Invited Abstracts
Alphabetical order by presenter
Ambient Mass Spectrometry Imaging: Recent Developments and Opportunities
Julia Laskin, PhD
Purdue University, West Lafayette, Indiana
Mass spectrometry imaging (MSI) is a powerful technique for studying the localization of lipids, metabolites, and proteins in biological samples. We have developed nanospray desorption electrospray ionization (nano-DESI), an ambient ionization technique that enables label-free molecular imaging of biological samples with minimal sample pretreatment. Nano-DESI relies on the localized liquid extraction of analyte molecules from the sample into a liquid bridge formed between two glass capillaries. The extracted analytes are transferred to a mass spectrometer inlet and ionized by electrospray ionization. Quantification is performed by adding an internal standard to the solvent and normalizing the signals of endogenous analytes to the corresponding adduct of the standard. Recent developments of nano-DESI MSI instrumentation have enabled quantitative imaging of lipids, metabolites, and proteins in tissues with high sensitivity and spatial resolution down to 8-10 microns using finely pulled capillaries. Furthermore, we have developed a microfluidic nano-DESI probe, which greatly simplifies the experimental setup and demonstrates similar performance to the capillary-based probe. We have enhanced the chemical specificity of nano-DESI MSI experiments by coupling it with ion mobility spectrometry and online photochemical derivatization of the extracted analytes. These complementary approaches have been used for isomer-selective imaging of biomolecules in tissue samples. Collectively, these new spatial omics approaches provide unique insights into the biochemical pathways in biological systems.
Assembling a Consensus Serum Metabolome
Shuzhao Li, PhD
The Jackson Laboratory for Genomic Medicine, Farmington, CT
Reference metabolomes will be critical for reproducible research, effective annotation and guiding scientific investigations, but their construction is currently hindered by many technical challenges. We report here the assembly of a consensus serum metabolome by large-scale data integration using our recently developed asari suite of tools. By reprocessing over 100,000 LC-MS metabolomic acquisitions (>100 studies) from the public domain, consensus mass registries were extracted using kernel density estimation and pre-annotations in each dataset were combined. Additional GC-MS and MS/MS data were integrated from ADAP-KDB. The results indicate that major gaps exist in the current metabolite databases; annotation can be greatly enhanced by cross–study information.
Spatially-Resolved-Omics Analysis of Molecular Aging in Ocular Tissues
Kevin Schey, PhD
Vanderbilt University, Nashville, TN
Spatially-resolved -Omics methodologies have been developed to analyze molecular (protein & lipid) aging processes in ocular lens and retina tissues. Both laser capture microdissection coupled with LC-MS/MS analysis as well as imaging mass spectrometry (IMS) have been used to define age-related changes in human lens proteins and lipids as well as to identify lipid constituents of retina deposits. Both age-related and cataract-specific protein modifications have been identified in aged human lenses. In retina tissues, multimodal imaging methods, including high resolution IMS, allowed lipid components of drusen and subretinal drusenoid deposits (SDD) to be characterized. This presentation will discuss the methods developed for these studies, the results obtained, and the biochemical insights generated.
Sponsored Abstracts (Lunch and Learn)
Alphabetical order by presenter
Chiral Ion Mobility: Strategies for Small Molecule Enantiomer Separations with High Resolution Ion Mobility-Mass Spectrometry (sponsored by Agilent)
Benjamin K. Blakley, Jody C. May, John A. McLean
Vanderbilt University, Nashville, TN
The pairing of noncovalent complexation with structural selective ion mobility-mass spectrometry (IM-MS) represents a powerful development in the field of enantiomer separations. Due to their nearly identical structural connectivity and physical properties, small molecule enantiomers remain among the most challenging classes of isomers to resolve using IM-MS. The IM-based separation of enantiomers necessitates interaction of enantiomeric analytes with a chiral selector (CS). The resulting diastereomeric complexes exhibit structural differences which can often be elucidated with high-resolution ion mobility (HRIM) instrumentation. Various chiral selection strategies have been reported in the literature, but transition metal-amino acid complexes are frequently utilized for broad enantiomeric selectivity across a range of drug structures. Herein, we report the ion mobility separation of drug enantiomers complexed within a ternary copper-amino acid (AA) complex of the form [(M)(AA)(CuII) – H]+. While enormous challenge remains, the promise of these results is immense—with applications in drug quality control, metabolomics, and beyond.
Rethink What is Possible with the Orbitrap Astral Mass Spectrometer (sponsored by Thermo Fisher Scientific)
Daniel Hermanson, PhD
Thermo Fisher Scientific, Waltham, MA
*coming soon*
Mass Spectrometry Imaging and High Throughput Single Cell Characterization to Unravel Unusual Cell to Cell Signaling Pathways (sponsored by Bruker)
Jonathan V. Sweedler, PhD
University of Illinois Urbana-Champaign, Urbana, IL
While the need for single cell chemical measurements of heterogeneous brain cells has been well established, the large number of cell types suggests that thousands to tens of thousands of measurements are required to characterize representative cell types in a brain region. Approaches for assaying the chemical content within populations of individual brain cells are highlighted, including mass spectrometry imaging (MSI) and direct single cell measurements. For the high throughput measurements, the cells of interest are scattered across a microscope slide, the exact cell positions determined via optical microscopy, and mass spectra are acquired only at the cell positions. Because the spaces between the cells are not measured, the approach is efficient and rapid. Using both the Bruker timsTOF Flex MALDI-2 and SolariX 7T FT-ICR, high acquisition rates and high information content is possible. The single cell assays allow differences in the metabolome and peptidome from supposedly homogeneous populations of cells to be explored. Using these approaches, we can measure lipids, fatty acids and neuropeptides, among others. By obtaining information from tens of thousands of individual cells, rare cells are found and unusual neurochemicals are discovered. For select cells, follow-up capillary electrophoresis-mass spectrometry is performed. We have adapted the approach to work with individual cellular organelles such as dense core vesicles and mitochondria providing us with unique details on organelle heterogeneity. Our overarching goal is to uncover the complex chemical mosaic of the brain and pinpoint key cellular players involved in a range of physiological and pathological processes.
Oral Presentations (Submitted Abstracts)
Alphabetical order by presenter
Investigation of n3-Polyunsaturated Fatty Acid Driven Differentiation of H3K27M Glial Progenitors in Diffuse Midline Glioma Using Mass Spectrometry Imaging
Julia R. Bonney1,2, Pruthvi Gowda3,4, Sina Neyazi5,6, Rishov Goswami3,4, Joana G. Marques5,6, Jennifer Gantchev1,2, Gerard Baquer1,2, Sylwia Stopka1,2, Mariella G. Filbin5,6, Nika N. Danial3,4, Nathalie Y. R. Agar1,2,3
1Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
2Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
3Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA
4Department of Medicine, Harvard Medical School, Boston, MA
5Department of Pediatric Oncology, Dana-Farber Boston Children’s Cancer and Blood Disorders Center, Boston, MA
6Broad Institute of Harvard and MIT, Cambridge, MA
Histone mutant diffuse midline glioma (H3K27M DMG) is a fatal malignant pediatric brain tumor with a survival rate of less than 10% two years after initial diagnosis. It’s been shown that the growth of these tumor cells is driven by a subpopulation of cells resembling oligodendrocyte progenitor cells (OPC-like), however, these cells have the potential to partially differentiate into cells that resemble astrocytes (AC-like) and oligodendrocytes (OC-like). In this work, we used mass spectrometry imaging (MSI) to study dietary n3-polyunsaturated fatty acid (PUFA) enrichment strategies in tumor progression using a patient-derived H3K27M DMG PDX model. DMG tumor models were generated by stereotactic injection of luciferized BT869 cells into the right pons of the mice. Mice were switched to custom diets 22 days post injection and were divided into three groups with different isocaloric custom diets enriched with monounsaturated fatty acids (MUFAs), n6-PUFAs, and n3-PUFAs. Dissected mouse brains were sectioned on a cryomicrotome at 10 µm thickness and a 9-aminoacridine MALDI matrix was applied using an HTX robotic sprayer. MSI experiments were performed on a 15T solariX XR FT-ICR mass spectrometer operating in negative ion mode using 100 µm spatial resolution. Analysis of lipids and free fatty acids showed that each diet led to the enrichment of the corresponding lipid species, illustrating that administration of the diets resulted in increases in the lipid species of interest in the brain and within the tumor. Cyclic immunofluorescence (CyCIF) was performed and tumor cell populations in the n3-PUFA diet cohort showed a decrease in OPC-like cells and an increase in AC-like cells, while MUFA and n6-PUFA diet cohorts had a larger population of OPC-like cells compared to AC-like cells. MSI was also performed on patient samples and future work includes CyCIF analysis to determine if there is an n3-PUFA associated OPC-like to AC-like shift.
Astrocytes and Sleep: The Effect of Sleep Deprivation and Recovery on the Astrocyte Proteome
Andrew D. Brown1,2, Caroline M. Jipa1, Ashley M. Ingiosi1,2
1Department of Neuroscience, The Ohio State University, Columbus, OH
2Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, OH
Sleep is a homeostatically regulated process where sleep need accumulates during periods of wakefulness and declines during sleep. Currently, the cellular and molecular mechanisms behind how sleep need is encoded, interpreted, and acted upon in the brain are incompletely understood. Recent work showed brain cells called astrocytes respond dynamically to sleep/wake states and disrupting astroglial signaling impairs the brain’s ability to encode and accumulate sleep need. To better define the interplay between astrocyte signaling and sleep homeostasis we conducted a proteomic analysis of cortical mouse astrocytes after sleep, sleep deprivation, and recovery. We split 8-week-old male mice into homecage control (HC; n=33) and sleep deprived (SD; n=36) groups. SD mice were sleep deprived for 6 hours starting at light onset and then either sacrificed immediately (n=18) or after 3 hours of recovery sleep (n=18) and brains collected. HC mice were left undisturbed but similarly sacrificed at 6 (n=18) or 9 (n=15) hours after light onset. Samples were pooled, producing 5-6 biological replicates per condition, and cortical astrocytes were isolated from each sample using a magnetic bead-based approach. Ultra performance liquid chromatography tandem mass spectrometry detected over 6,000 proteins, including over 2,500 proteins above a minimum intensity threshold of 50,000 for all samples. Removal of Unwanted Variance analysis was used to normalize data and identify proteins differentially expressed between treatments. We found 1086 differentially expressed proteins: proteins related to mRNA processing and lipid synthesis were upregulated after recovery sleep whereas proteins related to glycolysis, actin binding, and trafficking were downregulated. Next, we will conduct phosphoproteomics on these samples to see how proteins activate and deactivate in relation to homeostatic sleep processes. We will also replicate this study using mice with impaired astrocyte calcium signaling – a pathway shown to mediate sleep homeostasis – to determine which of these protein changes may be calcium-dependent.
Using Global and Targeted Proteomics to Match Biofluids to Biomarker Candidates
Katelyn B. Brusach¹, Ariana E. Shannon², Alex W. Joyce², Jessica M. Quimby¹, Brian C. Searle²
1Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, Columbus, OH
2Department of Chemistry, The Ohio State University, Columbus, OH,
Selecting the optimal biofluid for accurate biomarker assessment is vital to an informative clinical assay. However, in the initial stages of candidate biomarker discovery, the biologically appropriate biofluid might be unclear. To resolve this dilemma, we demonstrate a mass spectrometry-based workflow where paired urine, plasma, and serum samples are processed in parallel, permitting the comparison of relative protein abundance across biofluids. Additionally, we ran a targeted parallel reaction monitoring assay on proteins of interest in diseased and healthy models to evaluate biofluid biological relevance. Using this workflow and preliminary data, we explore the surrogacy and superiority of blood biofluid proteins compared to urinary proteins relating to sample processing, relative protein quantification, and clinical application. Our results suggest that urine is a uniquely interesting biofluid that represents blood biofluids without requiring venipuncture or sample depletion of high abundant proteins.
A 2D Microfluidic Paper-Based Analytical Device for Diagnosis of Canine Visceral Leishmaniasis via Mass Spectrometry-Based Immunoassay
Hianka J.C. de Carvalho1,2, Ayesha Seth¹, Ruth Speidel¹, Ana Lúcia Abreu Silva3, Hélida M. de Andrade4, Maria A. Miglino5, Abraham K. Badu-Tawiah¹
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH
2Faculty of Veterinary Medicine and Anima Science, University of São Paulo, São Paulo-SP, Brazil
3Department of Veterinary Pathology, State University of Maranhão, São Luís-MA, Brazil
4Department of Parasitology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte-MG, Brazil
5Department of Veterinary Medicine, University of Marília, Marília, Brazil
Canine visceral leishmaniasis (CVL) is a life-threatening neglected disease caused by Leishmania infantum, which can be transmitted to humans. Affordable and sensitive diagnostic tools are needed for CVL prophylaxis. This work represents the first indirect immunoassay using our paper-based microfluidic platform for capturing the CVL biomarker. We use on-chip Paper Spray Mass Spectrometry (PS-MS) analysis to achieve sensitive detection of captured markers. In our assay, recombinant K39 anti-L. infantum was mobilized in the bioactive paper chip as capture antigen (cAg). Positive serum containing the biomarker (canine IgG anti-L. infantum), and control samples were added to the spots for 20 minutes. Detection antibody (dAb) Goat anti-canine IgG conjugated to pH-sensitive ionic probes synthesized in our lab was added, completing the assay. The probes were then cleaved, releasing the mass tags, which were picked up analyzed by the on-chip PS-MS method. Immunoassay establishment included optimization of type of blocking buffer, cAg concentration, and incubation time of cAg, blocking, and serum. Calibration curve was provided using a positive serum sample with antibody titer of 1:1280, performing 13 different dilutions. We calculated the detection limit (LOD) of our device to be 2×104, corresponding to 1:4000 dilution. Analysis of 20 clinical samples with our 2D paper device demonstrated its capacity in differentiating between CVL positive and negative canine serum samples previously analyzed by immunofluorescence assay (IFA), a gold standard serological method for CVL diagnosis. We applied Pearson’s statistical analysis to determine correlation between IFA and our method, and analysis yielded a strong correlation coefficient (r = 0.76). Finally, stability studies showed that our device can be stored at room temperature for at least 30 days without affecting the clinical outcome. The sensitivity, stability, and low-cost of our device will allow miniature MS to be used as point-of-care for large-scale CVL diagnosis even in remote areas.
Lipidome Isotope Labelling of Gut Microbes (LILGM): A Method of Discovering Endogenous Microbial Lipids
Li Chen1,2, Rui Xu¹, Jiangjiang Zhu1,2
1Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, OH
2James Comprehensive Cancer Center, The Ohio State University, Columbus, OH
Lipidomics analysis of gut microbiome has become critical in recent surge of extensive human disease studies that investigate microbiome contributions. However, challenges remain in comprehending the origins of thousands of lipid species produced by the diverse microbes. Here, we proposed the development and utilization of a liquid chromatography-mass spectrometry-based approach, named lipidome isotope labelling of gut microbes (LILGM), which enables confident detection and identification of endogenous gut microbial lipidome via 13C/15N labeling strategy and high-resolution mass spectrometry. Our method leveraged in vitro microbial cultures and stable isotope-labeled 13C and 15N, allowing a reasonable degree of isotope incorporation into microbial lipids over short-term of inoculation. We then systematically detected the mass spectral patterns of 182 labeled lipid species by our in-house data analysis pipeline. Further bioinformatics analyses confidently identified biologically relevant microbial lipids from lipid classes such as diacylglycerols (DGs), fatty acids (FAs), phosphatidylglycerols (PGs), and phosphatidylethanolamines (PEs) that may have profound impacts to human physiology. Our study also demonstrated the application of LILGM by showcasing the confident detection of dysregulated microbial lipids post antibiotic perturbation. The debiased sparse partial correlation analysis provides insights into lipid metabolism intricacies. Overall, our method can provide unambiguous analyses to the endogenous microbial lipids in given biological context, and can also instantly reflect the lipidomic changes of gut microbes in response to environmental factors. We believe our LILGM approach has the potential to provide new body of knowledge by combining promising analytical approaches for sensitive and specific lipid detection to support functional microbiome studies.
Mass Spectrometry Imaging of Pimonidazole as a Hypoxia Marker in 3D Cell Cultures
Andrew T Freeman1, Arbil Lopez1, Amanda B. Hummon2
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH
2Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, and Comprehensive Cancer Center, The Ohio State University, Columbus, OH
Cancer is a leading cause of death worldwide. The United States is projected to have an estimated 2,001,140 new cases and 611,720 deaths in 2024. Thus, cancer treatment is an active area of research, including research with tumor models. Spheroids are three-dimensional cell cultures that model early-stage tumors that have not yet vascularized. Spheroids are created by aggregation of immortalized cells, which then proliferate in a radially symmetric manner. Over time, chemical diffusion gradients for oxygen, nutrients, and metabolic waste result in phenotypic layers: an outer proliferating cell layer, a middle quiescent cell layer, and a central necrotic core. The cells are in a hypoxic environment when the partial pressure of oxygen is lower than 10 mmHg. Hypoxic cells are more chemoresistant and have been linked to angiogenesis and aggressive tumor progression. There is a need to develop and evaluate new therapeutics that will treat these resistant cells. 2-Nitroimidazole compounds are not commonly found in nature and are metabolized mainly by nitroreductases under hypoxia conditions, and their metabolites are used to track hypoxia in tumors. Pimonidazole specifically is a 2-nitroimidazole used in clinical settings and the metabolism of this compound can be tracked by matrix–assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). We are utilizing MALDI-MSI to image pimonidazole as a marker of cellular hypoxia in spheroids. Four unique mass to charge (m/z) values corresponding to five metabolites have been observed in spheroids dosed with pimonidazole and harvested after 24, 48, and 72 hours of treatment. Co-localization of pimonidazole’s metabolites with metabolites of chemotherapy drugs will be examined to determine their effectiveness in hypoxic cells. In the future, we plan to utilize vascularized spheroids as later-stage tumor models to determine how the models differ in hypoxia regions and drug metabolism. The results will allow for more targeted chemotherapy treatments.
mmCloud: A Unified Cloud-Based Platform for Oligonucleotide and Protein Analysis
Dr. Michael A. Freitras1,2, Andrii Albatov1, G. Hall Johnson1, Herman Singh1, John Rose1, Mike Kolich1
1MassMatrix, Inc., Columbus, OH 43220
2The Ohio State University, College of Medicine, Columbus OH, 443210
MassMatrix’s mmCloud is a comprehensive cloud-based platform that integrates mmOligo and mmSearch, providing researchers with powerful oligonucleotide and protein analysis tools. Designed for scalability and accessibility, mmCloud streamlines workflows by offering unified access to these advanced analysis tools. With an API-driven backend, mmCloud supports custom solution development, making it adaptable to various research needs. This flexibility allows seamless integration with existing systems and customization to meet specific project requirements. Critical features of mmCloud include its ability to provide unified access to oligonucleotide and protein data, simplifying research processes, and improving efficiency. The platform’s API-driven customization allows users to tailor the platform to their needs, enabling innovative solutions that align with their specific research goals. With universal data access, researchers can access and share data from any location, enhancing collaboration and productivity. The mobile-friendly design ensures that users can stay connected to their research on the go. Additionally, mmCloud’s scalability makes it suitable for projects of all sizes, from individual studies to large-scale collaborative efforts. This presentation will highlight the mmCloud’s platform’s automated intact mass and oligonucleotide sequencing capabilities. The integration of mmOligo within mmCloud is particularly valuable for developing oligonucleotide-based therapeutics. mmOligo leverages advanced mass spectrometry (MS)–based data analysis to enable accurate sequencing of standard and hybrid nucleotide sequences. This capability is crucial for the direct sequencing, quality assurance, and quality control of complex and highly modified oligonucleotides, essential for developing new therapeutic solutions. By providing semi-automated direct sequencing, mmOligo improves reliability and accelerates the development process.
Spatial and Diurnal Variations in Sodium Content in Intervertebral Disc Tissues
Jordin Marshall1, A. Lutton2, A. Joenathan3, M. Grinstaff3, J. Olesik2, B.A. Walter1,4
1Department of Biomedical Engineering, The Ohio State University, Columbus, OH
2School of Earth Sciences Trace Element Research Laboratory, The Ohio State University, Columbus, OH
3Department of Biomedical Engineering, Boston University, Boston, MA
4Department of Orthopaedics, The Ohio State University Wexner Medical Center, Columbus, OH
Cyclic changes in tissue osmolarity resulting from diurnal loading of the charged and hydrated extracellular matrix (ECM) are a biophysical signal through which mechanical loading regulates cellular metabolism within the intervertebral disc (IVD). However, the magnitude of the diurnal osmotic cycle cells experience remains unknown as the composition of the ECM and its fixed charge density (FCD) varies spatially; regionally between the nucleus pulposus (NP) and annulus fibrosus (AF) and on the microscale Spatial between the pericellular matrix (PCM) and the surrounding ECM. The objectives of this study were to: 1) validate a novel application of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to assess FCD via measurement of intra-tissue sodium and 2) to utilize LA-ICP-MS to assess temporal and spatial changes in FCD over a diurnal loading cycle to inform the magnitude of the osmotic cycle in-situ. Following equilibration dialysis of NP tissue in 5-25% polyethylene glycol solutions, LA-ICP-MS measurements of intra-tissue sodium were validated against paired measurements of FCD via inductively coupled plasma optical emission spectroscopy (ICP-OES) and via LA-ICP-MS measurements of iodine after staining with an iodine-based cationic contrast agent. A high linear correlation (R>0.90) was observed between all measurements. Bovine caudal motion segments were loaded over a 40 (n = 4) or 48 (n = 4) hour period under simulated physiologic loading and sodium was measured via LA-ICP-MS and ICP-OES. In the NP, 40h FCD (0.52 mEq/g wet wt) was greater than 1.5x the 48h FCD (0.34 mEq/g wet wt) via LA-ICP-MS, with no diurnal change observed in the AF. Lastly, the ratio of PCM to ECM at the end of day (2.3) was ~1.5x higher than at the end of night (1.5). Overall, this technique is capable of accurately measuring intra-tissue sodium, as well as diurnal and spatial differences in sodium content and osmotic cycles.
Unprecedented Alkylation of the Catalytic Histidine in the Aging of Cholinesterases after Inhibition by Organophosphorus Pesticides
Kevin A. Miller1, Yiran He1, Stacey K. Allen1, Craig A. McElroy2, Christopher S. Callam1, Christopher M. Hadad1
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210
2InfinixBio, 1507 Chambers Road, Columbus, OH 43212
Organophosphorus compounds, including many pesticides, covalently inhibit the serine residue of the essential enzyme acetylcholinesterase (AChE). As a result, inactive AChE leads to cholinergic crisis and potentially death, if not treated. Current oxime therapeutics are capable of reactivating inhibited AChE; however, they provide limited efficacy on many fronts. Most dramatically, if inhibited AChE is left untreated for too long, a second spontaneous dealkylation event can occur in a process termed aging which results in a form of the enzyme that is recalcitrant to oxime reactivation. For decades, the mechanism of aging has been generally accepted to be a spontaneous dealkylation event via a carbocation-mediated mechanism or by water hydrolysis of the phosphylated serine adduct. The result is a phosphylated serine adduct with an oxyanion that is stabilized through a salt bridge between the nearby catalytic histidine residue. Here we report, for the first time, that aging of certain pesticides can result in an alkyl transfer to the catalytic histidine in the active site via an SN2-like mechanism. This result was predicted computationally and experimentally confirmed using bottom-up proteomics. Using this proteomics approach, we show the alkyl transfer is pH dependent in which at lower pH, less histidine alkylation is observed while at higher pH, more alkylation is observed. We also demonstrate that the alkyl transfer occurs in butyrylcholinesterase (BChE), a sister enzyme to AChE, though to a lesser extent in BChE. Furthermore, the Hadad lab has synthesized quinone methide precursors (QMPs) which are capable of recovering the aged form of AChE in a process termed “resurrection”. The work presented here also highlights the effect of this novel aging pathway on the recovery of aged-AChE by QMPs, and its broader impact on the design of future therapeutics in the treatment of organophosphorus exposure.
Coupling Capillary Zone Electrophoresis with Native MS and Surface-Induced Dissociation for Native Proteomics
Zihao Qi1,2, William J. Moeller1,2, Qianjie Wang3, Qianyi Wang3, Liangliang Sun3, Vicki H. Wysocki1,2
1The Ohio State University; Columbus, OH
2Native Mass Spectrometry Guided Structural Biology Center, Columbus, OH
3Michigan State University, East Lansing, MI
Native proteomics samples endogenous proteoforms. Capillary zone electrophoresis (CZE) is as an appealing separation technique for proteomics due to its high sensitivity and resolution. Native mass spectrometry (nMS) uses electrospray ionization (ESI) to transfer large biomolecules into the gas phase while retaining their non-covalent interactions. Due to the recent development of robust and sensitive electrospray interfaces, coupling CZE with nMS became feasible. CZE-nMS enables the efficient separation of intact protein complexes from a mixture, followed by accurate mass measurements. Here, for an E.coli cell lysate, we successfully separated and detected about 100 protein complexes from a range of masses spanning 20-400 kDa. Surface-induced dissociation (SID) of protein complexes separated by online CZE was also performed. SID directly cleaves interfaces of protein complexes in the gas phase such that SID fragmentation products reflect the native structures of protein complexes. To develop native CZE-SID MS, we first used a simple mixture system containing streptavidin (SA) and C-reactive protein (CRP) under both normal-charge and charge-reduced conditions. The SID fragmentation pattern of each protein complex matches observed patterns from previous nMS experiments, suggesting that CZE does not disrupt native-like topology. Using that information as the foundation, we then applied SID as an MS/MS method for a complex mixture. For example, a highly abundant hexameric protein complex in E.coli cell lysate was observed, and SID suggested it is a trimer of dimers; combined with top-down proteomics, we were able to identify this protein as glutamate decarboxylase beta with high confidence. By combining CZE-nMS-SID and CZE-based top-down proteomics, we were able to unravel the whole hierarchy of protein structures, from primary sequences and potential post-translational modifications (PTM) to assembly, topology, and even binding partners. Collectively, adding SID to the CZE-based native proteomics toolbox provides an additional layer of structural information and facilitates high-confidence proteome identification.
In Droplet Hydrogen-Deuterium Exchange and Computational Markov State Modeling for Understanding the Local Conformational Landscape of a Ligand-Protein System
Mohammad Rahman1, MST Nigar Sultana1, Kevin Courtney2, Blake Mertz1,3, Stephen Valentine1
1Department of Chemistry, West Virginia University, Morgantown, WV 26506
2Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, WV 26505
3Modulus, Inc.
Intrinsically disordered regions (IDRs) within proteins often undergo rapid conformational changes upon binding ligands such as drug candidates. Mapping such rapid solution state conformational dynamics is challenging through conventional biophysical techniques. Recently, the novel ionization technique, capillary vibrating sharp spray ionization (cVSSI) was developed and shown to provide superior performance in many native mass spectrometry (MS) experiments. In this work, an in-droplet hydrogen-deuterium exchange (HDX)-MS foot-printing method is performed on the sub-millisecond timescale. In conjunction with experiments, Markov-State Modelling (MSM) based on Molecular dynamics (MD) simulations enables the capture of local folding/unfolding events. In preliminary studies, human FK506 binding protein (FKBP12) is examined upon incubation with two well-characterized ligand molecules (i.e., FK506 and rapamycin) to map binding/release events, competitive binding, and conformer heterogeneity associated with folding/unfolding. His-tagged RNA sequence has been obtained from a commercial source and then cloned into the pET-sumo vector domain where the protein is subsequently expressed and purified as the SUMO-FKBP12 protein complex. Next, the protein is separated from the SUMO-domain and further purified using Fast Protein Liquid Chromatography (FPLC). This is followed by buffer exchange to provide an MS-quality sample for in-droplet HDX-MS measurements. MD simulations have been performed using NAMD3 precompiled binary and Markov-state modeling has been performed as described in the MSM modeling method described elsewhere. MSM provides a conformational ensemble with global minima as well as local minima. Computational modeling will be utilized for each conformational ensemble from MSM and compared with experimental HDX-MS results to provide solution state folding/unfolding information upon ligand binding.
Charge Transfer Dissociation Mass Spectrometry (CTD-MS) for the Structural Characterization of Polyethylene Glycol (PEG) Polymers with Complex End Groups
Emily A. Ruiz1, Inès Aloui2, William Buchman2, Jean-Yves Salpin2, Glen P. Jackson1,3
1C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506
2Université Paris-Saclay, Univ Evry, CY Cergy Paris Université, CNRS, LAMBE, 91025, Evry-Courcouronnes, France
3Department of Forensic and Investigative Science, West Virginia University, Morgantown, WV 26506
In this project, we describe how a novel method of tandem mass spectrometry called charge transfer dissociation-mass spectrometry (CTD-MS) can be used to elucidate the chemical composition of polymers. The characterization of polymers and their end groups is important when determining the mechanism of polymerization and their chemical properties. Tandem mass spectrometry (MS/MS) is commonly used for the characterization of polymers at the oligomer level and has been used to determine end-groups and monomer connectivity along the polymer backbone. One of the most common MS/MS techniques used for these purposes is collision induced dissociation (CID). Unfortunately, CID is not effective for all polymers or adduct types and can exclude important information such as end group functionalities.
Ultraviolet photodissociation (UVPD) is gaining popularity for polymer analysis because UV photons lead to both high and low energy fragmentation. CTD-MS, wherein precursor ions are bombarded with keV helium cations, has produced tandem mass spectra similar to UVPD and extreme-UVPD. As a proof of concept, the current work compares CID, UVPD, and CTD activation of a poly(ethylene glycol) polymer with a nonylphenyl end group. All three activation techniques provided ample monomeric structural information, but CID failed to produce product ions within the polymer end groups. Unlike CID, UVPD and CTD both produced product ions with an additional charge and radical relative to the precursor. Neutral losses from this oxidized radical species provide structural information about the end group of the polymer. For example, peaks at m/z 723, 716, 709, 702, and 695 are sequential losses along the alkane branch of the nonylphenyl endgroup. Whereas these peaks are present in both CTD and UVPD product spectra, CTD provides superior signal intensities by a factor of approximately 600x relative to UVPD spectrum, and CTD does not require the use of a synchrotron radiation source.
An In-Vitro Approach to Identify Key Urine Metabolomic Features in Response to a Tomato-Soy Intervention
Maria J. Sholola1, Jenna L. Miller1, Emma A. Bilbrey2, Molly A. Torok3, Janet A. Novotny4, David M. Francis5, Thomas A. Mace3, Jessica L. Cooperstone1
1Department of Food Science and Technology, The Ohio State University, Columbus, OH
2Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH
3Division of Gastroenterology, Hepatology, and Nutrition, The Ohio State University Wexner Medical Center, Columbus, OH
4Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD
5Department of Horticulture and Crop Science, The Ohio State University, Wooster, OH
Consumption of tomatoes and soybeans (separately and combined) are linked to improved health outcomes such as lower risk of prostate cancer and cardiovascular disease. Lycopene from tomatoes and soy isoflavones are often thought to produce these effects, however, there are numerous other probable compounds derived from these foods that could play a role. To elucidate which plant compounds may contribute, we aimed to understand metabolic changes with tomato-soy consumption. In a randomized, crossover clinical trial, 12 subjects with obesity consumed daily servings of a high-lycopene juice enriched with soy isoflavones (tomato-soy) or a control, yellow tomato juice without lycopene, each for 4 weeks. Plasma and 24-hour urine samples collected before and after each intervention were analyzed. Urinary metabolites were comprehensively assessed with UHPLC-QTOF-MS (ESI +/-) based metabolomics. Principal component analysis (PCA) was used to uncover natural clustering in the datasets. Features associated with tomato-soy intake were significantly altered (FDR p < 0.05, absolute fold change > 1.5) pre- vs. post-tomato-soy, and post-control vs. post-tomato-soy. Based on previous knowledge and MS/MS investigation, several standards along with hydrolyzed juice extracts were obtained to help biosynthesize and identify these features. Additionally, extracts were incubated with pig liver enzymes and co-factors to biosynthesize combinations of metabolism products. This in vitro approach allowed identification of 39 metabolites, many of which are soy isoflavone metabolites. Features not identified with in vitro standards were either confirmed with authentic standard or annotated based on MS/MS fragmentation patterns. PCA showed soy isoflavones as key drivers in differentiating post-tomato-soy samples, especially in negative ionization modes. These data show that soy is absorbed and metabolized after consumption of tomato-soy juice and could play a larger role than their parent compounds in health outcomes. Biosynthesis techniques prove valuable for identifying in vivo metabolites of food components for which authentic standards are lacking.
Beyond Volatile Organic Compounds (VOC) for Ignitable Liquid Residue Detection: Comparison of Results Using DART-MS and GC/MS
Mengliang Zhang1, Shurthi Perna2 , Ngee Sing Chong2
1Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio, 45701
2Department of Chemistry, Middle Tennessee State University, Murfreesboro, Tennessee, 37132
The detection of ignitable liquid (IL) residues is critical to the arson investigation process, which may potentially identify the cause of the fire and the ILs used to initiate the fire. The most commonly used technique to analyze IL is GC-MS; however, it has a major limitation of analyzing only the volatile organic compounds (VOC) in IL. The non-volatile or less volatile components in IL are likely to be contained in the fire debris and hence could yield corroborating evidence on the use of specific ILs in the investigation. Direct analysis in real time mass spectrometry (DART-MS) is an emerging analytical technique in the forensic community that has shown promising capability in drug analysis. However, its potential in other forensic fields, such as IL analysis, has not been fully explored. In this study, the IL, such as gasoline, paint thinner, and biodiesel were analyzed by DART-MS, and the results were compared with the traditional GC-MS method.
In comparison to the DART-MS data, the GC-MS profiles changed significantly among weathered IL samples because the weathering process alters the relative quantities of the IL. The DART-MS data of ILs provided less variable profiles and, therefore, may not be used to predict the weathering percentage of IL. However, the capability of DART-MS in detecting the less volatile IL constituents, such as fuel additives in gasoline and glycol ethers in paint thinner, imparts its unique ability to discriminate among different types of ILs.