with statistical and bioinformatics tools. We have developed the Comprehensive Lipidomic Automated Workflow (CLAW) platform with integrated workflow for parsing, detailed statistical analysis and lipid annotations based on custom multiple reaction monitoring (MRM) precursor and product ion pair transitions. CLAW contains several modules including identification of carbon-carbon double bond position(s) in unsaturated lipids when combined with ozone electrospray ionization (OzESI)-MRM methodology. To demonstrate the utility of the automated workflow in CLAW, large-scale lipidomics data was collected with traditional and OzESI-MRM profiling on biological and non-biological samples. Specifically, a total of 1497 transitions organized into 10 MRM-based mass spectrometry methods were used to profile lipid droplets isolated from different brain regions of 18-24 month-old Alzheimer's disease mice and age-matched wild-type controls. Additionally, triacyclglycerols (TGs) profiles with carbon-carbon double bond specificity were generated from canola oil samples using OzESI-MRM profiling. We also developed an integrated language user interface with large language models using artificially intelligent (AI) agents that permits users to interact with the CLAW platform using a chatbot terminal to perform statistical and bioinformatic analyses. We envision CLAW pipeline to be used in high-throughput lipid structural identification tasks aiding users to generate automated lipidomics workflows ranging from data acquisition to AI agent-based bioinformatic analysis.

Lipid Isobar and Isomer Imaging Using Nanospray Desorption Electrospray Ionization Combined with Triple Quadrupole Mass Spectrometry.

Mass spectrometry imaging (MSI) is widely used for examining the spatial distributions of molecules in biological samples. Conventional MSI approaches, in which molecules extracted from the sample are distinguished based on their mass-to-charge ratio, cannot distinguish between isomeric species and some closely spaced isobars. To facilitate isobar separation, MSI is typically performed using high-resolution mass spectrometers. Nevertheless, the complexity of the mixture of biomolecules observed in each pixel of the image presents a challenge, even for modern mass spectrometers with the highest resolving power. Herein, we implement nanospray desorption electrospray ionization (nano-DESI) MSI on a triple quadrupole (QqQ) mass spectrometer for the spatial mapping of isobaric and isomeric species in biological tissues. We use multiple reaction monitoring acquisition mode (MRM) with unit mass resolution to demonstrate the performance of this new platform by imaging lipids in mouse brain and rat kidney tissues. We demonstrate that imaging in MRM mode may be used to distinguish between isobaric phospholipids requiring a mass resolving power of 3,800,000. Additionally, we have been able to image eicosanoid isomers, a largely unexplored class of signaling molecules present in tissues at low concentrations, in rat kidney tissue. This new capability substantially enhances the specificity and selectivity of MSI, enabling spatial localization of species that remain unresolved in conventional MSI experiments.

Digital Quadrupole Isolation and Electron Capture Dissociation on an Extended Mass Range Q-TOF Provides Sequence and Structure Information on Proteins and Protein Complexes.

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).

3DMolMS: prediction of tandem mass spectra from 3D molecular conformations.

MOTIVATION: Tandem mass spectrometry is an essential technology for characterizing chemical compounds at high sensitivity and throughput, and is commonly adopted in many fields. However, computational methods for automated compound identification from their MS/MS spectra are still limited, especially for novel compounds that have not been previously characterized. In recent years, in silico methods were proposed to predict the MS/MS spectra of compounds, which can then be used to expand the reference spectral libraries for compound identification. However, these methods did not consider the compounds' 3D conformations, and thus neglected critical structural information. RESULTS: We present the 3D Molecular Network for Mass Spectra Prediction (3DMolMS), a deep neural network model to predict the MS/MS spectra of compounds from their 3D conformations. We evaluated the model on the experimental spectra collected in several spectral libraries. The results showed that 3DMolMS predicted the spectra with the average cosine similarity of 0.691 and 0.478 with the experimental MS/MS spectra acquired in positive and negative ion modes, respectively. Furthermore, 3DMolMS model can be generalized to the prediction of MS/MS spectra acquired by different labs on different instruments through minor fine-tuning on a small set of spectra. Finally, we demonstrate that the molecular representation learned by 3DMolMS from MS/MS spectra prediction can be adapted to enhance the prediction of chemical properties such as the elution time in the liquid chromatography and the collisional cross section measured by ion mobility spectrometry, both of which are often used to improve compound identification. AVAILABILITY AND IMPLEMENTATION: The codes of 3DMolMS are available at https://github.com/JosieHong/3DMolMS and the web service is at https://spectrumprediction.gnps2.org.

Ion Manipulation in Open Air Using 3D-Printed Electrodes.

Ambient ionization techniques provide a way to sample materials via creation of ions in the air. However, transferring and focusing of these ions is typically done in the reduced pressure environment of the mass spectrometer. Spray-based ambient ionization sources require relatively large distances between the source and mass spectrometer inlet for effective desolvation, resulting in a small fraction of the ions being collected. To increase the efficiency of ion transfer from atmosphere to vacuum, 3D-printed focusing devices made of conductive carbon nanotube doped polymers have been designed and evaluated for ion focusing in air. Three main classes of electrodes are considered: (i) conic section electrodes (conical, ellipsoidal, and cylindrical), (ii) simple conductive and non-conductive apertures, and (iii) electrodes with complex geometries (straight, chicane, and curved). Simulations of ion trajectories performed using the statistical diffusion simulation (SDS) model in SIMION showed a measure of agreement with experiment. Cross-sectional images of ion beams were captured using an ion detecting charge-coupled device (IonCCD). After optimization, the best arrangements of electrodes were coupled to an Agilent Ultivo triple quadrupole to record mass spectra. Observations suggest that electrode geometry strongly influences ion trajectories in air. Non-conductive electrodes also assisted in focusing, due to charge buildup from ion deposition. We also observed minimal spreading of the ion packet after exiting the focusing electrodes indicating that atmospheric collisions do not reduce collimation of the beam. The study suggests that high pressures need not be viewed as a hindrance to ion transport, but as a potentially useful force.

Multiple reaction monitoring (MRM)-profiling with biomarker identification by LC-QTOF to characterize coronary artery disease.

Metabolite profiling by mass spectrometry (MS) is an area of interest for disease diagnostics, biomarker discovery, and therapeutic evaluation. A recently developed approach, multiple reaction monitoring (MRM)-profiling, searches for metabolites with precursor (Prec) and neutral loss (NL) scans in a representative sample and creates a list of ion transitions. These are then used in an MRM method for fast screening of individual samples and discrimination between healthy and diseased. A large variety of functional groups are considered and all signals discovered are recorded in the individual samples, making this a largely unsupervised method. MRM-profiling is described here and then demonstrated with data for over 900 human plasma coronary artery disease (CAD) samples. Representative pooled samples for each condition were interrogated using a library of over a hundred Prec and NL scans on a triple quadrupole MS. The data from the Prec and NL experiments were converted into ion transitions, initially some 1266 transitions. Each ion transition was examined in the individual samples on a time scale of milliseconds per transition, which allows for rapid screening of large sample sets (<5 days for 1000 samples). Use of univariate and multivariate statistics allowed classification of the sample set with high accuracy. The metabolite profiles classified the CAD female, CAD male, and peripheral artery disease (PAD) samples relative to controls with an accuracy of 90%, 78%, and 85%, respectively. The compounds responsible for informative ion transitions were identified by chromatography and high resolution MS; some have been previously reported and found to be associated with coronary artery disease metabolism, indicating that the methodology generates a meaningful metabolite profile while being faster than traditional methodologies.

Global mass spectrometry based metabolomics profiling of erythrocytes infected with Plasmodium falciparum.

Malaria is a global infectious disease that threatens the lives of millions of people. Transcriptomics, proteomics and functional genomics studies, as well as sequencing of the Plasmodium falciparum and Homo sapiens genomes, have shed new light on this host-parasite relationship. Recent advances in accurate mass measurement mass spectrometry, sophisticated data analysis software, and availability of biological pathway databases, have converged to facilitate our global, untargeted biochemical profiling study of in vitro P. falciparum-infected (IRBC) and uninfected (NRBC) erythrocytes. In order to expand the number of detectable metabolites, several key analytical steps in our workflows were optimized. Untargeted and targeted data mining resulted in detection of over one thousand features or chemical entities. Untargeted features were annotated via matching to the METLIN metabolite database. For targeted data mining, we queried the data using a compound database derived from a metabolic reconstruction of the P. falciparum genome. In total, over one hundred and fifty differential annotated metabolites were observed. To corroborate the representation of known biochemical pathways from our data, an inferential pathway analysis strategy was used to map annotated metabolites onto the BioCyc pathway collection. This hypothesis-generating approach resulted in over-representation of many metabolites onto several IRBC pathways, most prominently glycolysis. In addition, components of the "branched" TCA cycle, partial urea cycle, and nucleotide, amino acid, chorismate, sphingolipid and fatty acid metabolism were found to be altered in IRBCs. Interestingly, we detected and confirmed elevated levels for cyclic ADP ribose and phosphoribosyl AMP in IRBCs, a novel observation. These metabolites may play a role in regulating the release of intracellular Ca(2+) during P. falciparum infection. Our results support a strategy of global metabolite profiling by untargeted data acquisition. Untargeted and targeted data mining workflows, when used together to perform pathway-inferred metabolomics, have the benefit of obviating MS/MS confirmation for every detected compound.

Benzyl benzoate glycosides from Oligoneuron rigidum.

Two new benzyl benzoate glycosides were isolated from the leaves and stems of the native North American prairie plant Oligoneuron rigidum (stiff goldenrod). The glycosides were isolated as a mixture of benzoate and acetate esters, which were subjected to mild base hydrolysis to facilitate full structural characterization using LCMSMS and 1D and 2D NMR data.

The catalase activity of diiron adenine deaminase.

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

Simultaneous separation and identification of limonoids from citrus using liquid chromatography-collision-induced dissociation mass spectra.

Limonoids are considered as potential cancer chemopreventive agents and are widely distributed in the Citrus genus as aglycones and glucosides. In the present study, reversed-phase HPLC coupled with CID mass spectra was developed for the simultaneous separation and identification of aglycones and glucosides of limonoids from citrus. Five aglycones such as limonin, deacetyl nomilin, ichangin, isolimonoic acid and nomilin were identified by positive ion CID MS/MS, whereas five glucosides, viz. limonin glucoside, isoobacunoic acid glucoside, obacunone glucoside, deacetyl nomilinic acid glucoside and nomilinic acid glucoside were analyzed by negative ion CID mass spectra. The developed method was successfully applied to complex citrus samples for the separation and identification of aglycones and glucosides. Citrus seeds were extracted with methanol and partially purified and analyzed by LC-CID mass spectra. The separation was achieved by C-18 column; eight limonoids were identified by comparing the retention times and mass spectral fragmentation. To the best of our knowledge, this is the first report on the identification of citrus limonoids using CID technique.

Variation in and responses to brood pheromone of the honey bee (Apis mellifera L.).

The 10 fatty acid ester components of brood pheromone were extracted from larvae of different populations of USA and South African honey bees and subjected to gas chromatography-mass spectrometry quantitative analysis. Extractable amounts of brood pheromone were not significantly different by larval population; however, differences in the proportions of components enabled us to classify larval population of 77% of samples correctly by discriminant analysis. Honeybee releaser and primer pheromone responses to USA, Africanized and-European pheromone blends were tested. Texas-Africanized and Georgia-European colonies responded with a significantly greater ratio of returning pollen foragers when treated with a blend from the same population than from a different population. There was a significant interaction of pheromone blend by adult population source among Georgia-European bees for modulation of sucrose response threshold, a primer response. Brood pheromone blend variation interacted with population for pollen foraging response of colonies, suggesting a self recognition cue for this pheromone releaser behavior. An interaction of pheromone blend and population for priming sucrose response thresholds among workers within the first week of adult life suggested a more complex interplay of genotype, ontogeny, and pheromone blend.

A mechanistic study of the H/D exchange reactions of protonated arginine and arginine-containing di- and tripeptides.

The gas-phase H/D exchange reactions of arginine (R) and arginine-containing di- and tri-peptide (gly-arg (GR), arg-gly (RG), gly-gly-arg (GGR), gly-arg-gly (GRG) and arg-gly-gly (RGG)) [M + H]+ ions with deuterated ammonia (ND3) were investigated by using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR), ion mobility-mass spectrometry (IM-MS), ab initio and density functional theory-based molecular orbital calculations and molecular modeling. Three exchanges are observed for arginine and arginine-containing tri-peptide [M + H]+ ions, whereas the di-peptide [M + H]+ ions undergo a single H/D exchange. In addition, C-terminal methylation blocks H/D exchange of arginine and the arginine-containing peptide [M + H]+ ions, and a single H/D exchange is observed for N-terminal acetylated arginine [M + H]+ ions. A general mechanism for H/D exchange involving a collision complex that is best described as a "solvated salt-bridge" structure is proposed.

Ion mobility-mass spectrometer interface for collisional activation of mobility separated ions.

An ion mobility-mass spectrometer (IM-MS) interface is described that can be employed to perform collisional activation and/or collision-induced dissociation (CID) with good transmission of mobility separated ions to the MS analyzer. The IM-MS interface consists of a stacked-ring ion guide design, where the field strength and pressure ratio can be operated such that structural rearrangement reactions and/or CID are achieved as a function of the effective ion temperature. The ion dynamics and collisional activation processes in the IM-MS interface are described as a function of the ion-neutral collisions, ion kinetic energies, and effective ion temperature. The applicability of the IM-CID-MS methodology to studies of peptide ion fragmentation is illustrated using a series of model peptides.

Unimolecular dissociation reactions of methyl benzoate radical cation.

The blackbody infrared radiation induced dissociation of methyl benzoate (C8H8O2(+*)) radical cation was investigated by using a Fourier transfer ion cyclotron resonance mass spectrometer equipped with a resistively heated (wire temperatures of 400-1070 K) wire ion guide. We observed product ion branching ratios that are strongly dependent upon wire temperature. At low temperatures (670-890 K) the major product ion C7H8 (+*) (m/z 92), which is formed by loss of CO2, and at higher temperatures (above 900 K), loss of methoxy radical ((*)OCH3) competes with loss of CO2. The energies of the various reactant ions and transition states for product ion formation were estimated by using density functional theory molecular orbital calculations, and a proposed mechanism for the dissociation chemistry of C8H8O2 (+*) involving a multistep rearrangement reaction is tested using the Master Equation formalism.

Synthesis and cellular effects of cycloterpenals: cyclohexadienal-based activators of neurite outgrowth.

An unusual class of diterpenoid natural products, 'cycloterpenals' (with a central cyclohexadienal core), that arise in nature by condensation of retinoids and other isoprenes, have been isolated from a variety of organisms including marine sponges as well as from the human eye. A milk whey protein has also demonstrated the formation of a cycloterpenal derived from beta-ionylidineacetaldehyde. Here, we generate a synthetic library of these molecules where we detail reaction conditions required to effect cross condensation of alpha,beta-unsaturated aldehydes as opposed to homodimerization. The ability of this class of molecules to activate neurite outgrowth activity is reported.

Structure and function of the virulence-associated high-temperature requirement A of Mycobacterium tuberculosis.

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.

Structure and function of the sterol carrier protein-2 N-terminal presequence.

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, and fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2's affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts and caveolae (AF488-CTB); 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488 antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.

UC/MALDI-MS analysis of HDL; evidence for density-dependent post-translational modifications.

The purpose of this study is to determine whether the nature of the post-translational modifications of the major apolipoproteins of HDL is different for density-distinct subclasses. These subclasses were separated by ultracentrifugation using a novel density-forming solute to yield a high-resolution separation. The serum of two subjects, a control with a normolipidemic profile and a subject with diagnosed cardiovascular disease, was studied. Aliquots of three HDL subclasses were analyzed by MALDI and considerable differences were seen when comparing density-distinct subclasses and also when comparing the two subjects. A detailed analysis of the post-translational modification pattern of apoA-1 shows evidence of considerable protease activity, particularly in the more dense fractions. We conclude that part of the heterogeneity of the population of HDL particles is due to density-dependent protease activity.

Establishing the kinetic competency of the cationic imine intermediate in nitroalkane oxidase.

The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2-(2H2)-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent with cyanide trapping this electrophilic intermediate.

Gas-phase reactivity of charged pi-type biradicals.

Four pi,pi-biradicals, 2,6-dimethylenepyridinium and the novel isomers N-(3-methylenephenyl)-3-methylenepyridinium, N-phenyl-3,5-dimethylenepyridinium, and N-(3,5-dimethylenephenyl)pyridinium ions, were generated and structurally characterized in a Fourier transform ion cyclotron resonance mass spectrometer. Their gas-phase reactivity toward various reagents was compared to that of the corresponding monoradicals, 2-methylenepyridinium, N-phenyl-3-methylenepyridinium, and N-(3-methylenephenyl)pyridinium ions. The biradicals reactivity was found to reflect their predicted multiplicity. The 2,6-dimethylenepyridinium ion, the only biradical in this study predicted to have a closed-shell singlet ground state, reacts significantly faster than the other biradicals, which are predicted to have triplet ground states. In fact, this biradical reacts at a higher rate than the analogous monoradical, which suggests that to avoid the costly uncoupling of its unpaired electrons, the biradical favors ionic mechanisms over barriered radical pathways. In contrast, the second-order reaction rate constants of the isomeric biradicals with triplet ground states are well approximated by those of the analogous monoradicals, although the final reaction products are sometimes different. This difference arises from rapid radical-radical recombination of the initial monoradical reaction products. The overall reactivity toward the hydrogen-atom donors benzeneselenol and tributylgermanium hydride is significantly greater for the radicals with the charged site in the same ring system as the radical site. This finding indicates that polar effects play an important role in controlling the reactivity of pi,pi-biradicals, just as has been demonstrated for sigma,sigma-biradicals.