Posters | Ohio Mass Spectrometry Symposium

Poster Listing (submitted abstracts)

Note: Odd numbered posters will present during Session 1 (9:05-9:45 am); Even numbered posters will present during Session 2 (1:15-1:45 pm).

Posters are clustered by general topic: MS Fundamentals (posters 1-12); Diagnostics and Metabolomics (posters 13-28); Native MS (posters 29-36); Proteomics/Glycomics (posters 37-47); RNA (48-56)

Rebekah Strong; OSU Department of Chemistry and Biochemistry; Direct Synthesis of Primary Amines from Alcohols via Uncatalyzed Plasma-Microdroplet Chemistry
Adam Reed; OSU Department of Chemistry and Biochemistry; Enhanced Contained Electrospray Ionization and Fragmentation of Sterol and Nonpolar Analytes using Silver Coated Microcapillaries
Purva S. Damale; OSU Department of Chemistry and Biochemistry; Plasma-In-Droplet Ionization with Dynamic Power Supply for Liquid Chromatography-Mass Spectrometry
Alex Zumock; OSU Department of Chemistry and Biochemistry; Development of a Conductive Emitter for Dynamic Spray Mass Spectrometry
Keerthana Ravi; OSU Department of Chemistry and Biochemistry; Panoptic Mass Spectrometry: A Novel High-Throughput Ion Source for All States of Samples and Complex Mixtures
Shixiang Xu; OSU Department of Chemistry and Biochemistry; Development of Advanced Database Tools for Characterization of Saccharides Isomers with Negative-Ion Mode Direct Infusion Mass Spectrometry
Calum Bochenek; The University of Akron Department of Chemistry; Characterization of Crosslinked Networks by Thermal Desorption/Pyrolysis Interfaced to Direct Analysis in Real Time Mass Spectrometry (TDPy-DART-MS)
Santosh Raman Acharya; OSU Department of Chemistry and Biochemistry; Characterization of Human Milk Oligosaccharides (HMOs) Using Halid Adduction Mass Spectrometry
Owen Looker; OSU Department of Chemistry and Biochemistry; Site Selective Chlorination of Arenes and Heteroarenes Using Hypervalent Iodine Catalysis via the use of Microdroplet Chemistry
Luciana Rivera Molina; The University of Akron; MALDI and DART Mass Spectrometry Analysis of a Single Entity Microorganism Attached to a Disk Electrode
Courtney LaPointe; West Virginia University; Charge Transfer Dissociation (CTD) of Phospholipids with Alkali Metal Adducts
Herman Singh; MassMatrix, Inc.; Advancing Mass Spectrometry Data Management through Numerical Compression
Randy Arnold; Sciex; Scout triggered MRM (stMRM): A Method to Acquire Large Numbers of MRM without Predefined Retention Time
Chao Guo; OSU Comprehensive Cancer Center & Department of Human Sciences; Investigating PFAS Induced Perturbation to Gut Microbiota and their Metabolism in a Host-Free Human Colonic Model
Huan Zhang; OSU Comprehensive Cancer Center & Department of Human Sciences; Discovery of Noninvasive Biomarkers for Radiation Exposure via LC-MS-based Hair Metabolomics
Xinru Pang; OSU Comprehensive Cancer Center & Department of Human Sciences; Adaptive Metabolic Responses to Single-Gene Deletions in Escherichia coli: A Non-targeted LC-MS Analysis
Sophia K. Wu; OSU Radiation Oncology; UPP1 Contributes to the Maintenance of Cancer Stem Cells in Ovarian Cancer via Metabolism Reprogramming
Ziqi Li; OSU Food Science and Technology; LC-MS Metabolomics Reveals How Food Proteins Affect Iron Absorption Using In Vitro Digestion Coupled with a Caco-2 Cell Intestinal Model
Manpreet Kaur; OSU Food Science and Technology; Effect on Volatile Profile of Growth Temperature, Tissue Type and Sex in Fresh Atlantic Salmon (Salmo salar) using SIFT-MS
Victoria Hermes de Vargas; OSU Food Science and Technology; Plant-Based Milk: A Metabolomic Approach to Evaluate Chemical Profile Changes Induced by Innovative Processing Technologies
Niraj Panday; OSU Department of Chemistry and Biochemistry; Deep Lipidomics Mass Spectrometry Analysis of Complex Mixtures Enabled by In-Situ Droplet Reactions Flowing Liquid Chromatographic Separation
Octavion Spears; OSU Department of Chemistry and Biochemistry; Online Fractionation of Complex Lipid Mixtures on SPE Cartridge followed by In-Situ Mass Spectrometry Analysis
Ruth Speidel; OSU Department of Chemistry and Biochemistry; University of São Paulo, Brazil; Development of Mass Spectrometry-Based Immunoassay for Microfluidic Paper-Analytical Device for the Early Prediction of Severe Acute Pancreatitis 
Shiqi Zhang; OSU Comprehensive Cancer Center & Department of Human Sciences; Distinct Plasma Molecular Profiles between Early-Onset and Late-Onset Colorectal Cancer Patients Revealed by Metabolic and Lipidomic Analyses
Kyungsuh Lee; OSU Department of Chemistry and Biochemistry; Enhanced Lipidomic Profiling of Pseudomonas aeruginosa through Advanced NMR and MS Techniques
Cristian (Daniel) Quiroz-Moreno; OSU Horticulture and Crop Science; Quantitative Analysis of Plant Phenolics by LC-MS/MS, and PhenolicsDB: A Publicly Available High-Resolution MS/MS Spectral Library
Aaron Wiedemer; OSU Food Science and Technology; Arugula Grown With Elevated Environmental Sulfur Produces More Glucosinolates and Isothiocyanates
Lydia Balogh; OSU Department of Horticulture and Crop Science; Linking Apple Fruit Phenolics to Human Taste Perception
Philip Lacey; OSU Department of Chemistry and Biochemistry; Non-Canonical Inflammasome LPS-mediated Oligomerization Probed by Electron Capture Charge Reduction
Luis Andres Casavilca Ramirez; OSU Department of Chemistry and Biochemistry; Molecular Dynamics Modeling of Gas-Phase Compaction of a Flexible Enzyme
Yuan Gao; OSU Department of Chemistry and Biochemistry; Native MS Characterization of the Binding of Substrate and Inhibitors to Salmonella FraB Delycase, a Drug Target
Mst Nigar Sultana; West Virginia University; Mapping Protein-Ligand Ion Stability Landscapes Using a cVSSI Approach
Kristie Baker; OSU Department of Chemistry and Biochemistry; Study of a Surface Salt Bridge’s Effect on Structure and Stability of a Four Helix Bundle
Zhixin Xu; OSU Department of Chemistry and Biochemistry; Determination of Glycoform Masses of SARS-COV-2 Spike Protein Variants by Electron Capture Charge Reduction (ECCR) Mass Spectrometry
Evan Whitford; OSU Department of Chemistry and Biochemistry; Probing the Structure of Covalently Labeled Proteins in the Gas Phase by Native MS
William Moeller; OSU Department of Chemistry and Biochemistry; Variable Temperature Electrospray Measurement of Enthalpy and Entropy of Tryptophan Binding to Ring Shaped Protein TRAP
Madalyn Moore; OSU Department of Chemistry and Biochemistry; Using Pediatric Urine Proteomics to Identify Potential Chronic Pancreatitis Biomarkers
Gautam Ghosh; OSU Department of Chemistry and Biochemistry; Investigating MBNL1 Splicing Variants in Glioblastoma Using DDA and PRM Techniques
Melanie Chen; OSU Biomedical Informatics; Investigating GRP94 Functionality in Protein Folding Through Thermal Proteome Profiling
Alex Joyce; OSU Biomedical Informatics; Exploring Biofluid Protein Expression with CATalog, an Interactive Proteomics Dashboard
Jacelyn Greenwald; OSU Department of Chemistry and Biochemistry; Surface Proteomics to Identify Therapeutic Targets in Search for Therapeutic Targets in ALK Positive Lung Cancer
Dijana Vitko; Bruker; Increasing Throughput While Maintaining Coverage Depths in Single Cell Proteomics Using the timsTOF Ultra2
Eman Mohamed; Cleveland State University; Study the Mechanisms of Releasing Dynorphin B in Mice Cerebrospinal Fluid and Cochlear Fluid
Regina Edgington; OSU Department of Chemistry and Biochemistry; Identifying Peptides Outside the Mass Spectrometry Search Space through Mining of Evolutionary Peptide Variation
Andrew Arslanian; OSU Department of Chemistry and Biochemistry; Roughhousing with Ions: Surface-Induced Dissociation and Electron Capture Dissociation as Diagnostics of Q-Cyclic IMS-TOF Instrument Tuning Gentleness
Martha Ortega Zepeda; OSU Department of Chemistry and Biochemistry; Surface-Induced Dissociation (SID) for Comprehensive Glycopeptide Analysis
Madeline Schuch; West Virginia University; Charge Transfer Dissociation Mass Spectrometry (CTD-MS) for the Structural Characterization of Glycopeptides
WITHDRAWN
Anju Sheregar; OSU Department of Chemistry and Biochemistry; Facilitating Disease Diagnosis Detection of RNAs by Ambient Ionization Mass Spectrometry
Emily Fairchild; Ohio University; Novel RNA-Ligand Complex Formation for Antibiotic Drug Design: Method Design and Initial Results
Bibek Hamal; University of Cincinnati; Mapping tRNA Modifications Using LC-MS/MS
Jennifer Kist; University of Cincinnati; Offline HPLC Fractionation and LC-MS/MS Characterization of tRNAs
Aastha Gyawali; University of Cincinnati; Simplifying Eukaryotic Transfer RNA Modification Mapping via FPLC
Hina Zain; University of Cincinnati; Liquid Chromatography-Mass Spectrometry Analysis of Total tRNA using RNase 4
Asif Rayhan; University of Cincinnati; Nucleoside Modification Mapping by Genome-Independent Universal Mass Exclusion List of Unmodified Oligonucleotides During LC-MS/MS
Kaylee Grabarkewitz; OSU Department of Chemistry and Biochemistry; Role of Transcriptional Start-Site Differences in the HIV-1 Genomic RNA 5’UTR on Gag Binding and RNA Dimerization

Core Facility and Resource Posters

Campus Chemical Instrument Center Mass Spectrometry and Proteomics Facility

The Campus Chemical Instrumentation Center (CCIC) Mass Spectrometry and Proteomics (MSP) Facility provides advanced mass spectrometry based proteomics (bottom-up, top-down, PTM analysis and quantitation), metabolomics (targeted and untargeted, with multiple modes of separation and ionization), characterization of protein complexes (protein-protein, protein-RNA/DNA, and protein-ligand), mass spectrometry imagining, and general mass spectrometry services (including ultrahigh resolution MS analysis) to OSU members, other universities and industry. We provide consultation with interested researchers prior to project initiation to aid in experiment design and assistance in grant and manuscript preparation.

Native MS-guided Structural Biology Center

The Native Mass Spectrometry Guided Structural Biology (nMS→SB) Center is a Biomedical Technology Development and Dissemination (BTDD) Center funded by the NIH National Institute of General Medical Sciences with a RM1 grant. As a national BTDD Center, our purpose is to develop and disseminate robust technologies throughout the broader biomedical research community. The nMS→SB Center will produce robust, vendor neutral, technology for characterizing the assembly and disassembly of macromolecular protein complexes, including the definition of their compositions, PTMs, stoichiometry, structure/architecture/topology, flexibility/stability, and their conformational/binding-induced changes under different conditions. Methods are being developed for protein-protein, protein-ligand, and protein-DNA/RNA complexes. Our mission is to develop improved native MS as a routine tool and disseminate the technology to the biomedical research community through vendor partnerships and training.

Poster Abstracts (submitted abstracts)

In alphabetical order by presenter

Characterization of Human Milk Oligosaccharides (HMOs) using Halide Adduction Mass Spectrometry
Santosh Raman Acharya, Enoch Amoah, Abraham Badu-Tawiah
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210

Human milk oligosaccharides (HMOs) play a critical role in neonatal immune development and the modulation of the infant gut microbiome. The complete biosynthesis of HMOs and the fortification of infant milk products are increasing the demand for methods capable of in-depth structural characterization. Their analysis, however, is complicated by low ionization efficiency and structural complexity, including variations in linkage and stereoisomerism. Traditional mass spectrometry (MS) methods often involve extensive sample preparation and advanced instrumentation, which can limit their widespread application. In this study, we introduce a novel, streamlined method for HMOs quantification using nano electrospray ionization (nESI) coupled with tandem mass spectrometry (MS/MS). This technique involves the generation and analysis of halide adducts of isomeric HMOs, producing diagnostic ions that enable precise isomer identification. For initial experiments we used set of 8 different structural isomers of HMOs which includes three lacto-N-fucopentose (LNF-I, II, III and IV), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexalose (LNH) and lacto-N-neohexalose (LNnH). For example, LNF-I and LNF-II being isomers provides [M+Cl]– peak at 888 m/z but under collision induced dissociation (CID) generates diagnostic ions at 307 and 330 m/z respectively. Our method eliminates the need for prior chromatographic separation and is compatible with portable MS systems, providing a cost-effective and efficient solution for HMO characterization in milk supplements. We will present data for the analysis of for fortified infant formula.

Scout Triggered MRM (stMRM); A Method to Acquire Large Numbers of MRM without Predefined Retention Time
Randy Arnold, Holly Lee, David Cox, Yves Le Blanc
SCIEX

Using a Scout triggered multiple reaction monitoring (stMRM) acquisition method, it was possible to automatically schedule the acquisition of over 300 MRM traces across five different LC workflows offered on the Evosep One system (60 to 500 SPD) using a single acquisition method.

The selectivity and sensitivity of triple quadrupole systems make them the instrument of choice for quantitative LCMS/MS. In MRM mode, triple quads offer the highest duty cycle and large numbers of analytes (>100) can be monitored. This number can be significantly increased by scheduling the analysis and acquiring signal during a narrow time window at the expected retention time (RT). This concept, referred to as Scheduled MRM, implies reproducible and stable LC conditions for all analytes. Therefore, any changes in LC conditions require the adjustment of the time windows. This can be a daunting task for a large MRM cohort. Here we propose the use of stMRM which is designed to dynamically adapt to any changes that are associated with LC conditions.

Using Scout triggered MRM (stMRM) offers the opportunity to have a single acquisition method that can be used for large panels of peptides across different LC workflows provided by the Evosep One system. This provides greater flexibility in managing sample throughput by eliminating the need to adjust MRM scheduling for each LC analysis. Scout triggered MRM provides the added benefit of maximizing the number of data points across the LC peak, thus providing more confidence in the analyte detection and quantitation as demonstrated in other work.

Roughhousing with Ions: Surface-Induced Dissociation and Electron Capture Dissociation as Diagnostics of Q-Cyclic IMS-TOF Instrument Tuning Gentleness
Andrew J. Arslanian^1,2, Vicki H. Wysocki^2,3
¹ The Ohio State University
²Native MS Guided Structural Biology Center
³Georgia Institute of Technology

Native mass spectrometry can characterize a range of biomolecular features pertinent to structural biology, including intact mass, stoichiometry, ligand-bound states, and topology. However, when an instrument’s ionization source is tuned to maximize signal intensity or adduct removal, it is possible that the biomolecular complex’s tertiary and quaternary structures can be rearranged in a way that no longer reflects its native-like conformation. This could affect downstream ion activation experiments, leading to erroneous conclusions about the native-like structure. One such activation strategy is surface-induced dissociation (SID), which generally causes native-like protein complexes to dissociate along the weakest subunit interfaces, revealing critical information about the complex’s native-like topology and subunit connectivity. This makes SID an excellent tool to diagnose quaternary structure rearrangement and help tune an instrument’s source and other upstream transmission regions to strike the balance between signal intensity, adduct removal, and conserving the native-like structure. Complementary to SID results, electron-capture dissociation (ECD) was also used after quaternary structure rearrangement to confirm that the subunit interfaces were rearranged, opening the structure to electron capture and subsequent dissociation. These strategies were applied to the standard protein complexes streptavidin, C-reactive protein, and TRAP to fine-tune the voltage gradients of the recently developed Waters SELECT Series Cyclic IMS (Q-IM-TOF), ensuring its suitability for native mass spectrometry. When these complexes were subject to high degrees of in-source activation the SID results reflected rearranged quaternary structures, and evidence for secondary fragmentation due to the high amount of internal energy the ions absorbed during in-source activation and SID. The ECD results for streptavidin also revealed that in-source activation led to rearrangement of the subunit interfaces. These results provide a valuable guide for new practitioners to native mass spectrometry and highlight the importance of using standard protein complexes when tuning new instrument platforms for optimal native mass spectrometry performance.

Study of a Surface Salt Bridge’s Effect on Structure and Stability of a Four Helix Bundle
Kristie L. Baker^1,2, Anusha Kumar¹, Aniruddha Sahasrabuddhe^1,2, Hossein Ashrafian¹, Thomas J. Magliery¹, Vicki H. Wysocki^1,2
¹ The Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH
²Native MS Guided Structural Biology Center, Columbus, OH

Rop is a small, antiparallel, homodimeric, four-helix bundle protein used as a model system for understanding protein stability and structure. The structure of wild-type Rop shows a salt bridge between D32 and R55 at each end of the dimer interface, which may be important for stability or structural specificity. To characterize the salt bridge’s importance, R55 has been mutated to different acidic, basic, and nonpolar residues. Gas phase stability is determined by using surface-induced dissociation (SID) and surface induced unfolding (SIU). Solution phase stability is measured with a variable temperature electrospray ionization (vT-ESI) device. SID and SIU data are quantified by determining the transition energy, this is defined by the point where 50% of the precursors has been deleted for SID50 and the point where half of the precursor structure has been rearranged for SIU50. The complementary data from gas phase and solution phase measurements provide key information to understand the role of salt bridges in interfacial strength for Rop. The variants with an intact salt bridge show a large difference between their SID50 and SIU50 energies, with compaction occurring at a lower energy than dissociation. The variants with nonpolar mutations show a smaller difference in transition energy between SID50 and SIU50. R55E, with an acid-acid interaction, has dissociation and compaction occurring at nearly the same voltage. Variable temperature results show the general trend that variants with hydrophobic mutations, like R55L, are more stable than AV, which has two salt bridges, one at each end of the anti-parallel dimer. The increased stability of R55L may be due to hydrophobic interactions at the dimer interface, since position 55 is expected to be a core position based on the periodicity of helix.

Linking Apple Fruit Phenolics to Human Taste Perception
Lydia Balogh¹, Cristian D. Quiroz-Moreno¹, Diane D. Miller¹, Chris Simons², Jonathan Fresnedo Ramírez¹, Jessica Cooperstone^1,2
¹ Department of Horticulture and Crop Science, The Ohio State University
²Department of Food Science and Technology, The Ohio State University

Apples (Malus × domestica Borkh.) are the most consumed fruits by Americans. The familiar proverb “an apple a day keeps the doctor away” has received scientific support, much of which has been attributed to its polyphenols. Variation in phenolic profile in apples is influenced by genetics, therefore nutritional quality of apple fruits is modifiable. However, some phenolic compounds are hypothesized to contribute to impart bitterness in apples, which may reduce their acceptability by consumers. The goal of this work is to better understand the relationship between putatively bioactive phenolics in apples and consumer taste perception.

We profiled a diverse set of 35 apple selections for phenolics, including 5-O-caffeoylquinic acid, cyanidin-3-O-glucoside, quercetin-3-O-rutinoside, and quercetin-3-O-rhamnoside using UHPLC-MS/MS. We assessed these apples using descriptive analysis and consumer hedonic testing. In descriptive analysis, panelists were trained to produce quantitative data on specific sensory attributes to characterize the appearance, flavor, and texture of apples. In hedonic consumer testing, liking data were obtained via consumers rating apples using standardized scales. We found red color to be positively correlated to cyanidin-3-O-glucoside content, an expected finding as this anthocyanin is a red pigment. We found no significant relationship between the taste sensory traits (i.e., sweetness, sourness, bitterness, and astringency) and any phenolic content of these apples, suggesting that polyphenol content does not alter flavor perception at the concentrations present in our apples. The apples with the highest content of 5-O-caffeoylquinic acid and quercetin-3-O-rhamnoside were liked equivalently to Honeycrisp (a top commercial apple), consistent with our finding of no relationship. This work suggests that apples relatively high in phenolics may not been disliked by consumers since flavor does not seem to be negatively impacted. Future work will include a wider variation of apples to further investigate the relationship between fruit phenolics and taste.

Characterization of Crosslinked Networks by Thermal Desorption/Pyrolysis Interfaced to Direct Analysis in Real Time Mass Spectrometry (TDPy-DART-MS)
Calum Bochenek¹, Chrys Wesdemiotis^1,2
¹ The University of Akron Department of Chemistry
²The University of Akron School of Polymer Science and Polymer Engineering

Tires are complex industrial products composed of rubbers (both natural and synthetic), fillers, steel wire, textiles, and a range of antioxidant and curing systems. These constituents are distributed differently among the various tire parts, which are classified based on their function and proximity to the rim. Here, we present a rapid and sensitive approach for the characterization of tire components, using mild thermal desorption/degradation (TDPy) coupled to direct analysis in real time mass spectrometry (DART-MS). This ambient technique was applied to five important tire components: the tread, the sidewall, the inner liner, the body piles and the bead chafer, to determine the composition and structural information accessible through a combination of TDPy-DART-MS and concurrent tandem mass spectrometry (MS/MS) and ion mobility (IM) analysis of select ions produced in the DART source. The tread provides traction, turning grip, and abrasion resistance; the sidewall and the body plies provide weathering resistance and structural stability; Rapid the inner liner retains inflation inside the tire and the bead chafer ensuring the tire’s durability, performance, and safety. The tread and sidewall showed very similar TDPy-DART-MS data. At lower ionRocket temperatures (~200°C), the dominant ions corresponded to the antiozonants CPPD and 6PPD, plasticizer dodecyl adipate, and silane crosslinker TMS#1. At higher temperatures (~400°C), styrene butadiene (SB) rubber oligomers were the dominant products. The inner liner initially (~140°C) produced the plasticizer dodecyl adipate and silane crosslinker TMS#1, whereas higher temperatures (~400°C) yielded CPPD, the antioxidant trimethyl-quinoline (TMQ), and large amounts of PEG; no SB oligomers were observed from this tire part. The body piles gave rise to PPO and SB oligomers at higher temperatures (~450°C), together with CCPD and 6PPD; while the bead chafer led to dominant ions from various silanes, antioxidants and antiozonants CPPD and 6PPD.

Molecular Dynamics Modeling of Gas-Phase Compaction of a Flexible Enzyme
Luis A. Casavilca¹, Yuan Gao^1,2, Vicki H. Wysocki^1,2
¹The Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH
²Native MS Guided Structural Biology Center, Columbus, OH

Native mass spectrometry (nMS) has emerged as an alternative technique for probing the structure of proteins. However, whether conclusions obtained from nMS studies can be extrapolated to a macromolecule’s behavior in water depends on how “native” a protein structure is in the gas-phase or vacuum environment of nMS instrumentation. The transition of a protein’s solution state to a kinetically trapped gas-phase state after desolvation is typically in the order of nanoseconds. Due to this short timescale and the unavailability of high-resolution techniques that probe protein dynamics in the gas phase, this transition can only be investigated by molecular dynamics. We investigated the solution-to-gas-phase state transition of adenylate kinase (AKe), a protein involved in cellular energy homeostasis. Unlike compact monomeric proteins and protein complexes stabilized by interface interactions, AKe undergoes significant conformational changes to perform its function and has a high degree of flexibility. AKe’s flexibility and lack of a strong hydrophobic core complicated modeling its gas-phase dynamics and structure. Different MD modeling approaches (including different force fields, integrators, and whether to model protons as mobile) did not make much of a difference in the observed gas-phase dynamics of compact proteins used as standards in nMS, which maintained their overall 3D solution structure. On the other hand, AKe’s native structure was not preserved in most cases. Ultimately, a dampened molecular dynamics approach using a Langevin integrator was necessary to simulate AKe’s transition in the gas phase using mobile protons while preserving most of its native structure. Rapid compaction and increased numbers of salt bridges, expected events of protein dynamics in the gas phase, were observed. Overall, these results underscored the difficulty of modeling gas-phase behavior in very flexible monomeric proteins, and suggest using Langevin dynamics to model such behavior in this type of proteins while preserving most of their native structure.

Investigating GRP94 Functionality in Protein Folding Through Thermal Proteome Profiling
Melanie Chen, Chase P. Rezelmann, Ariana E. Shannon, Rachael N. Teodorescu, Brian C. Searle
Department of Biomedical Informatic, Pelotonia Institute for Immuno-Oncology, OSU

GRP94 is a chaperone protein crucial in proteins’ tertiary and quaternary folding stages, particularly under stress conditions such as low oxygen and pH. Based on previous work in our lab, we believe that GRP94 plays a role in modulating protein folding in immune cells in the tumor microenvironment. By identifying GRP94 client proteins, we aim to provide insights into protein folding and stability. We utilize Thermal Proteome Profiling (TPP) to measure the relative intensity of proteins by generating melting curves under varying temperatures. This allows us to detect protein stability in potential chaperone clients, measure protein-protein interactions, and identify potential for drug target engagement. We hypothesize that client proteins interacting with GRP94 will exhibit increased thermostability when GRP94 is active. We intend to infer this from individual protein melting curves across samples treated with increased heat conditions. To accomplish this, we are treating cells with PU-WS13 to disrupt the functionality of GRP94. We anticipate that proteins with the drug treatment would display lower thermal stability, as indicated by shifts in their melting curves. GRP94 potential clients will be identified through altered thermostability by utilizing TPP and drug treatments that disrupt its functionality.

Plasma-In-Droplet Ionization with Dynamic Power Supply for Liquid Chromatography–Mass Spectrometry
Purva S. Damale, Alexander P. Zumock, Dmytro S. Kulyk, and Abraham K. Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States

Liquid chromatography (LC)–mass spectrometry (MS) is widely used for analyzing complex mixtures, but a single ionization source remains incapable of ionizing samples of varying physicochemical properties (polar/non-polar and small/big molecules). Recent advancements have led to dual sources combining electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), enabling ionization switching without hardware modifications. However, simultaneous ESI and APCI use compromises ion transfer, leading to sensitivity loss. Additionally, minimizing mass spectrometer idle time is essential for cost-effective operation. Therefore, the switch to the appropriate ion source (ESI or APCI) for a given eluent is often pre-determined to enable the ionization of polar and nonpolar compound with the correct source. Herein, we describe a single, novel ion source that allows the fusion of nonthermal plasma into charged microdroplets (termed plasma-in-droplet) to enable sequential ionization of polar and nonpolar analytes via ESI and APCI mechanism, respectively, without any source switching. This plasma-in-droplet ionization (PIDI) source does not require typical usage of nebulizing gas (unlike ESI), external heating (unlike APCI), and different power supplies for ESI and APCI while operating at high flowrates. The PIDI source is developed simply by inserting ESI emitter into a sharp stainless-steel needle. We then apply a novel dynamic power supply to the stainless-steel needle, which employs voltage ramping mechanism with voltages ranging from 0 to ±8 kV. When coupled to LC-MS, this dynamic PIDI source facilitates rapid and sequential ionization of analytes that might co-elute in a single chromatogram via several cycles of ESI and APCI ionization mechanisms. Optimization data showing the ability to ionize mixture of polar (nucleotides, sugars, amino acids, proteins, charged lipids) and nonpolar (vitamins, dietary supplements, drugs, steroids, fatty acids, hydrophobic lipids) biomolecules will be presented. We will also discuss data demonstrating the differentiation of co-eluting lipid isomers differing only by C=C bond positions.

Identifying Peptides Outside the Mass Spectrometry Search Space through Mining of Evolutionary Peptide Variation
Regina Edgington_,Damien B. Wilburn
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210, USA

Mass spectrometry-based proteomics is a widely used technique for identifying, quantifying, and characterizing proteins in complex biological samples. In these analyses, peptides are identified through database searches against reference proteomes. However, this approach overlooks peptides that fall outside the defined search space, such as those arising from allelic variants or unconsidered post-translational modifications. These peptides, which may have significant biological implications—especially in cases where allelic variations are pathogenic—pose a challenge for accurate identification. Without appropriate strategies to account for these peptide variations, similar peptides can be misidentified due to shared fragment ions. Therefore, developing models or algorithms to mine additional peptide variation, starting from a reference proteome, is essential to reduce the risk of misidentification and better capture biologically relevant modifications and mutations.

To address this issue, we leveraged published data from Ba et al., which includes data from fibroblast cells from 11 different mammalian species. In these experiments, paired global DIA, phospho-DIA, and transcriptomics data were acquired using identical acquisition schemes. Global DIA data was searched using a standardized pipeline in EncyclopeDIA, resulting in an average of ~40k identifications per species, with a total of ~200k unique precursors detected. Of these, ~9k precursors were detected in all species, while ~70k precursors were unique to a single species. This dataset – with its subtle sequence variations acquired over evolutionary time and species divergence – provides a valuable opportunity to investigate how mass spectrometry signals shift in response to specific chemical modifications across phylogenetic space. Our computational pipelines aim to disentangle the chemical features of these peptide variations, and how it effects properties such as proteotopicity and fragmentation fingerprints. These findings could ultimately aid in identifying peptides that fall outside the proteomics search space to improve peptide-spectrum matching.

Novel RNA-Ligand Complex Formation for Antibiotic Drug Design: Method Design and Initial Results
Emily Fairchild¹, Dylan Carter¹, Sophie Harvey², Sebastian Schmutzler³, Ralf Hoffmann³, Jennifer Hines¹
¹ Department of Chemistry & Biochemistry, Ohio University
²Mass Spectrometry & Proteomics Facility, Ohio State University
³Department of Chemistry, Leipzig University

The T-box riboswitch is a novel Gram positive antibacterial target found at the 5´- end of many genes related to amino acid and protein synthesis and which uses tRNA as a ligand. The riboswitch is composed of the Stem 1 region, that binds to the codon of the cognate tRNA and the antiterminator region, which binds to the 5´-NCCA-3´ end of the tRNA. Previous work has identified small molecules that bind to the antiterminator selectively, but there is currently no defined structural data for what makes the small molecules effective. Therefore, we selected to use peptides to probe ligand interactions for the antiterminator and develop a more predictive methodology for future small molecule work. Peptides were selected for this method development due to their readily synthesized backbone, variety of functional R- groups and good water solubility. An initial training set of 16 peptides was prepared. In functionally relevant screening assays, 5 out of 16 peptides interacted with the antiterminator model RNA and several modulated the T-box riboswitch function in vitro. In order to characterize the RNA interactions for these ligands, we optimized an ESI-MS analysis method initially using tRNA as the model RNA. We applied this optimized method using nanospray ESI- MS and identified native folding of the antiterminator RNA and antiterminator RNA- peptide complexes formation in the positive and negative mode.

Native MS Characterization of the Binding of Substrate and Inhibitors to Salmonella FraB Deglycase, a Drug Target 
Yuan Gao^1,2, Jamison Law¹, Venkat Gopalan^1,3, Vicki H. Wysocki^1,2,3
¹ Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH 43210
²Native Mass Spectrometry Guided Structural Biology Center, The Ohio State University, Columbus, OH 43210
³Center for RNA Biology, The Ohio State University, Columbus, OH 43210

Salmonella enterica serovar Typhimurium is the second leading cause of death from foodborne illness in the United States. Salmonella FraB is a deglycase that catalyzes the final essential step during the catabolism of fructose-asparagine and converts 6-phosphofructose-aspartate (6-P-F-Asp) to L-aspartate and glucose-6-phosphate (P). FraB is a promising drug target since its inhibition results in 6-P-F-Asp build-up and Salmonella intoxication. However, the FraB-substrate (enzyme-substrate, ES) binding sites have yet to be well characterized. As FraB functions as a homodimer, we leveraged native mass spectrometry (nMS) to characterize the stoichiometry of substrate and inhibitor binding and electron capture dissociation (ECD) to characterize the potential conformational changes during catalysis. We first used nMS of a reaction consisting of FraB (E) and its substrate (S) showed peaks corresponding to enzyme-substrate (ES) and enzyme-product (EP) complexes, confirming that FraB was functional under the conditions tested. The results also suggest each monomer in the dimer can bind to the substrate and generate the product independently. The strong binding of glucose-6-phosphate agrees with the previous hypothesis that the enzyme binds to the sugar site of the substrate instead of the amino acid. Furthermore, we isolated the different compounds E/ES/EP for WT and E/ES for the E214A mutant with narrow m/z window selection and performed native top-down ECD experiments. We observed fragment pattern differences from multiple regions, such as H23-Y25, Y33-A35, and V73-T83, which are likely due to the substrate binding and potential conformational changes. Finally, we tested uncompetitive inhibitors and found substrate can bind the enzyme in the presence of inhibitors and form enzyme-inhibitor-substrate (EIS) complexes. We even observed enzyme-inhibitor-product (EIP) complexes and enzyme-inhibitor-substrate-product (EISP) complexes, suggesting the binding of these inhibitors to a functional FraB, albeit not at the active site. Moreover, preliminary data suggests that the substrate enhances inhibitor binding, as expected for uncompetitive inhibition.

Investigating MBNL1 Splicing Variants in Glioblastoma Using DDA and PRM Techniques 
Gautam Ghosh^1,2, Brian C. Searle^1,2,3
¹ Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, USA
²Pelotonia Institute for Immuno-Oncology, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, USA
³Department of Biomedical Informatics, The Ohio State University Medical Center, Columbus, Ohio, USA

Glioblastoma (GBM) is an aggressive brain tumor characterized by rapid growth and resistance to conventional therapies. Research suggests that aberrant alternative splicing plays a pivotal role in GBM progression, potentially giving rise to tumor-specific protein isoforms that may serve as biomarkers or therapeutic targets. This study focuses on the RNA-binding protein Muscleblind-like 1 (MBNL1), a key regulator of alternative splicing, and investigates its isoforms in GBM. Previous findings using Western Blots revealed distinct MBNL1 isoforms in GBM samples, with differential splicing patterns that were not observed in normal brain tissue. These results suggest that MBNL1 isoforms could serve as potential biomarkers for GBM. For this study, we analyzed protein extracts from gel bands for three tumor and two normal brain samples.

Both Data-Dependent Acquisition (DDA) and Parallel Reaction Monitoring (PRM) were utilized to explore these isoforms. DDA involves the simultaneous acquisition of fragment ion spectra from multiple precursor ions in a single run, providing a comprehensive overview of the proteome. However, due to its untargeted nature, DDA can sometimes miss low-abundance isoforms or those overshadowed by more abundant proteins, leading to incomplete detection of splicing events. To overcome these limitations, PRM was employed as a complementary technique. PRM is a targeted mass spectrometry method that monitors specific precursor ions and their corresponding fragment ions with higher resolution and accuracy. This allows for the precise quantification of specific MBNL1 isoforms, ensuring the detection of even subtle splicing variations that might be missed by DDA.

This research will not only enhance our understanding of MBNL1’s role in GBM but also highlight the importance of high-resolution proteomics methods like DDA and PRM in identifying and characterizing cancer-specific splicing events. Future work will also involve the use of alternative digestion enzymes to expand the range of detectable isoforms, which will further refine this study.

Role of Transcriptional Start-Site Differences in the HIV-1 Genomic RNA 5´UTR on Gag Binding and RNA Dimerization 
Kaylee Grabarkewitz^1,2,3, Vicki Wysocki^1,2,3, and Karin Musier-Forsyth^1,2,4
¹ Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH
²Center for RNA Biology, The Ohio State University, Columbus, OH
³Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, OH
⁴Center for Retroviral Research, The Ohio State University, Columbus, OH

Human Immunodeficiency Virus type 1 (HIV-1) has become one of the world’s deadliest pathogens. During the HIV-1 retroviral lifecycle, reverse transcription converts the genomic RNA (gRNA) into double-stranded DNA, which is incorporated into the host genome. Following transcription, the full-length viral RNA has two fates: it serves as mRNA for translation of viral polyproteins Gag and Gag-Pol, and as gRNA, which is packaged as a dimer into new virions. The latter is orchestrated by the HIV-1 Gag protein through interactions with the 5´UTR. Due to transcription start-site heterogeneity, multiple full-length viral RNA species with a variable number of guanines at the 5´end of the gRNA (1G, 2G, and 3G) are produced. Surprisingly, several studies have shown that the number of 5´ G residues affects the localization of the gRNA: 1G RNA is selectively packaged into the virion even though 3G RNA is the most abundant transcript in the cell. RNA structural differences between 1G and 3G 5´UTR RNAs were recently reported. The 1G RNA has an exposed dimerization initiation site and exposed G-rich Gag binding sites; these motifs are sequestered in the 3G RNA. Gag specifically recognizes and binds to a packaging signal (Psi) within the 5´UTR; previous work suggested that the packaging selectivity can’t be explained by differences in binding affinity alone. To understand other factors that may contribute to selective gRNA packaging, we used mass photometry (MP) to investigate Gag-RNA interactions. The MP data show higher order binding to Psi RNA relative to a non-Psi region of the 5´UTR. MP was used to investigate RNA dimerization and Gag-RNA binding stoichiometry using 2G and 4G 5´UTR RNAs designed to mimic 1G/3G RNA plus an m7G cap. From the MP data, Kd values were calculated for both the dimerization of RNA and Gag binding. Preliminary data are consistent with differences in the dimerization efficiencies, strength of dimer interactions, and the interaction between the 2G vs. 4G 5´UTR and the full-length Gag protein.

Surface Proteomics to Search for Therapeutic Targets in ALK Positive Lung Cancer 
Jacelyn Greenwald¹, Dharti Shantaram², Xilal Rima², Brian Searle³, Willa Hsueh², Vicki H Wysocki^1,4
¹ Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH
²Division of Endocrinology, Diabetes and Metabolism, The Ohio State University Wexner Medical Center, Columbus, OH
³Department of Biomedical Informatics, The Ohio State University Wexner Medical Center, Columbus, OH
⁴Resource for Native MS Guided Structural Biology, The Ohio State University, Columbus, OH

Extracellular vesicles (EVs) have come into recent focus as mediators of intercellular crosstalk and key regulators of metabolic and neurodegenerative diseases. EVs are endogenous nanoparticles shed from all cells that harbor and transmit bioactive cargo (i.e. nucleic acids, proteins, metabolites) to recipient cells. EVs can travel long distances and are known to cross the blood-brain-barrier (BBB), making them one of the few mechanisms by which peripheral proteins can be delivered to the central nervous system (CNS). Adipocyte EVs, or AdEVs, have been found to enter the CNS and preferentially target neurons. Crucially, the mechanism by which AdEVs breech the BBB is surface protein dependent.

We are interested in characterizing the AdEV surface proteome (surfaceome) and understanding the mechanisms by which AdEVs propagate peripheral signals to the CNS. The findings from this study will help us to define the surfaceome of AdEVs with the goal of using this information to understand the connection between obesity and AD risk.

Here we report on an initial panel of AdEVs from lean (n=3) and obese (n=3) donors. Total proteome and surfaceome data were collected for all samples by bottom-up proteomics. Previous data from obese adipose tissue demonstrate that we can identify adipocyte-specific proteins, including fatty acid binding protein 4 which has been linked with obesity status. These initial studies demonstrate the feasibility of expanding our approach to adipocyte EVs.

Better characterization of AdEVs may contribute essential information regarding obesity disease status and pave the way toward adipocyte-enriched protein candidate EV markers in peripheral circulation. The results of this research will help both in understanding how AD can be influenced by peripheral processes such as obesity, and in developing a liquid biopsy to probe for AD risk in obese patients.

Investigating PFAS Induced Perturbation to Gut Microbiota and their Metabolism in a Host-Free Human Colonic Model 
Chao Guo¹, Jiangjiang Zhu^1,2
¹ Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, OH 43210, USA
²James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA

Per- and polyfluoroalkyl substances (PFAS), commonly known as “forever chemicals,” are widespread environmental pollutants found in cookware, food packaging, drinking water, and seafood. These substances have been detected in the blood of 98% of adults in the general population and are linked to various health concerns, including tumorigenicity, immune dysfunction, endocrine disruption, metabolic syndrome (MetS), and inflammatory bowel disease (IBD). Increasing evidence suggests significant interactions between PFAS, gut microbial metabolism, and chronic metabolic diseases. Identifying the environmental factors that contribute to the perturbation of gut bacteria and metabolites is essential for managing and potentially treating these conditions. Metabolomics technology is widely employed to assess the toxic effects of environmental pollutants by analyzing changes in endogenous metabolites, which reflect alterations in the physiological state of organisms. In this study, we employed microbiome and metabolomics analyses to explore key bacteria and metabolites associated with PFAS-induced bacterial dysbiosis and metabolic disorders in a host-free HCM system. We observed that the PFAS exposure phase exhibited a higher relative abundance of the CRC-associated genus Morganella compared to the pre-treatment phase. Moreover, PFAS exposure significantly altered fatty acid profiles, upregulating oleic acid, tetradecenoic acid, α-linolenate, linoleic acid, and palmitoleic acid, while notably reducing levels of bile acids such as taurodeoxycholic acid and taurocholic acid, and the amino acid leucine. Additionally, PFAS exposure markedly increased the production of short-chain fatty acids (SCFAs), notably acetic acid and butyric acid, which are predominant in the gut. These findings advance our understanding of the link between PFAS exposure and gut microbiota dysbiosis, metabolic dysregulation, and their impact on host health, thereby addressing a significant gap in current knowledge.

Simplifying Eukaryotic Transfer RNA Modification Mapping via FPLC 
Aastha Gyawali, Scott Abernathy, Patrick Limbach
University of Cincinnati, Cincinnati Ohio

Transfer RNA (tRNA) undergoes post-transcriptional modifications and these modified residues play important roles in RNA structure formation and stability. Moreover, tRNA modifications are known to have correlated and causal effects with a variety of human diseases. Eukaryotes can transcribe hundreds or even thousands of different tRNA sequences. Out of 150 total RNA modifications discovered, 93 modifications are found in tRNAs. Organisms typically express around 30-50 tRNA genes. However, many eukaryotic organisms may express hundreds or even thousands of different tRNA genes – even if some of these tRNAs only differ from one another by a single nucleotide change. Current LC-MS/MS approaches are unable to differentiate this complexity of tRNAs in the sample.  Given the complexity of tRNA expression patterns, mass spectrometry-based RNA modification mapping, demonstrated for bacterial and archaeal organisms, can be complex. Thus, the goal of this project is to reduce the complexity of eukaryotic tRNA mixtures in a fashion that would mimic bacterial tRNAs. We selected FPLC as a fast, efficient, and reproducible fractionation method that can handle relatively large sample amounts using a strong anion exchange column with mobile phase A(20mM tris HCl, pH 8) and mobile phase B(1M NaCl + 20mM tris HCl) and weak anion exchange column with mobile phase A(20mM HEPES-KOH pH7.5) and mobile phase B (1MNaCl +20mM HEPES-KOH pH7.5). The standard sample used was 90 micrograms of total yeast tRNA for all studies. We investigated a variety of different mobile phase gradients – including both starting and ending %B – to identify those conditions that lead to appropriate and reproducible fractionation of the tRNA mixture. For each fraction, LC-MS/MS was used to identify the constituent tRNAs in each fraction, and LC-MS/MS sequencing using RNase T1 will be used in further study to verify the predictions of different yeast tRNAs present in each fraction.

Mapping tRNA Modifications Using LC-MS/MS
Bibek Hamal¹, Asif Rayhan², Patrick A. Limbach²
¹Biological Mass Spectrometry, Department of Chemistry, University of Cincinnati, Cincinnati, OH

tRNAs serve as temporary transporters of amino acids, assisting the ribosome in assembling the correct amino acids during translation. Over 160 post-transcriptional modifications have been reported in tRNAs, involving the addition of chemical groups to the ribose sugar, nucleobase, or both. In RNA modification mapping, tRNAs are digested into smaller oligonucleotides. If modifications are present, the digested product will have a higher molecular weight than the unmodified oligonucleotide. The UMEL method was developed to selectively eliminate unmodified oligonucleotides, allowing only modified ones to be sequenced using MS/MS. This research focuses on employing multiple ribonucleases and the UMEL technique to characterize organisms with high tRNA content, such as Spinacia oleracea (spinach). The tRNAs from spinach were extracted using a Phenol-Chloroform and LiCl extraction protocol, optimized for higher eukaryotes like spinach.

After purification, the tRNAs were digested using a combination of ribonucleases (RNase T1, MC1, Cusativin, RNase A, and RNase 4) to ensure confident modification mapping. For oligonucleotide analysis, HPLC with Ion Pair Reverse Phase (IPRP) Chromatography and an LTQ-XL Mass Spectrometer were utilized. The UMEL approach played a key role in this procedure. For nucleoside analysis, an Orbitrap Mass Spectrometer and Quantiva-QQQ Mass Spectrometer with RP-HPLC were used. Nuclease P1 and BAP were employed for nucleoside digestion. To validate the UMEL method and establish the technique, total tRNAs from Saccharomyces cerevisiae were utilized. The tRNA sequence modification mapping was carried out using RAMM software. Additionally, small RNA sequencing is in progress for the spinach sample to identify highly expressed tRNA sequences.

This study advances the methodology for tRNA modification mapping in organisms with high tRNA content, providing valuable insights into RNA modifications.

Plant-Based Milk: A Metabolomic Approach to Evaluate Chemical Profile Changes Induced by Innovative Processing Technologies
Victoria Hermes de Vargas^1,2, Maria Sholola¹, Sai Sasidhar Guduru¹, V.M. Balasubramaniam¹, Ligia Damasceno Ferreira Marczak², Eliseu Rodrigues³,Giovana Domeneghini Mercali³,Jessica Cooperstone^1,4
¹Department of Food Science and Technology, The Ohio State University, Columbus, OH
²Department of Chemical Engineering, Federal University of Rio Grande do Sul, Brazil
³Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Brazil
⁴Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH

As the demand for plant-based milk continues to rise, the need for effective processing technologies to ensure their safety and quality becomes increasingly important. This study aimed to evaluate the impact of innovative non-thermal processing technologies on the chemical composition of almond milk using an untargeted metabolomics approach. A single batch of almond milk was prepared from raw almonds and water (1:3). Subsequently, samples were subjected to different processing methods, including ultra-shear technology (UST) (300 and 400 MPa), high-pressure processing (HPP) (100, 300, 400, and 600 MPa for 0 and 5 minutes), and conventional thermal treatment (C72) (72 °C for 15 seconds). All processing runs were conducted in quintuplicate, and raw milk was used as a control. Data were acquired from methanol-water extracts using UHPLC-QTOF-MS. All data were log2-transformed, and differential analysis was performed using unpaired t-tests (p < 0.05). After pre-processing and filtering, 1502 molecular features were obtained. Principal component analysis revealed clustering of samples within the same group and overlap of the pooled quality control samples indicating good data quality. Hierarchical clustering of samples identified two main groups: one consisting of the raw sample, C72, and the lower levels of HPP (100 and 300 MPa), and the other comprising both UST treatments (300 and 400 MPa) and the higher levels of HPP (400 and 600 MPa). The clear separation between these groups suggests that pressure-based technologies induce more significant changes in the chemical profile of almond milk compared to thermal treatment. For example, 923 features differed by at least two-fold between the raw and UST at 400 MPa, whereas 635 significant features were found between the raw and C72 samples. Our next steps involve identifying the key metabolites and conducting further comparisons among the different technologies to better understand the chemical changes resulting from each processing method.

Exploring Biofluid Protein Expression with CATalog, an Interactive Proteomics Dashboard
Alex W. Joyce¹, Katelyn B. Brusach², Jessica M. Quimby², Brian C. Searle^1,3
¹Department of Biomedical Informatics, The Ohio State University Medical Center, Columbus, OH, United States
²Veterinary Clinical Sciences, The Ohio State University College of Veterinary Medicine, Columbus, OH, United States  
³Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States  

Biofluids contain many different potential biomarkers, providing valuable insight into both normal and aberrant biological processes. However, it is not always apparent which biofluid to choose when evaluating biomarker candidates as specific proteins are detected at different concentrations among various biofluids. Pursuing the wrong biofluid can result in limited detection or results with lower significance. To aid in the process of selecting the correct biofluid for investigation, we have developed CATalog, an interactive application written in R using the Shiny framework. CATalog displays baseline relative intensities of identified proteins from paired urine, serum, and plasma samples measured using mass spectrometry-based proteomics. These baseline intensities of healthy individuals aids biomarker discovery by providing a reference point to determine abnormal protein levels in disease. Additionally, users of this application have the option to filter out proteins with highest relative intensities in any given biofluid, view boxplots to visualize data, identify outliers that correspond to demographic factors, and obtain gene ontology (GO) annotations to be viewed for every protein. The integration of GO annotations into the database allows the end user to quickly assess both function and subcellular localization of a given biomarker, allowing the application to assist in both biofluid and biomarker selection. We demonstrate this software tool using a core database of feline proteomic data collected from nine healthy cats. We believe that this application is a useful tool for both biofluid selection and biomarker exploration in the field of proteomics.

Effect of Growth Temperature, Tissue Type and Sex on the volatile profile in Raw Fresh Atlantic Salmon (Salmo salar) using SIFT-MS
Manpreet Kaur¹, Konrad Dabrowski², Kevin Fisher², Sheryl Barringer¹
¹Department of Food Science and Technology, The Ohio State University, Columbus, OH
²School of Environment and Natural Resources, The Ohio State University, Columbus, OH

Atlantic salmon (Salmo salar) is one of the most consumed seafood products in the USA. Flavor acceptability is a crucial determinant of consumer preference. The volatiles present in the product are key factors in shaping the sensory experience for consumers as indicators of quality leading to overall consumer acceptance. The purpose of this study is to investigate the effect of different rearing temperatures, muscle vs skin tissue, and sex of Atlantic salmon on the volatile compounds that contribute to the flavor profile of salmon. Fish were filleted and samples of muscle with the skin-on or skin-off as well as the skin alone were compared. Muscle and skin samples (2 g) in 20ml of 0.5% ethanol were processed for analysis. The samples were held at 42°C in a shaking waterbath for 30 minutes. Six replicates of each fillet type were analyzed using SIFT-MS headspace method. The fish grown at 23°C had higher concentrations of 38 volatiles as compared to the fish grown at 13°C, likely due to greater growth rate and metabolism. The fish at 23°C were 328 g ± 23.9 g and fish at 13°C were 184 g ± 9.6 g in weight and the fish at 23°C were 30.9 cm ± 0.17 cm and fish at 13°C were 27.3 cm ± 1.04 cm. Both skin-off and skin individually showed higher concentrations of all the volatiles than the sample with skin-on muscle. Additionally, female salmon had higher concentrations of 20 volatiles than male fish suggesting that male and female fish produce distinct volatile profiles under the same rearing conditions. This research provides a baseline on how the volatiles in fresh fish are affected by sex, tissue type and growth temperature which will be used to determine the effect on off odor production during storage.

Offline HPLC Fractionation and LC-MS/MS Characterization of tRNAs
Jennifer A. Kist, Cassandra Herbert, Scott Abernathy, Patrick A. Limbach
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati

Transfer ribonucleic acids (tRNAs) are adapter molecules acting as a carrier of amino acids to the growing polypeptide chain. Among all RNAs, tRNAs exhibit the largest number and the widest variety of modifications to ensure proper structure and function. Mass spectrometry methods are becoming increasingly important for the characterization of post-transcriptional modifications on ribonucleic acids. RNA Modification Mapping by LC-MS/MS is applicable to mixtures of RNAs (in particular, tRNAs) from bacterial and archaeal organisms. However, many organisms, especially eukaryotes, can transcribe hundreds or more individual tRNA sequences within the cell. Such mixtures are inherently difficult to accurately map to particular sequences given current technologies. In this work, a reverse phase (RP) HPLC method for fractionating tRNAs compatible with RNA modification mapping was developed. Yeast tRNA as well as tRNA from WM793 primary melanoma cells were both successfully fractionated into 4 fractions. The fractions were collected, washed, and ran on LC-MS for nucleoside analysis.

Non-Canonical Inflammasome LPS-mediated Oligomerization Probed by Electron Capture Charge Reduction
Philip Lacey^1,2, Chengliang Wang³, Jianbin Ruan³, Vicki Wysocki^1,2
¹Chemistry and Biochemistry Department, The Ohio State University, OH
²Native MS-Guided Structural Biology Center, The Ohio State University, OH
³Department of Immunology, University of Connecticut Health, CT

Understanding inflammatory responses can help prevent or manage many inflammation-based conditions like septic shock, which has been found to be mediated by non-canonical inflammasome activation. This process initiates when caspase-11 binds to a heterogeneous class of lipopolysaccharides (LPS) released from gram-negative bacteria, causing oligomerization and activating inflammatory cascades. Despite efforts to use cryo-EM to determine the caspase-11/LPS oligomeric state, it remains unconfirmed. Thus, native mass spectrometry (nMS), was used to maintain the crucial noncovalent interactions between subunits and reveal caspase-11/LPS oligomeric states. This was accomplished using collision induced dissociation (CID), quadrupole selection, and electron capture charge reduction (ECCR). Two samples of caspase-11, one purified without LPS (-LPS) and one with LPS (+LPS), were analyzed. The -LPS sample only exhibited well-resolved monomers with a deconvolved mass of ~54.6 kDa and several slightly truncated proteoforms. The caspase-11/LPS oligomerization only happened in the presence of bacterial LPS. The +LPS sample showed similarly resolved full-length and truncated monomers but with several unresolved and overlapping peaks, which spanned a range of several thousand m/z each. CID resulted in the formation of monomer and complementary (n-1)mer, however, the +LPS (n-1)mer remained too heterogeneous to determine the oligomeric state. This suggests that caspase-11/LPS oligomerization is stochastic, involving full and truncated caspase-11 forms binding to the many heterogeneous oligosaccharide forms of LPS. To address this heterogeneity, we used the quadrupole to select 15 m/z-wide windows, or “slices”, within the broad MS1 peaks, which were then subjected to ECCR. Charge reduction resulted in new, well-resolved charge state distributions that were recognized by deconvolution software. This process was repeated across all unresolved peaks, generating an oligomer mass profile for the caspase-11 non-canonical inflammasome. The oligomer mass profiles, which range mostly from 350 kDa-550 kDa, display a strong propensity towards an octameric complex with heptamers and nonamers present as well.

Charge Transfer Dissociation (CTD) of Phospholipids with Alkali Metal Adducts
Courtney E. LaPointe, Glen P. Jackson
C. Eugene Bennett of Chemistry, West Virginia University, Morgantown, WV 26506, USA
Department of Forensic and Investigative Science, West Virginia University, Morgantown, WV 26506, USA

Lipids are critical components of cellular and sub-cellular membranes, and their structural characterization is essential for explaining lipid biochemistry. Lipids are classified by the head group, fatty acyl chain length(s), unsaturation degree and location, and any stereochemistry. Electrospray ionization (ESI) is a soft ionization technique that preserves the molecular mass of lipid samples, and combining ESI with tandem mass spectrometry (MS/MS) enables their structural characterization. For example, collision induced dissociation (CID) is useful for determining the head group and acyl chains. Although informative, low-energy techniques like CID do not provide enough detail for full characterization.

Charge transfer dissociation (CTD) mass spectrometry is a high-energy, radical-driven activation technique in which precursor ions are activated with a beam of ~5.5 keV helium cations. Previously, CTD has been used to successfully elucidate biomolecules including phospholipids. In this study, phospholipid standards were ionized with ESI, and singly charged H+, Na+, K+ adducted precursors were selected for isolation before ~30 ms activation with CTD.

CID of the protonated species yielded more product ions than CID of the sodiated and potassiated precursors, and all adducts yielded head group information and acyl chain lengths. Regardless of the adduct, CID did not provide enough evidence for full structural characterization. CTD of all precursors provided even-electron and odd-electron fragments. Like CID, CTD of the protonated lipid precursor produced a wider range of informative fragments than of the sodiated and potassiated species. The protonated precursor fragmented along the head group, sn-1 and sn-2 sites, and fatty acyl chain, which led to complete structural characterization of the lipid. CTD of the sodiated and potassiated species resulted in fewer fragments than the protonated precursors, with the most abundant product ion being M+•. This product indicates the neutral losses of radical metal atoms.

Enhanced Lipidomic Profiling of Pseudomonas aeruginosa through Advanced NMR and MS Techniques
Kyungsuh Lee
Department of Chemistry and Biochemistry at The Ohio State University

Pseudomonas aeruginosa is a formidable opportunistic pathogen resistant to various types of antibiotics, leading to intense and chronic infections in immunocompromised individuals.[1] P. aeruginosa infections are challenging to treat because the colonies of P. aeruginosa form biofilms, which are complex extracellular gel-like matrices that contribute to resist treatment. Biofilm formation, coupled with specific virulence mechanisms mediated through enzymes and bacterial lipids, requires the need for a deeper understanding of the mechanisms of lipids in P. aeruginosa-host interaction including bacterial colonization and survival from antibiotics.[2]

In response, our study employs an untargeted NMR and MS-based lipidomics approach for a better understanding of biofilm formation. However, conventional NMR-based untargeted lipidomics research of complex mixtures encounters difficulties because of the spectral complexity causing severe peak overlaps. Furthermore, the MS-based untargeted approach in lipids analysis is mostly focused on combined instrumental analysis with liquid chromatography.[3]

Recently, non-uniform sampling (NUS) techniques applied to 2D NMR real-time pure shift (BIRD) HSQC spectra led to substantially improved spectral resolution with a simultaneous gain in sensitivity.[4] Direct infusion (DI)-MS with Fourier transformation (FT) equipped showed 23 different lipid classes.[5] Our recent research allowed the unique identification of key reductive metabolites including steroid derivatives by 2D pure-shift NUS HSQC experiments. These results will be discussed along with their integration with DI-MS, thereby further enhancing lipid identification and quantification. The combination of these powerful instrumental analysis techniques helps understand P. aeruginosa infections at the molecular level and may guide future targeted therapeutic research strategies.

The NMR and MS spectra of complex lipid mixtures extracted by P. aeruginosa will be demonstrated first in OMSS2024.

[1] Leggett, A.; Li, D.-W.; Bruschweiler-Li, L.; Sullivan, A.; Stoodley, P.; Brüschweiler, R. Sci Rep 2022, 12 (1), 17317.

[2] Constantino-Teles, P.; Jouault, A.; Touqui, L.; Saliba, A. M. Front. Immunol. 2022, 13, 931027.

[3] Gerhardtova, I.; Jankech, T.; Majerova, P.; Piestansky, J.; Olesova, D.; Kovac, A.; Jampilek, J. IJMS 2024, 25 (4), 2249.

[4] Wang, C.; Timári, I.; Zhang, B.; Li, D.-W.; Leggett, A.; Amer, A. O.; Bruschweiler-Li, L.; Kopec, R. E.; Brüschweiler, R. J. Proteome Res. 2020, 19 (4), 1674–1683.

[5] Nielsen, I. Ø.; Vidas Olsen, A.; Dicroce-Giacobini, J.; Papaleo, E.; Andersen, K. K.; Jäättelä, M.; Maeda, K.; Bilgin, M. J. Am. Soc. Mass Spectrom. 2020, 31 (4), 894–907.

LC-MS Metabolomics Reveals How Food Proteins Affect Iron Absorption Using In Vitro Digestion Coupled with a Caco-2 Cell Intestinal Model
Ziqi Li¹, Agnes Josylyn Komey², Rachel E. Kopec^3,4,*
¹Department of Food Science and Technology, The Ohio State University, Columbus, OH, 43210, USA 
²Interdisciplinary Nutrition Graduate Program, Department of Human Sciences, The Ohio State University, Columbus, OH, 43210, USA 
³Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, OH, 43210, USA 
⁴Foods for Health Discovery Theme, The Ohio State University, Columbus, OH, 43210, USA

Background: Iron chlorophyll derivatives (ICDs) can serve as a plant-based iron fortificant, better delivering iron to intestinal cells when the protein albumin is co-digested, as compared to ICDs alone. In contrast, other food proteins recently tested inhibit iron uptake from ICDs, however ascorbic acid (AA) reversed the whey-based inhibition. This inhibition, and the reversal with AA, has never been characterized.

Aim: This study leverages LC-MS metabolomics to investigate how protein metabolites in digesta may be influencing iron uptake from ICDs.

Methods: A range of whey, soybean, and pea protein isolate doses (0-154 mg protein) were co-digested in vitro with ICDs (providing 0.5 mg Fe). This digestion was performed both with and without AA. Post-digestion, the samples were centrifuged, filtered, diluted 16x, and applied to confluent Caco-2 cells. Metabolites in the filtered digesta were separated via a HILIC column and analyzed using a UHPLC-QTof with ESI+ Masses were scanned over 50-500 m/z. Iterative fragmentation was used to produce MS2 spectra. Deconvolution, alignment, and feature grouping were performed. Quality control cutoffs (CV>25%) were applied, with remaining peaks manually inspected. Metabolites were annotated with predicted chemical formula, and tentatively identified via comparison with the METLIN database.

Results: Among the 631 polar metabolites detected, significantly higher levels of the plant alkaloid trigonelline and asparagine-containing dipeptides were found in soybean and pea digesta. In contrast, higher levels of glutamine, cysteine, and lysine-containing tripeptides were found in the whey digesta. The addition of AA significantly increased the concentrations of tripeptides rich in tryptophan and arginine.

Conclusions: Trigonelline and/or asparagine-rich dipeptides in legumes may inhibit iron absorption. In contrast, better release of tryptophan and arginine-rich tripeptides when whey protein is co-digested with AA, may assist in iron uptake by Caco-2 cells. The results herein highlight how UHPLC-MS metabolomics can provide insight into nutrient-nutrient interactions which occur under physiological digestion conditions.

Site Selective Chlorination of Arenes and Heteroarenes Using Hypervalent Iodine Catalysis via the use of Microdroplet Chemistry
Owen L. Looker, Rebekah E. Strong, Abraham K. Badu-Tawiah
The Ohio State University, Columbus, OH

This presentation will showcase the use of charged microdroplets to accelerate selective chlorination of aromatic compounds. Site-selective methods are a valuable tool in the synthesis of industrially relevant compounds, particularly within medicinal chemistry. Simple late-stage synthesis allows chemists to make necessary conversions to near-complete pharmaceutical agents. However, direct reactions that are regioselective are limited. Available reactions are currently limited to reactions relying on hazardous and expensive heavy metal catalysts that are environmentally detrimental. Typically, organic reactions are multi-step, and time consuming, taking hours or even days. Herein, we describe a contained electrospray ionization (cESI) reaction platform, utilizing etched silica capillaries for the generation of non-thermal plasma during the generation of charged microdroplets that facilitate chemical reactions in a single step, and run at the time scale of microseconds. Products ensuing from the plasma-microdroplet fusion platform are analyzed in real-time using a proximal mass spectrometer. We have taken advantage of the green catalytic nature of hypervalent iodanes (e.g., phenyliodine(III) diacetate) to probe selective chlorination of heteroarenes (isoquinoline), using acetyl chloride as our chlorine source. All compounds were analyzed using negative-ion mode and tandem MS has been used for structural characterization of products and intermediates. We will show NMR data that validate the regioselectivity of reactions. Radical scavengers will be used to further probe the mechanism, which is currently believed to proceed via an arene radical cation.

Variable Temperature Electrospray Measurement of Enthalpy and Entropy of Tryptophan Binding to Ring Shaped Protein TRAP
William Moeller^1,2, Mark Foster¹, Vicki Wysocki^1,2
¹Department of Chemistry and Biochemistry, The Ohio State University
²Native Mass Spectrometry Guided Structural Biology Center

Accurate measurement of thermodynamics is essential for a complete understanding of structure-function relationships in biological systems. Native mass spectrometry (nMS) is a valuable tool for quantifying protein-ligand interactions in part because of its ability to quantify differently liganded species. The populations measured by MS can be directly related to the binding free energies, ΔG. Variable-temperature electrospray ionization (vT-ESI) instruments allow for control of sample solution temperature prior to introduction into the mass spectrometer, enabling measurement of the temperature dependence of populations, and therefore the binding enthalpy, ΔH. However, validation of thermodynamic values obtained from vT-ESI against established solution phase techniques is essential for enabling the implementation of vT-ESI nMS as a standard tool for characterizing protein-ligand thermodynamics. Here we used the homo-undecameric trp RNA-Binding Attenuation Protein (TRAP) and its ligand Trp as a model system. Protein and ligand were incubated at selected temperatures using a custom vT-ESI source (Russell group, TAMU). Populations of TRAP oligomers with varying numbers of bound Trp were quantified as a function of added Trp by peak intensity, and thermodynamic values, including free energy, enthalpy, and entropy, were obtained by Van’t Hoff analysis. We also observed a decrease in ligand binding as a function of temperature, suggesting that the affinity is weakened at higher temperatures (55 °C, compared to 25 °C). We also observed that tighter binding is reversibly restored upon reducing the temperature (~25 °C). However, upon incubation at very high temperatures (85 °C) we observed that ligand binding is irreversibly weakened, likely because of temperature-induced protein restructuring. We compare these results against previously published solution-based isothermal titration calorimetry results.

Study the Mechanisms of Releasing Dynorphin B in Mice Cerebrospinal Fluid and Cochlear Fluid
Eman Mohamed, David Anderson
Cleveland State University

The long-term goal of our research is to investigate the mechanisms behind permanent sensorineural hearing loss (SNHL) resulting from exposure to loud sounds, often known as acoustic overstimulation (AOS). Multiple mechanisms by which AOS causes damage to the inner ear, including the increase of cytokines, and oxidative stress leading to an upregulated inflammation and immune response. We hypothesize that dynorphins (DYNs), which are peptides secreted in the inner ear, may be the initiating agent to the increased inflammatory/immune response, leading to the damage caused by AOS. Our hypothesis suggests that the locus coeruleus (LC), a brainstem nucleus responsible for the stress response, might be implicated in the release of detrimental DYNs. To examine this, our approach will be electrostimulation of locus coeruleus (LC) of mice and quantifying the amounts of dynorphins (DYNs) in the cerebrospinal fluid (CSF) using a highly sensitive LC-MS/MS method comparing the results of non-stimulated control mice. Confirming an increased DYNs in CSF release from LC stimulation would suggest a neuro pathway for DYNs release. To support a hypothesis that this pathway is operative in the ear, LC electrostimulation will be done, anticipating an increase in DYNs measured in the cochlear fluid of the ear compared to that measured in non-LC-stimulated mice. There is a possible connection of AOS with the LC pathway DYNs release by considering that the stress of excess noise leads to the firing of the LC pathway, which leads to DYNs release in the inner ear.

Using Pediatric Urine Proteomics to Identify Potential Chronic Pancreatitis Biomarkers 
Madalyn G. Moore^1,2,3, Ariana E. Shannon^1,2,3, Zobeida Cruz-Monserrate^1,4,5, Maisam Abu-El-Haija^6,7*, Brian C. Searle^1,2,3*
¹Pelotonia Institute for Immuno-Oncology Comprehensive Cancer Center, The Ohio State University
²Department of Biomedical Informatics, The Ohio State University Medical Center
³Department of Chemistry and Biochemistry, The Ohio State University
⁴Department of Internal Medicine, Division of Gastroenterology, Hepatology, and Nutrition, The Ohio State University, Wexner Medical Center
⁵The Comprehensive Cancer Center–Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University
⁶Division of Pediatric Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital
⁷Department of Pediatrics, College of Medicine, University of Cincinnati

Background: Chronic pancreatitis (CP) is characterized by decreased pancreatic function and morphological changes that can lead to pancreatic cancer. Adult CP cases result from chronic alcohol consumption, which causes interfering clinical factors that make studying the disease and identifying biomarkers difficult. We propose using a pediatric population to search for urine proteins with diagnostic biomarker potential that differentiate CP from acute pancreatitis (AP) cases and other controls without alcohol and age-related variables.

Methods: Urine samples (150: AP [28], CP [50], healthy [31], acute orthopedic injury [41]) were collected upon presentation. The median age of the population was 12.4, where 54% was female. Following a bottom-up proteomics workflow, data-independent acquisition (DIA) was used for collection on a Thermo Orbitrap Fusion mass spectrometer and samples were analyzed using a sample-generated chromatogram library. The sample population was designed to help identify biomarkers that differentiate chronic pancreatitis from acute pancreatitis as well as other generally painful conditions, so external fracture controls were included.

Results: We identified 2,252 proteins (13,842 peptides) in the pooled sample chromatogram library, or the urine proteome, and quantified 1,760 proteins across individual samples. Pairwise comparisons (FDR<0.05) were used to evaluate the CP group against control groups. 34 proteins were significantly upregulated in CP. Specifically, isocitrate dehydrogenase (IDH1) and S100 calcium-binding protein P (S100P) both show above a ten-fold increase in expression when compared to controls.

Conclusions: S100P has previously been found to be upregulated in patients with pancreatic cancer and precancerous lesions, while mutations in IDH1 have been associated with pancreatic cancer. Both manifestations are established symptoms of late CP. Using least discriminatory analysis (LDA), IDH1 was found to have an area under the curve (AUC) of 96 +/- 0.2 when compared to control groups. For these reasons, we believe both proteins are promising candidates for diagnostic pediatric CP biomarkers.

Surface-Induced Dissociation (SID) for Comprehensive Glycopeptide Analysis
Martha Ortega Zepeda¹, Vicki H. Wysocki^1,2
¹The Ohio State University, Columbus, OH
²The Ohio State University Department of Chemistry & Biochemistry1 Resource for Native MS Guided Structural Biology 

The study of glycoproteins and their associated glycan structures has emerged as a critical field in understanding cellular processes, disease mechanisms, and potential therapeutic targets. However, glycoproteins’ inherent complexity and heterogeneity pose significant analytical challenges, particularly in the fragmentation and accurate characterization of glycopeptides. Traditional collision-induced dissociation (CID) often leads to the loss of glycan structural information, while electron-based dissociation preserves labile glycan modifications but suffers from lower efficiency. Surface-induced dissociation (SID), alternatively, fragments a given ion population via collision against a surface. SID enables the fragmentation of glycopeptides, producing comprehensive fragmentation patterns that preserve both glycan and peptide backbone information.  In this work, glycopeptides from sialoglycopeptide (SGP), prostate-specific antigen (PSA), fetuin, and avidin were used to compare fragmentation patterns generated by both SID and CID. Preliminary results with SGP indicate that SID provides additional sequence coverage at lower activation energies compared to CID.  This enhanced coverage is critical to  accurately characterize the glycan heterogeneity present in complex glycoproteins. SID presents a powerful complementary technique to CID in these studies, allowing detailed characterization of glycoproteins. This method enhances our understanding of glycan-related biological processes and aids in the development of glycan-targeted diagnostics and treatments. 

Deep Lipidomics Mass Spectrometry Analysis of Complex Mixtures Enabled by In-Situ Droplet Reactions flowing Liquid Chromatographic Separation
Niraj Panday, Alexander Grooms, Abraham K. Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH

Lipids comprise of diverse organic compounds, encompassing fats, sterols, and soluble vitamins, each exhibiting distinct polarities and ionizable groups. Analyzing these intricate lipids at various isomeric levels, such as headgroup, acyl chain composition, and C=C bond position along acyl chains, poses significant analytical challenge. Acyl chains may possess different degrees of unsaturation and pinpointing the exact location of C=C is crucial in biomarkers identification for clinical applications. Analyzing these lipids at multiple isomeric levels usually necessitates sample derivatization before MS analysis, instrument modifications, and high-resolution instrumentation (e.g., Orbitrap), thus limiting analyses to select laboratories. To address this challenge, we propose an innovative liquid chromatographic method, combined with online reactive (co-axial-contained) electrospray ionization (ESI) mass spectrometry (MS). We coupled reverse-phase liquid chromatography (LC) with a custom-built coaxial contained ESI source on an ion trap mass spectrometer (LTQ Velos Pro). This approach provides concurrent lipid class separation and C=C isomeric analysis for both polar (e.g., Glycerophosphoglycerol, phosphatidylcholines, phosphoethanolamines) and neutral (triglycerides) lipids in a single LC run using a conventional mass spectrometer without modification. The co-axial contained ESI source was able to form epoxides of the separated and co-eluted lipids in microdroplets in the cavity provided by the source. The epoxide precursor ion was then subjected to CID MS/MS to form diagnostic ions corresponding to the position of C=C bonds along the acyl chain. We observed 2 diagnostic ions for each C=C position with a difference of 16 Da indicating the oxidized fragments and allyl fragment allowing effective C=C localization. Thus far, we have analyzed 17 lipids standards in a mixture with 3 containing their positional (C=C) isomers. Results for complex mixtures from plant sources (e.g., olive oil) will be presented, from which at least two new lipid isomers have been identified and characterized. Thus, the coupling of liquid chromatography with the co-axial contained ESI source allows accelerated microdroplet chemistry for the comprehensive analysis of isomeric lipids via online analysis on conventional mass spectrometers.

Adaptive Metabolic Responses to Single-Gene Deletions in Escherichia coli: A Non-targeted LC-MS Analysis
Xinru Pang^1,2, Li Chen^1,2, Shiqi Zhang^1,2, Huan Zhang^1,2, Jiangjiang Zhu^1,2
¹Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
²Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, Ohio 43210

Central carbon metabolism, encompassing glycolysis, tricarboxylic acid cycle (TCA) and pentose phosphate pathway (TCA), is crucial for the survival and growth of all organisms, with disruption in these pathways have been extensively studied in the context of pathogenesis and cellular death. However, the impact of gene dysfunction or deletion – particularly for genes encoding non-enzymatic functions- on metabolite levels remains largely unexplored. This knowledge gap hampers our ability to predict how genetic disruptions propagate through interlinked metabolic networks and affect the global metabolic state, as well as how cells can bypass inactivated pathways to survive. In this study, we investigated the adaptive metabolic responses to single-gene deletions in Escherichia coli, a model prokaryotic organism. We utilized liquid chromatography-mass spectrometry (LC-MS) for non-targeted metabolomic analysis across a selected collection of E. coli mutant strains. Our systematic approach aimed to map gene-metabolite associations and decipher potential metabolic functions for genes with unknown/unexpected roles. Our findings revealed that while the deletion of specific enzymes caused localized metabolic effects, there were also significant gene-metabolite associations that could not be explained by classical pathway proximity, which suggests the presence of previously uncharacterized metabolic interactions. Furthermore, analysis of metabolomic signatures demonstrated how E. coli compensates for the loss of specific genes by redistributing substrates and altering metabolic states. The observed metabolic profile similarities and empirical gene-metabolite associations enabled us to predict the functions of genes with unknown metabolic roles and identify potential genetic interactions. Our study provides new insights into the adaptive mechanisms of E. coli under genetic perturbations, advancing our understanding of metabolic network robustness and gene function prediction.

Quantitative Analysis of Plant Phenolics by LC-MS/MS, and PhenolicsDB: A Publicly Available High-Resolution MS/MS Spectral Library
Cristian (Daniel) Quiroz-Moreno¹, Jessica Cooperstone^1,2
¹Horticulture and Crop Science Department, Ohio State University, Columbus
²Food Science Department, Ohio State University, Columbus

Consumption of plant phenolics is linked to human health promotion. Therefore, developing methods for identifying and quantifying these compounds is crucial to characterize matrices, including foods, beverages, and crops. Apples (Malus x domestica Borkh) are the third most produced and the first most consumed fruit in the US. Additionally, apple consumption accounts for one-third of total dietary polyphenol intake. This work presents PhenolicsDB, a publicly available high-resolution MS/MS library with 320 reference MS/MS spectra, and a multi-panel quantitative extraction and LC-QqQ for 26 phenolics commonly found in plants. Fragmentation patterns for 73 authentic standards were collected using electrospray ionization in positive and negative polarity and at multiple collision energies (20, 40, 60, and 80 CE) using an LC-QTOF (6545 or 6546, Agilent, Santa Clara, CA, USA) instrument. The resulting 320 reference MS/MS patterns are presented in PhenolicsDB and used for compound identification in apple fruit of 26 phenolics. Next, we developed an LC-QqQ (TQD, Waters, Milford, MA, USA) method to quantify these phenolics across diverse apple fruits. Parameters such as chromatographic gradient, product ions, dwell times, cone voltages, and collision energies were optimized. Our database includes flavonols, flavonoids, anthocyanins, dihydrochalcones, phenolic acids, organic acids, and sugars. This library is under a CC-BY-4.0 license and is publicly available for download and use at https://cooperstonelab.github.io/PhenolicsDB/. PhenolicsDB has been successfully used to identify 26 compounds in apple fruit at a level 1 identification. This library could be used for broader phenolic identification across the plant kingdom. Additionally, we developed an UHPLC chromatographic method that is 10 min long, minimizes co-elution, and enables quantification of all analytes using negative ionization mode. The dwell time ranges from 0.025 to 0.115 ms. Finally, the limits of detection and quantification range from 0.04 – 0.09 and 0.23 – 0.13 ng/mL, respectively.

Panoptic Mass Spectrometry: A Novel High-Throughput Ion Source for All States of Samples and Complex Mixtures
Keerthana Ravi, Dmytro Kulyk, Riley Ferguson, Anju Sheregar, Abraham Badu-Tawiah
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH

Mass spectrometry has proven to be a useful research tool to identify and quantify analytes in various settings, such as different industries and research laboratories. With the first stage of analysis being ionization, several techniques have been developed to ionize various sample types. Nano-electrospray ionization (nESI) and atmospheric pressure chemical ionization (APCI) are examples of commonly used ionization techniques. Currently, samples of solid, liquid or gaseous states could be ionized separately by different experimental set-ups and platforms. However, there is no platform available commercially with the ability to hold three states of samples – solids, liquids and gases, as well as complex solutions within the same device for ionization. Without this, the efficiency of analysis and the range of compounds that could be analyzed is limited. To overcome this challenge, we have developed a novel panoptic ion source. This prototype was designed and developed with the goal of rapidly and accurately ionizing all states of samples with various chemical and physical properties, such as acidity, polarity and volatility within the same device. By doing so, this source makes it possible to achieve an efficient and high-throughput analysis. The ionization chamber was designed to hold samples within a nano-electrospray capillary tube and/or several vials at a particular time. With this source, analytes will be ionized via nESI and/or APCI. The ion source set-up, approach and preliminary data will be presented. Using samples of various chemical and physical properties, such as cocaine, limonene and lauric acid, data from optimization studies and complex mixture analysis will be presented. Future directions of high-throughput studies and limit of detection studies will also be highlighted.

Nucleoside Modification Mapping by Genome-Independent Universal Mass Exclusion List of Unmodified Oligonucleotides during LC-MS/MS
Asif Rayhan, Balasubrahmanyam Addepalli, Patrick A. Limbach
University of Cincinnati

Identification of the location of nucleoside modification in an RNA sequence is essential to understand its biochemical significance. Excluding the mass values of unmodified oligonucleotides during liquid chromatography-tandem mass spectrometry (LC-MS/MS) data acquisition can improve the signals for modified oligonucleotides. Since all RNAs are made from four canonical nucleotides (A, U, C, and G), combinations of these nucleotides in a defined oligonucleotide length can be computed as an exclusion list. The goal of this study is to develop a genome-independent universal mass exclusion list (UMEL) of unmodified oligonucleotides for a defined size range and test the exclusion efficiency during tandem mass spectra (MS/MS) acquisition. We show that the exclusion list preparation requires the knowledge of cleavage behavior of ribonuclease/s employed in bottom-up RNA-sequencing approaches. Our results indicate that the employment of UMEL in combination of hydrophilic interaction chromatography improve the detection of low abundant nucleoside modifications during RNA modification mapping procedures. We will also report the applicability of this enzyme-specific single exclusion list for modification mapping of the tRNA population of a well-characterized model organism, Escherichia coli and poorly characterized fission yeast, Schizosaccharomyces pombe.

Enhanced Contained Electrospray Ionization and Fragmentation of Sterol and Nonpolar Analytes using Silver Coated Microcapillaries
Adam Reed and Abraham Badu-Tawiah
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH

Contained electrospray ionization (cESI) sources typically utilize either polymer coated glass capillaries or etched glass capillaries to facilitate microdroplet reactions. Standard glass capillaries suffer from poor conductivity and while etched capillaries do allow for a sufficient conductivity increase to generate plasma, they have a markedly increased fragility, making them challenging to work with. Herein, we describe the optimization of silver coated glass capillaries for use in contained electrospray systems. Taking inspiration from the classic Tollens’s test for reducing sugars, aqueous silver salts can be chemically reduced to silver metal with a high affinity for glass surfaces. Modifications to the solvent, temperature, reducing agent, stoichiometry, and concentration allow for a controlled reduction of the silver salts to produce nanoparticles of a given size with even distribution over the glass surface. Preliminary analysis of cholesterol shows silver adduct formation with fragments corresponding to the loss of the alkyl chain and hydroxyl groups. Further investigation of this fragmentation with cholesterol and other sterol systems will be reported as well as further characterization of this system relative to a standard cESI source. Lastly, potential mechanistic pathways for the silver mediated fragmentation will be explored.

MALDI and DART Mass Spectrometry Analysis of a Single Entity Microorganism Attached to a Disk Electrode
Luciana Rivera Molina, Gabriela Campos, Gabriel Gemadzie, Chrys Wesdemiotis, Aliaksei Boika
University of Akron

Small-single-celled organisms like bacteria are found almost everywhere on Earth and are vital to the ecosystem and the human body. The human body is full of bacteria, with most being symbiotic, but some being pathogenic and thus detrimental to health. In this study, we couple electrochemistry with mass spectrometry (MS) with the creation of a band electrode to which individual bacterial cells can be attached and quantitatively detected due to favorable electrokinetic phenomena. E. Coli K-12 was the chosen strain to be analyzed. Raw E. coli analysis was done at first with α-cyano-4-hydroxycinnamic acid (CHCA) as matrix and no ionizing salt for MALDI-TOF/TOF characterization. All ions detected were identified through searches in general databases, including EcoCyc and LIPID MAPS. Initially, regular MALDI-MS analysis of bacterial samples was performed to establish the conditions needed for the bacteria to give rise to peaks characteristic of their biological components. Since the objective of this study was to work with low concentrations of bacteria (1.2 pm -1.4 pm), due to the size of the electrode, the lipid signatures of the bacteria were probed; 3 signature peaks (~ m/z 91, 146, and 285) in reflectron negative mode, and 3 peaks (~ m/z 375, 439, and 666) in linear negative mode, were chosen to determine the limit of MS detection of the instrument and will be used to identify the bacteria in the band electrode using imaging. This work is currently being expanded to include DART-MS for bacterial identification, as this method is ambient and requires no sample preparation. Combining such qualitative analysis by MALDI-MS and DART-MS with electrochemical quantitation offers a new route for complete and sensitive bacterial characterization in various environments and samples.

Charge Transfer Dissociation Mass Spectrometry (CTD-MS) for the Structural Characterization of Glycopeptides
Madeline Schuch¹, Glen Jackson^1,2
¹C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, USA
²Department of Forensic and Investigative Science, West Virginia University, Morgantown, WV 26506-6121, USA

The heterogeneity of protein glycosylation makes it a difficult type of post-translational modification (PTM) to characterize; the modification can occur at various amino acid positions and with multiple different glycan structures. Tandem mass spectrometry is a leading technique to elucidate the structure of glycoproteins and glycopeptides. Current fragmentation methods include collision induced dissociation (CID) and electron transfer dissociation (ETD), but they are typically unable to obtain full structural characterization of intact glycopeptides. In this work, we used a radical-driven fragmentation technique called charge transfer dissociation (CTD) for structural analysis of glycopeptides. We analyzed several glycopeptide standards, including sialylglycopeptide (SGP), Erythropoietin (117-131) (EPO) with N-acetylgalactosamine (GalNAc), and MUC5AC3 (MUC) with GalNAc. Experiments were conducted on a modified Bruker AmaZon quadrupole ion trap using either CTD or CID. We analyzed the most abundant charge state of the glycopeptide using electrospray ionization (ESI). For CTD, a pulse width of 20 ms of ~5.5 keV helium cations was used. CID data of the same precursors were collected for comparison. CID could not determine the linkage pattern of the sialic acid because it generated no cross-ring cleavages and only a partial array of glycosidic cleavages. In contrast, CTD determined the sialic acid linkage of an α-2,6 linkage and not an α-2,3 linkage for SGP from unambiguous cross-ring cleavages. CTD also determined full monomeric sequencing through a full array of glycosidic cleavages. For the O-linked glycopeptides, CID generated mostly b/y peptide ions, whereas CTD generated a/x, b/y, c/z peptide ions. CTD produced a higher sequence coverage for the O-linked glycopeptide compared to CID. The positionings of the glycans for the O-linked glycopeptides could both be determined by CTD, but CID could only localize the glycan of EPO.

Facilitating Disease Diagnosis Detection of RNAs by Ambient Ionization Mass Spectrometry
Anju Sheregar, Rachel Roberston, Ayesha Seth, Keerthana Ravi, Venkat Gopalan, Abraham Badu-Tawiah
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH

We present the design and validation of a paper-based platform for detecting RNA, with the long-term objective of developing noninvasive blood tests for genetic and infectious diseases.1 For example, many messenger RNAs and long noncoding RNAs1 are upregulated in the blood of cancer patients highlighting their potential to be used in cancer diagnosis.2 The detection in blood of cell-free DNA/RNA released from tumor cells offers new prospects for exploiting them as biomarkers in diagnostics. To this end, we leverage a mass spectrometry (MS)-based platform to detect specific RNAs.

For proof-of-concept, we used the Methanobrevibacter smithii (Msm) RNase P RNA (RPR), a ribozyme, that has been engineered with 5′ and 3′ extensions. A biotin-streptavidin–based system was used to pull-down the RPR; specifically, the RPR was incubated with a DNA oligo complementary to its 3′ extension and having an overhang. A biotinylated DNA oligo that binds the overhang was then used to capture the RPR on streptavidin beads. The tight non-covalent interactions between streptavidin and biotin provide the foundation for this assay3. Once captured, the RPR is ready for interrogation with a second DNA probe that is complementary to the RPR’s 5′ extension.

In parallel with the pull-down optimizations, we developed a detection conjugate by attaching an in-house-synthesized ionic probe to a dendrimer, which in turn was attached to a DNA oligo that is complementary to the RPR’s 5′ extension. The ionic probe can be hydrolyzed by alkali to release a mass tag, which is detected by MS. To ensure signal amplification and thus high assay sensitivity, the G4-PAMAM (polyamidoamine) dendrimer was used in the bioconjugation process to facilitate attachment of multiple probes. The ionic probe-dendrimer-DNA conjugate when annealed to the RPR sets the stage for specific detection of the RPR.

This work will showcase proof-of-concept experiments for the pull-down assay and MS analysis of the cleaved mass tags to demonstrate successful RNA detection.

References:

1. 1. Chivers, C. E., Koner, A. L., Lowe, E. D., & Howarth, M. (2011). How the biotin–streptavidin interaction was made even stronger: Investigation via Crystallography and a chimaeric tetramer. Biochemical Journal, 435(1), 55–63. https://doi.org/10.1042/bj20101593 
  2. Nam, G.-H., Mishra, A., Gim, J.-A., Lee, H.-E., Jo, A., Yoon, D., Kim, A., Kim, W.-J., Ahn, K., Kim, D.-H., Kim, S., Cha, H.-J., Choi, Y. H., Park, C.-I., & Kim, H.-S. (2018). Gene expression profiles alteration after infection of virus, bacteria, and parasite in the olive flounder (paralichthys olivaceus). Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-36342-y 
  3. Struyf, T., Deeks, J. J., Dinnes, J., Takwoingi, Y., Davenport, C., Leeflang, M. M., Spijker, R., Hooft, L., Emperador, D., Dittrich, S., Domen, J., Horn, S. R., & Van den Bruel, A. (2020). Signs and symptoms to determine if a patient presenting in primary care or hospital outpatient settings has COVID-19 disease. Cochrane Database of Systematic Reviews. https://doi.org/10.1002/14651858.cd013665

Advancing Mass Spectrometry Data Management through Numerical Compression
Herman Singh^1*, John Rose^1*, Michael A. Freitas^1,2
¹MassMatrix Inc., Columbus, OH 43220he Ohio State University, College of Medicine, Columbus, OH, 43210
²MassMatrix Inc., Columbus, OH 43220he Ohio State University, College of Medicine, Columbus, OH, 43210
^*Co-first authors

High-resolution mass spectrometry data, such as m/z and intensity information from LC-MS/MS runs, generates large datasets that require efficient storage solutions. Traditional general-purpose compression algorithms struggle with the specific patterns in numerical data, resulting in suboptimal performance. In this study, we utilized the PCodec Numerical Compression Algorithm (Pco) to losslessly compress centroid and profile data from LC-MS/MS runs, demonstrating substantial advantages in reducing data size and maintaining data integrity. Our preliminary results show that we achieve 2.1x greater compression on profile data and 1.3x greater compression on centroided data relative to existing mzML compression methods. We also surpass existing mzML compression methods in data integrity and compression speed. This translates to faster data transfers and lower data storage costs (e.g., $1-3K per year in AWS S3 savings)

We also evaluated the lossless nature of the compression, ensuring that the integrity of the original data was preserved during the compression and decompression processes. Data quality metrics, file size reductions, and compression/decompression throughput will be presented, illustrating the superior performance of modern numerical compression over traditional methods. Based on these findings, we propose adopting newer numerical compression approaches, like Pco, into the MzML standard to optimize file size and storage efficiency for mass spectrometry data.

The results highlight the potential for significant data management improvements in mass spectrometry workflows, emphasizing the critical need for tailored numerical compression methods to handle the increasing scale of modern mass spectrometry datasets.

Online Fractionation of Complex Lipid Mixtures on SPE Cartridge followed by In-Situ Mass Spectrometry Analysis
Octavion Spears, Alex Grooms, Benjamin Burris, Abraham Badu-Tawiah
Department of Chemistry and Biochemistry, The Ohio State University

Lipidomics, the study of the lipidome in bodily systems, has grown in prominence over the last decade due to an improved understanding of how altered metabolism impacts the early onset of many important diseases like diabetes, cardiovascular disease, and cancers. Matrix effects from complex samples (e.g., blood, plasma, or serum) often leads to the masking lipidomic information. Consequently, a robust, user-friendly, and accessible lipidomic platform designed with people-oriented care in mind is desired. Human blood, plasma, and serum contain many thousands of distinct lipids, and the physiological contributions of these diverse lipids are now beginning to emerge. Among the first steps toward the objective of complete characterization of these species is the development of analytical methods for in-depth lipidomic analysis. Until now, however, technologies developed for lipidomics rely on the enhanced resolution provided by high-field NMR and multi-dimensional mass spectrometry (MS) techniques requiring expensive specialization. With increasing interest to apply mass spectrometry (MS) in middle- and third-world countries, a multi-functional ion source that is widely applicable and accessible can transform simple standalone mass spectrometers, including portable instruments, into a powerful tool for applications in clinical chemistry, environmental analysis, agricultural, and biomedical research. Our novel lipidomic platform paired with an affordable solid phase extraction (SPE) syringe cartridge is designed to complete quantitative and selective online fractionation of a lipid mixture that can be directly injected into the MS for facile analysis. This analysis is designed for biological samples like serum blood and plasma, and so far, we’ve reached done separations of bovine heart extract and a polar lipid extract, with other biofluid trials coming in the future. This extraction has been completed using bare, unmodified silica, with wash solvents of hexane and methanol.

Development of Mass Spectrometry-Based Immunoassay for Microfluidic Paper-Analytical Device for the Early Prediction of Severe Acute Pancreatitis
Ruth Speidel¹, Ella Warner¹, Jona Kozyr-Verni¹, Sophie Miller¹, Georgios Papachristou², Peter Lee², Abraham Badu-Tawiah^1*
¹Ohio State University, Columbus, OH
²Ohio State University Wexner Medical Center, Columbus, OH

A paper-substrate device for the early prediction of severe acute pancreatitis is being developed using paper-based immunoassay followed by on-chip paper spray mass spectrometry. Acute pancreatitis (AP) accounts for over 300,000 hospital admissions in the U.S. annually. Approximately 15% of AP subjects develop severe AP (SAP), defined by presence of persistent organ failure (POF). The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) recognized a critical knowledge gap that is a barrier to advancing clinical care: establishment of a bedside, early prediction tool to identify which subjects will develop SAP during hospitalization. In our work, a pH-sensitive ionic probe is coupled to specific monoclonal antibodies that can capture a panel of five cytokine protein biomarkers during immunoassay. Afterward the probe is cleaved, and the mass tags with low molecular weight are analyzed via paper spray mass spectrometry. Therefore, instead of analyzing the large protein biomarker with expensive and big mass spectrometers, our method utilizes portable mass spectrometers to detect the small mass tags, making bedside analysis possible. To obtain high sensitivity in low sample volumes, a generation 4.0 polyamidoamine (PAMAM) dendrimer is used to amplify the signal by increasing the number of ionic probes that can attach to one antibody. In this presentation, data will be shown for manual immunoassay in two-dimensional wax-printed paper substrates for the five cytokine protein biomarkers where cross reactivity and antibody selectivity will be evaluated and reported. Data will include method optimization, calibrations, and bio-specificity of the monoclonal antibodies. Preliminary data supports detection limits in the pM range for most biomarkers, which is comparable to other immunoassay-based tests. The advantage of our method lies in our ability to utilize whole blood in microliter quantities without pre-treatment, which reduces turnaround time for diagnosing this time-sensitive diseases.

Direct Synthesis of Primary Amines from Alcohols via Uncatalyzed Plasma-Microdroplet Chemistry
Rebekah E. Strong, Owen L. Looker, Abraham K. Badu-Tawiah
The Ohio State University

Primary amines are one of the most versatile and sought after intermediates in synthetic chemistry. They are commonly used as starting materials or intermediates in total synthesis of pharmaceutically and industrially relevant compounds. Current methods rely on the use of protecting groups, multiple purification steps, and corrosive reducing agents to achieve this moiety. Recent research has led to more direct methods of primary amine synthesis, but they rely on expensive transition metal photocatalysts to do so. Therefore, there is still the need for simpler, greener, and less expensive means of direct synthesis of a primary amine. Using an in-house made contained electrospray ionization (cESI) source, fixed with a fused silica capillary and an etched silica capillary, we demonstrated the ability to convert alcohols (aromatic and aliphatic) to primary amines using hydrazine (i.e., H2NNH2). The alcohol was delivered through the fused silica capillary and the hydrazine via the etched silica capillary in a co-axial spray fashion. The use of etched silica capillary enables the generation of corona discharge in the wake of charged microdroplets, which provides energetic collisions in the electrospray to split H2NNH2 into two NH2^● radical species. The NH2^● groups are subsequently trapped in the charged microdroplets, which react with the alcohol to generate the final amine product in a single step. A wide range of alcohols have been investigated, with promising yields, and structure of the amine products were confirmed via tandem mass spectrometry analysis. All compounds were analyzed in positive-ion mode, except where necessary to use negative-ion mode. Our studies suggest this method is a useful alternative to bulk synthesis, achieving primary amines without the need for expensive and corrosive reagents. We plan to explore collection of these products and verification by NMR analysis. Mechanistic studies will be discussed using isotopically labeled reagents and radical scavengers.

Mapping Protein-Ligand Ion Stability Landscapes Using a cVSSI Approach
Mst Nigar Sultana¹, Mohammad Rahman¹, Kevin Courtney², Stephen Valentine¹*
¹Department of Chemistry, West Virginia University, WV-26505, USA
²Department of Biochemistry and Molecular Medicine, West Virginia University, WV-26505

Native mass spectrometry (MS) has emerged as a valuable tool in protein structure analysis, especially for examining large protein complex stabilities and protein-ligand interactions. The methodology provides the ability to analyze protein-ligand interactions in their native, non-denatured states, allowing for biological insight into the structures and stabilities of the complexes. However, the stability and dynamics of intrinsically disordered regions (IDRs) within proteins can be altered upon binding diverse ligands because of the various interactions formed by IDRs. In this study, controlled, in-droplet protein unfolding using an ultrasonic vibrating sharp-edge spray ionization (VSSI) method is utilized to map the stability landscape of a protein and a ligand (drug) in both bound and unbound states. The protein was expressed and purified according to the standard protein production procedures described elsewhere. A solution of FKBP12 protein and a mole ratio range (i.e., 0 to 10) of drug (i.e., FK506 and rapamycin) in ammonium acetate buffer has been examined. Initially, kD is determined from the different mole-ratio solutions through examination of bound and unbound states (ions) in the MS spectra. By applying voltage to the VSSI emitter tip in a stepwise fashion, controlled unfolding the disordered protein FKBP 12 as well as its binding with FK 506 is examined. Correlations between unfolding events of bound and unbound protein are drawn with changes in the radius of gyration obtained from molecular dynamics (MD) simulations. This expanded analysis aims to present a new methodology for mapping protein structure stabilities and conformer heterogeneity in drug-protein systems.

Increasing Throughput while Maintaining Coverage Depths in Single Cell Proteomics Using the timsTOF Ultra2
Christoph Krisp¹, Dijana Vitko², David Hartlmayr³, Anjali Seth³, Guilhem Tourniaire³, Thorsten Ledertheil¹, Jean-Francois Greisch⁴, Markus Lubeck¹
¹Bruker Daltonics GmbH & Co. KG, Bremen, Germany
²Bruker Daltonics Inc., Billerica, US
³Cellenion, Lyon, France
⁴Bruker Switzerland AG, Faellanden, Switzerland

For single cell proteome analysis, throughput while maintaining coverage depths is one of the bottlenecks in the field. The latest enhancements in 4D-proteomicsTM on the timsTOF platform pushes the limits of detection to the single cell level. Combined with automated single-cell isolation and sample preparation on the cellenONE® platform enables deep proteome coverage, with high throughput and reproducibility.

HeLa cells at different cell counts were isolated into the LF48 proteoCHIP®, directly lysed and digested using the cellenONE platform. Samples were either dried down in the chip or manually transferred into 96-well plates. Peripheral blood mononuclear cells (PBMCs) were FACS sorted into T-Cells (CD4+, CD8+) B-Cells (CD19+) and monocytes (CD14+), isolated with the cellenONE and prepared as HeLa. The LF48 proteoCHIP was placed into the nanoElute® 2 autosampler. Sample were picked up with the dissolve sample function, injected onto a 5 cm Aurora Rapid 75cm or a 25cm Aurora Ultimate C18 column (IonOpticks) and eluted into a timsTOF Ultra2 at 80 or 32 SPD. dia-PASEF® data were processed with Spectronaut 19 (Biognosys) using directDIA+. Processing of the dia-PASEF data with Spectronaut 19 demonstrated good quantitative reproducibility for individual concentration loads.

Sample pickup directly from the label-free proteoCHIP with the dissolve sample function of the nanoElute® 2 was compared to proteome depths obtainable with samples manually transferred to 96 well plates. Both setups performed equally well delivering a protein depth of about 4,000 proteins per single cell and increasing to >6,000 proteins for 20 cells. The PBMC analysis workflow from FACS sorted T-Cells (CD4+, CD8+) B-Cells (CD19+) and monocytes (CD14+) in the 80 SPD setup identifying over 2,000 protein groups across the different cell types, classified the two T-Cells classes from each other and also from the B-Cells and monocytes.

Probing the Structure of Covalently Labeled Proteins in the Gas Phase by Native MS
Evan Whitford^1,2, Elijah Day^1,2, Steffen Lindert^1,2 and Vicki H. Wysocki^1,2,3
¹Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States
²Native Mass Spectrometry Guided Structural Biology Center, The Ohio State University, Columbus, OH, United States
³Campus Chemical Instrumentation Center, The Ohio State University, Columbus, OH, United States

Drug discovery hinges on suitable methods for protein structure determination, which is critical for interpreting biomolecule function and reactivity. Mass spectrometry (MS) paired with covalent labeling (CL) uses reagents like diethylpyrocarbonate (DEPC) to modify solvent-accessible amino acids, adding a mass tag with minimal perturbation to the native structure. Modified residues are detected by tandem MS of peptides from enzymatic digestion and provide insight on the topology of the labeled protein. Our goal is to use native MS as a diagnostic tool to define over-labeling conditions, as slower-reacting reagents (DEPC) can cause structural perturbations.

Protein-DEPC reactions were confirmed using high-resolution native MS to observe the 72 Da mass addition from DEPC. Collision induced unfolding (CIU) experiments were used to observe differences in unfolding pathways of monomeric proteins (MEIN1, lysozyme, ubiquitin) and complexes (CRP, βLG). Differences in unfolding reveal whether structural integrity is significantly perturbed. Ion mobility data of native MEIN1 reveals an increase in the collision voltage required to unfold MEIN when it has been multiply labeled. Other monomeric proteins showed subtle changes, suggesting MEIN1 may be restructuring to a more stable conformation after labeling. RMSD changes in fingerprints reveal differences in unfolding pathways, with each added label slightly increasing RMSD compared to an unlabeled fingerprint.

Native MS experiments were used to investigate DEPC effects on interfaces of the complexes CRP and βLG. A 20:1 DEPC: βLG ratio decreased dimer and increased monomer populations, suggesting higher DEPC concentrations disrupt βLG’s dimer structure into monomers. Surface induced dissociation (SID) experiments provided comparison of fragmentation patterns between labeled and native proteins. SID of native samples showed native-like fragmentation where SID of extensively labeled samples showed a significant increase in monomer fragments indicating restructuring has occurred.

Arugula Grown With Elevated Environmental Sulfur Produces More Glucosinolates and Isothiocyanates
Aaron Wiedemer¹, Haley Kruest², Chieri Kubota², Jessica Cooperstone^1,2
¹Department of Food Science and Technology, The Ohio State University, Columbus, OH, United States
²Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH, United States

Consumption of Brassicaceae vegetables such as broccoli, kale, and arugula have been correlated to improved health outcomes, including reduced risk for developing cancer, obesity, and heart disease. To date, most work to alter crop phytochemistry for human health has focused on breeding new varieties. However, not all phytochemicals are under genetic control. Controlled environment agriculture offers a powerful avenue to alter phytochemical content by precisely controlling environmental conditions such as light, temperature, and nutrient levels. In addition to fiber and essential micronutrients, Brassicaceae vegetables also uniquely produce glucosinolates, a class of sulfur containing plant derived metabolites with the general structure of a sulfonated aldoxime domain bonded to a β-D-thioglucose and a variable R group. When consumed, glucosinolates are converted to bioactive isothiocyanates which are also for the characteristic pungent flavors of Brassicaceae vegetables. In this preliminary analysis, we hypothesize that arugula grown with higher concentrations of sulfur will produce more glucosinolates and thus more isothiocyanates than under lower levels. Four cultivars of arugula (‘Astro’, ‘Esmee’, ‘Sparkle’, ‘Wasabi’) were grown with either low (~ 0 mg/L) or high (40 mg/L) levels of environmental sulfur using mini deep water culture hydroponic systems in a Conviron reach-in growth chamber (Columbus, OH). Seven replicates of each treatment (cultivar $\times$ sulfur level) across four blocks were harvested after four weeks, flash frozen, and aliquoted for both glucosinolate and isothiocyanate analysis. Isothiocyanates were derivatized with N-acetyl cysteine (NAC) to decrease volatility for analysis. HILIC-UHPLC-ESI-QTOF-MS and MS/MS were used to identify and quantify glucosinolates and isothiocyanate-NAC derivatives. Glucosinolates and isothiocyanates were significantly higher in arugula grown with elevated sulfur (p < 0.05) and the specific metabolites produced varied across cultivars. These data suggest controlling environmental growing conditions can improve the health promoting capacity and flavor of arugula.

UPP1 Contributes to the Maintenance of Cancer Stem Cells in Ovarian Cancer via Metabolism Reprogramming 
Sophia K. Wu^1,2, Linzhou Wang¹, Ananya Banerjee¹, Na Li¹, Yajing Yang¹, Patrick Stevens³, Xiaoli Zhang³, Qi-En Wang¹
¹Department of Radiation Oncology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH
²Columbus Academy, Gahanna, OH
³Center for Bioinformatics, College of Medicine, The Ohio State University, Columbus, OH

Epithelial ovarian cancer (EOC) is the most lethal malignancy of the female reproductive tract with an only 30% five-year survival rate in advanced stages. Tumor relapse and acquired chemotherapy resistance are two major factors leading to the high mortality of EOC patients. Ovarian cancers contain a subpopulation of cancer stem cells (CSCs) with enhanced tumorigenicity and chemoresistance. These CSCs are believed to be responsible for treatment failure and tumor relapse. In addition, chemotherapeutic agents such as cisplatin can actually enrich CSCs. However, it remains unclear how cisplatin treatment enhances the stemness of ovarian cancer cells.

Using single cell RNA-sequencing (scRNA-seq), we identified several clusters of ovarian cancer cells that survive cisplatin treatment and gain a growth advantage. These cells exhibit elevated levels of Uridine phosphorylase 1 (UPP1), a crucial enzyme in pyrimidine metabolism that catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate. Further analysis revealed that reducing UPP1 expression diminishes the self-renewal capability of ovarian cancer cells. This suggests that cisplatin-induced increases in UPP1 expression may help maintain the stemness of these cells. Further untargeted metabolomics analysis by LC-MS/MS indicated that UPP1 knockdown impacts several potential metabolic pathways, such as “Superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass” and “Superpathway of pentose and pentitol degradation”. This implies that UPP1 may bolster the stemness of ovarian cancer cells by reprogramming their metabolism.

In summary, our findings uncover a new mechanism by which cisplatin treatment boosts the stemness of ovarian cancer cells and expands the CSC population. Targeting UPP1 could be a promising strategy to improve ovarian cancer treatment and reduce the risk of tumor recurrence following conventional platinum-based chemotherapy.

Development of Advanced Database Tools for Characterization of Saccharides Isomers with Negative-Ion Mode Direct Infusion Mass Spectrometry
Shixiang Xu, Santosh R. Acharya, Ruth Speidel, Abraham K. Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH

Saccharide plays a vital role in various living systems. A typical saccharide can be classified into four different types: monosaccharides, disaccharides, oligosaccharides and polysaccharides depending on the number of building blocks. Imposed on these different saccharide types are various isomeric forms, including composition, connectivity, configuration, and branching. These make characterization of saccharides very challenge. Preliminary experiment indicates that collisional–induced dissociation of saccharide in positive-ion mode only yield glycosidic cleavage, giving limited details on the isomer structure. This challenge has been resolved by negative-ion mode analysis with chloride adduct, which provides more detailed isomer differentiation due to dissociation of both glycosidic and cross-ring bonds. In our previous tandem MS experiments, we showed that sugar-chloride adduct dissociates to give distinct fragment ions for each structural isomers of sucrose. However, there is a challenge remaining, which involves manual interpretation of the tandem mass spectra. We have observed that disaccharide isomers undergo cross-ring breakage to produce common product ions via the loss of neutral species such as 60 Da (C2H4O2), 90 Da (C3H6O3), and 120 Da (C4H8O4). Therefore, this brings advantages to utilize database algorithm for high-throughput data analysis to avoid the burden of manual interpretation. Here, our database algorithm utilizes several statistical methods include principal component analysis (PCA) and cosine core similarity to compare the stored data to the tested sample and automatically assigns confidence score for complete structural characterization. With the assistance of database analysis, we will accurately identify the saccharide isomer and we envision that such method will be highly applicable in various fields, including pharmaceuticals, food safety and metabolomics studies.

Determination of Glycoform Masses of SARS-COV-2 Spike Protein Variants by Electron Capture Charge Reduction (ECCR) Mass Spectrometry
Zhixin Xu^1,2, Chen Du^1,2, Eduardo Olmedillas³, Regina M. Edgington¹, Sophie R. Harvey², Erica Ollmann Saphire^3,4, Vicki H. Wysocki^1,2
¹Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH
²Native Mass Spectrometry Guided Structural Biology Center, The Ohio State University, Columbus, OH
³Center for Vaccine Innovation, La Jolla Institute for immunology, La Jolla, California
⁴Department of Medicine, University of California, San Diego, La Jolla, California

SARS-CoV-2 spike protein has numerous glycan sites, resulting in more than 1010 possible glycoforms. Although other methods have shown information like glycan composition and percentage occupancy, the rapid characterization of this heterogenous protein is still challenging. We are solving this problem by using native mass spectrometry (nMS) combined with Electron Capture Charge Reduction (ECCR) and narrow mass-to-charge (m/z) window selection. A modified Thermo Q Exactive Ultra-High Mass Range mass spectrometer with a custom Electron-based Dissociation-Surface Induced Dissociation (ExD-SID) device (eMSion) was used for all experiments. The ExD-SID cell replaced the transfer multipole. We utilized the quadrupole to perform narrow-window mass-to-charge (m/z) selection and ECCR to deconvolve many overlapping glycoform signals in the selected m/z range. Typically, a spectrum of a heterogeneous sample appears as a single, broad peak, providing limited information about the charge states. After applying ECCR, several apexes corresponding to different charge states can be observed.  We collected resolved charge state peaks for spike proteins and determined the mass distribution of three variants of concern (VOC). The average mass for these three VOC (D614G, BA.4/5, and Brazil) are 509 kDA, 521 kDa, and 503 kDa, respectively. The results also were compared with glycoproteomic data by using Monte Carlo simulation. The simulation data shows a narrower distritbutions, which suggests the underestimation of glycan heterogeneity of glycoproteomics data. The results provided glycan masses from intact spike variants and revealed the differences in oligosaccharide chains between different VOCs, which will facilitate vaccine and drug development for this virus. The intact mass distribution can benchmark the glycan percentage occupancy to assist future omics studies. Our technique utilizes rapid sample preparation and accurately differentiates between glycosylation variants using nMS. This strategy allows the distinction of three spike protein variants by turning unresolvable spectra into distinct charge state distributions.

Liquid Chromatography-Mass Spectrometry Analysis of Total tRNA using RNase 4
Hina Zain, Bibek Hamal, Asif Rayhan, Patrick A. Limbach
Department of Chemistry, College of Arts and Science, University of Cincinnati, Cincinnati, OH USA

Tandem liquid chromatography-mass spectrometry (LC-MS/MS) is one of the best tools for the analysis of the RNA sequence without amplification and converting it into cDNA. Identifying the RNA sequence and locating the specific ribonucleoside modifications requires the digestion of RNA into oligonucleotides that can then be analyzed by LC-MS/MS. Various ribonucleases with different cleavage specificity can be used to generate these oligonucleotides. Recently, human RNase 4, which cleaves at the sequence motif Up(A/G), became available commercially. Past work has focused on the use of this enzyme for the digestion of messenger RNA (mRNA). However, transfer RNAs (tRNAs) contain far more modifications than are found in mRNAs. In the present study, different incubation periods and enzyme concentrations for human RNase 4 were optimized with a 78-nt tRNA mimic. Once the conditions were optimized, the reaction was applied to yeast total tRNA. We found that RNase 4 cleavage motif can include the modified uridines, pseudouridine and dihydrouridine. Additionally, a Cp(A/G) cleavage motif was also found. Optimization of reaction conditions for human RNase 4 digestion of complex tRNA mixtures will enable improved tRNA modification mapping by LC-MS/MS

Discovery of Noninvasive Biomarkers for Radiation Exposure via LC-MS-based Hair Metabolomics
Huan Zhang^1,2, Shruthi Kandalai^1,3,4, Haidong Peng^1,3,4, Rui Xu^1,2, Michael Geiman^1,3, Shuaixin Gao^1,2, Shiqi Zhang^1,2, Naduparambil K. Jacob^1,3, Qingfei Zheng^1,3,4, Jiangjiang Zhu^1,2
¹Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
²Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, Ohio 43210
³Department of Radiation Oncology, College of Medicine, The Ohio State University, Columbus, Ohio 43210
⁴Center for Cancer Metabolism, The Ohio State University, Columbus, Ohio 43210

Radiological exposures pose significant risks to human health, particularly with the growing use of nuclear technologies in modern society. While irradiation biomarkers have been extensively studied in biological specimens like urine and blood, research on less invasive samples such as hair and skin remain limited. Our study focuses on developing LC-MS-based metabolomic analyses of hair samples from animals subjected to low-dose irradiation (0, 1, 2, and 4 gray) to identify potential biomarkers for health monitoring. We considered different exposure doses and also assessed the impact of sex and post-exposure time as confounding factors. Using a sample of 240 irradiated C57BL/6 mice, we collected hair samples over a 180-day period and performed both untargeted and targeted metabolomics, analyzing the data with advanced bioinformatics tools. Our optimized workflow identified 284 metabolites, revealing significant variations based on time, gender, and irradiation dose. Partial least squares-discriminant analysis (PLS-DA) indicated that sample collection time, gender, and dose influence molecular profiles differently. Our novel machine learning method, Multiple & Optimal Screening Subset (MOSS), achieved high accuracy in differentiating samples based on post-exposure time points, with AUCs of 0.89 for short-term and 0.91 for long-term exposure, and an AUC of 0.75 for gender differentiation. Differentiating responses to varying irradiation doses proved challenging, with AUCs ranging from 0.55 to 0.71. Notably, fatty acyls like azelaic acid and palmitoylcarnitine emerged as potential timepoint-dependent biomarkers, while trimethylamine N-oxide (TMAO) was identified as gender-specific. Additionally, hippuric acid and 5-methoxy-3-indoleacetae were potential biomarkers for varying irradiation doses. Our study is among the first to use non-invasive, rapid, and sensitive mass spectrometry-based analyses of hair to evaluate irradiation exposure, uncovering novel metabolic biomarkers.

Distinct Plasma Molecular Profiles between Early-Onset and Late-Onset Colorectal Cancer Patients Revealed by Metabolic and Lipidomic Analyses
Shiqi Zhang¹, Rui Xu¹, Ming Hu¹, Fouad Choueiry¹, Ning Jin², Jieli Li³, Xiaokui Mo⁴, Jiangjiang Zhu^1,5
¹Human Nutrition Program, Department of Human Sciences, The Ohio State University, Columbus, OH 43210, USA
²Medical Oncology, The Ohio State University, Columbus, OH 43210, USA
³Department of Pathology, The Ohio State University, Columbus, OH 43210, USA
⁴Biomedical Informatics, The Ohio State University, Columbus, OH 43210, USA
⁵James Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA

Colorectal cancer (CRC) incidence in younger adults has been steadily rising, warranting an in-depth investigation into the distinctions between early-onset CRC (EOCRC, < 50 years) and late-onset CRC (LOCRC, ≥ 50 years). Despite extensive study of clinical, pathological, and molecular traits, differentiating EOCRC from LOCRC and identifying potential biomarkers remain elusive. We analyzed plasma samples from healthy individuals, EOCRC, and LOCRC patients using liquid-chromatography mass spectrometry (LC/MS)-based metabolomics and lipidomics. Distinct polar metabolite and lipid profiles with significant metabolites altered in CRC group (e.g., choline and DG 40:4) were identified. Notably, EOCRC exhibited distinct polar metabolomic and differential lipidomic profiles compared to LOCRC, with polar metabolites like aminoadipate and uridine contributing significantly to the difference and originating from pathways such as lysine biosynthesis and nucleotide metabolism. Furthermore, gene set enrichment analysis (GSEA) using independent TCGA gene expression data identified pathways significantly enriched in either EOCRC or LOCRC. Integrating gene expression and metabolomics data revealed numerous associations differentiating EOCRC and LOCRC. Our multi-omics integration underscores critical molecular distinctions, offers insights into the EOCRC development mechanisms and potential plasma biomarkers for diagnosis.

Development of a Conductive Emitter for Dynamic Spray Mass Spectrometry
Alexander P. Zumock, Purva S. Damale, Abraham K. Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, United States

Mass spectrometry relies heavily on the initial ionization step which influences metabolic coverage and detection limits in complex mixtures. Existing ionization methods struggle with samples that have diverse physicochemical properties (polar/non-polar and high/low molecular weight) using a single source. To overcome this, we present a novel single ionization source capable of sequential ionization across a wide molecular range without requiring sample preparation or chromatography. This is accomplished through dynamic voltage ramping, which allows different analytes to be ionized at specific voltages. Our dynamic spray source features an electrodeless design with a single emitter made of disposable silver-coated borosilicate glass capillaries, operating within a DC voltage range of 0 to ±8 kV. The voltage waveform increases or decreases linearly over time, enabling conventional nanoelectrospray ionization (nESI) at initial voltages (1-2 kV) and transitioning to microdroplet/gas-phase ionization for APCI. This method works in both positive- and negative-ion modes, allowing for the sequential detection of diverse analytes in complex mixtures and biofluids. At higher voltages (>4 kV), corona discharge is initiated at the pointed tips of the borosilicate glass capillaries, generating nonthermal plasma. Initially, commercially available conductively coated PicoTips were used, but their high cost and limited capillary size variation prompted the development of a more cost-effective and customizable solution. We successfully coated sharp borosilicate glass capillaries with a conductive silver layer using a silver nitrate solution in ethanol and butyl amine, yielding an even silver deposit. Detection of wide range of molecules (steroids, lipids, proteins, sugars, vitamins, and nucleotides) in a complex mixture without any manual voltage tuning has been accomplished. The application of this Dynamic Spray ionization source, coupled with a single conductive emitter, shows significant promise for the analysis of complex mixtures and biofluids, making it a versatile and robust method with potential applications in translational and biomedical research.