Poster Listing (submitted abstracts)
Note: Odd numbered posters will present during Session 1 (Wednesday); Even numbered posters will present during Session 2 (Thursday).
Posters are clustered by general topic: MS Methods (posters 1-10); Protein MS (posters 11-19); Diagnostics and Omics (posters 20-35)
- Kulyk, Dmytro S.; OSU Department of Chemistry and Biochemistry; Plasma-in-Droplet Ionization Mass Spectrometry
- Wairegi, Salmika G.; OSU ; Thread-Based Microfluidic Device for Real-Time Reaction Monitoring by Thread Spray Mass Spectrometry
- Seth, Ayesha; OSU Department of Chemistry and Biochemistry; Self-Aspirating Contained Electrospray Ionization (SAC-ESI) for Microsampling and Sensitive Diagnosis
- Acharya, Santosh Raman; OSU Dept of Chemistry and Biochemistry; High Performance Liquid Chromatography coupled to Contained Electrospray Ionization Mass Spectrometry (HPLC-cESI-MS) for High-Throughput Analysis of Isomeric Saccharides
- Panday, Niraj; OSU Department of Chemistry and Biochemistry; Separation and quantification of isomeric lipids of different classes via high flow rate plasma-droplet fusing using LC-Contained-Coaxial ESI-MS
- Grooms, Alexander; OSU; Programmable Carbon-Carbon Bond Formation via Uncatalyzed Michael Addition in a Plasma-Water Microdroplet Fusing Green Synthetic Platform
- Damale, Purva Shripad; OSU; Dynamic Electrospray Ionization Mass Spectrometry
- Baker, Kristie; OSU; Variable-Temperature Electrospray Ionization Coupled with Electron Capture Dissociation to Study Temperature Induced Solution Phase Structural Changes
- Khair, Mst Ummul; Cleveland State University; Muricholic Bile Acid Enantiomers Separation by C18 RPLC-MS as a Function of Polarity Values of the Mobile Phase Components
- Shaffer, Tyler; C. Eugene Bennett Department of Chemistry, West Virginia University; Modifaction of Commercial Capillary Electrophoresis Instrument to use Vibrational Sharp-Edge Spray Ionization
- Lacey, Philip ; OSU Chemistry and Biochemistry; Characterizing Caspase-Lipopolysaccharide Oligomeric States with Electron Capture Charge Reduction MS
- Xu, Zhixin; OSU Department of Chemistry and Biochemistry; Electron Capture Charge Reduction (ECCR) for Heterogeneous Glycoproteins
- Qi, Zihao; OSU Chemistry and Biochemistry; Implementing Surface Collision in native MS for Essential DNA Repair Biomolecules
- Xu, Shixiang; OSU Department of Chemistry and Biochemistry; Resolving the Challenges of Overlapping Charge States in Native MS and SID through Electron Capture Charge Reduction and Charge Detection Mass Spectrometry
- Ahmed, Ezaz; Case Western Reserve University; High-Throughput Structural Analysis via Synchrotron X-ray Footprinting at NSLS-II
- Ortega Zepeda, Martha; OSU Department of Chemistry and Biochemistry; Collision-Induced and Surface-Induced Unfolding of Protein Complexes
- Marathe, Ila; OSU Chemistry and Biochemistry; nMS and ECD characterize interactions in RAS-SOS complexes
- Whitford, Evan; OSU Department of Chemistry and Biochemistry; Probing Protein Structures Using Covalent Labeling Mass Spectrometry
- Arslanian, Andrew J.; OSU Dept of Chemistry and Biochemistry; How Rearranged Native Quaternary Structure Affects Surface-Induced Dissociation of Large Protein Complexes
- Speidel, Ruth; OSU CBC; Paper-Based Microfluidic Device for Early Diagnosis of Severe Acute Pancreatitis using Ambient Mass Spectrometry Detection
- Duarte da Silva, Monica; University of São Paulo, Brazil; Paper-based immunoassay for highly sensitive detection of SARS-COV 2 using Miniature Mass Spectrometry
- Zerrudo, Stephanee; OSU Department of Chemistry and Biochemistry; Development of Isobaric Peptide Probes for Multiplex Disease Detection Using Paper Spray Mass Spectrometry
- Carvalho, Hianka; OSU Department of Chemistry and Biochemistry; University of São Paulo, Brazil; A 2D Microfluidic Paper-Based Analytical Device For Diagnosis of Canine Visceral Leishmaniasis in Developing Countries
- Akkaya-Colak, Kubra; OSU; Analysis of metabolites through complementary mass spectrometry imaging and histological methods in mini guts
- Edgington, Catherine; OSU; The Proteomic Changes in HCT 116 Colon Cancer Spheroids During Growth
- Edgington, Regina; OSU Department of Chemistry and Biochemistry; Mass spectral properties of ubiquitylated peptides from non-tryptic proteolysis support alternative informatic strategies for probing the dark ubiquitylome
- Komey, Agnes Josylyn; OSU Department of Human Sciences; Utilizing HPLC-DAD and HPLC-high resolution mass spectrometry to identify iron chlorophyll derivatives synthesized from spinach
- Moore, Madalyn G.; OSU Department of Chemistry and Biochemistry; A Proteomic Analysis of Processed Proteins and Endogenous Peptides in Human Urine
- Patil, Akshay; Cleveland State University; Untargeted metabolomics analysis of LPS-stimulated mast cells by UHPLC-QTOF-MS
- Rayhan, Asif; University of Cincinnati; Universal Mass Exclusion List for Organism Independent Modification Mapping of RNA
- Renzelmann, Chase; OSU; Thermal Proteome Profiling for Characterizing Biophysical Properties of Chaperone Proteins
- Sholola, Maria; OSU; The effects of a lycopene-rich tomato juice enhanced with soy isoflavones on inflammation in individuals with obesity
- Yu, Peifeng; Ohio University; Defining the Developmental Dynamics of Ubiquitylation Proteomes Regulated by ASK1-Containing Ubiquitin Ligases
- Spears, Octavion; OSU; A Multi-Modal Approach for the On-Line Modification of Lipids for Efficient Analysis of Complex Mixtures
- Joyce, Alex; OSU Biomedical Informatics/Pelotonia Institute for Immuno-Oncology; Probabilistic Validation for Targeted Proteomics Using Parallel Reaction Monitoring
- Sledziona, James; OSU; Effects of Scd1 loss upon colon cancer cell metabolism
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
High Performance Liquid Chromatography coupled to Contained Electrospray Ionization Mass Spectrometry (HPLC-cESI-MS) for High-Throughput Analysis of Isomeric Saccharides
Santosh Raman Acharya1, Enoch Amoah1, Abraham Badu-Tawiah1
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210
Saccharides represents an important class of biomolecules with wide-ranging physiological significance. Currently there is considerable interest in characterization of saccharides in food and pharmaceutical industry. Nevertheless, the analysis of saccharides through Mass Spectrometry (MS) encounters substantial challenges due to their intrinsic attributes, such as low ionization efficiency and structural intricacies encompassing linkage and stereo isomerism. Consequently, comprehensive sample preparation or the utilization of sophisticated instrumentation becomes inevitable for successful analysis. In this context, we developed a High-Performance Liquid Chromatography (HPLC) coupled with contained Electrospray Ionization Mass Spectrometry (cESI-MS) platform that can be efficiently used to create halide adducts of isomeric saccharides. Such adducts, upon collision induced dissociation (CID), provided diagnostic ions that could be exploited to differentiate saccharide isomers. This innovative platform shows a promise of unpresented sensitivity via halide ion attachment and might facilitate the separation and complete characterization of complex saccharide isomers.
High-Throughput Structural Analysis via Synchrotron X-ray Footprinting at NSLS-II
Ezaz Ahmed1,2, Erik R. Farquhar1,2, Rohit Jain1,2, Michael Sullivan1,2, Mark R. Chance1,2
1Center for Synchrotron Biosciences, National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, NY, USA; 2Center for Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, OH, USA.
Structural mass spectrometry methods involving covalent labeling are valuable probes of protein structure and dynamics in structural biology, with hydrogen-deuterium exchange being a particularly well established such method. Hydroxyl radical protein footprinting (HRPF) has gain increasing attention in recent decades as an alternative covalent labeling mass spectrometry method. In HRPF, hydroxyl radicals generated by a variety of methods including X-ray radiolysis, laser photolysis, plasma, or Fenton chemistry react irreversible with solvent-accessible amino acid sidechains in proteins. Changes in solvent accessibility in response to a stimulus, such as protein folding, binding of a small molecule or biomolecule partner, or dynamics changes in response to a reactive transformation can be followed using this method. The technique is particularly well suited to structural biology problems where conventional structure determination methods such as crystallography, NMR, or Cryo-EM are less fruitful, including structure assessment of intrinsically disordered proteins in neurodegenerative diseases, probing RNA protection during virus assembly, or tracking protein/RNA interactions in vivo. The 17-BM X-ray Footprinting of Biological Materials beamline operated by Case Western Reserve University at the National Synchrotron Light Source II (Brookhaven National Laboratory, NY) offers access to synchrotron-based HRPF methods, including scientific and technical experimental support. Here, we present the capabilities that the resource offers the research community and introduce a new at-beamline mass spectrometry capability that allows the entire HRPF experiment from sample preparation to data analysis to be performed at a single facility. Opportunities for collaborative access to the resource will be described.
Analysis of metabolites through complementary mass spectrometry imaging and histological methods in mini guts
Kubra B. Akkaya-Colak1, 2*, Emily Sekera2*, Amanda Hummon2^, Maria Mihaylova1, 2^
1Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio
2Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio
3Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio
Organoids are self-organized clusters of cells containing differentiated and undifferentiated cells and are often referred to as mini organs. They can be generated from adult stem cells which are either isolated from healthy or diseased tissues. Organoid technology has been a powerful tool to answer scientific questions in a wide variety of disciplines such as in infectious and hereditary diseases, toxicology, cancer, personalized and regenerative medicine. Mass spectrometry imaging (MSI) is another powerful technique that allows researchers to identify many single molecules at once by preserving their spatiotemporal distribution on a single tissue cross-section. Previous studies have reported protocols to analyze spheroids and single-cell layers of organoids by MSI; however, some of the prior methods were not optimized to preserve 3D budding organoid structures such as those found in intestinal organoids or combined with complementary immunostaining techniques. Therefore, to fill this knowledge gap, we have tested different polymer solutions and conditions to design the most suitable matrices to study branching and budding intestinal organoids by MSI analysis. We generated mouse small intestinal organoids from wild-type C57BL/6 mice and embedded them in different hydrogels for MALDI-MSI. Single-layer budding organoid sections were analyzed by a timsTOF fleX MALDI-2 mass spectrometer in the negative ion mode and data was analyzed by using Scils Lab. In addition, we were able to couple MSI with immunofluorescent (IF) staining from consecutive sections of single-cell layer budding organoids. The combination of these two very important approaches will improve our understanding of disease initiation and progression. It will be of interest to many and will open avenues to study novel biomarkers that can be used to improve the treatment of a wide array of diseases.
How Rearranged Native Quaternary Structure Affects Surface-Induced Dissociation of Large Protein Complexes
Andrew J. Arslanian,1-2 Vicki H. Wysocki1-2
1Department of Chemistry and Biochemistry, The Ohio State University
2National Resource for MS-Guided Structural Biology, The Ohio State University
In-source activation of protein complexes is used for adduct removal to enable highly accurate m/z measurement. While this critical step provides accurate masses, in-source activation can rearrange the quaternary structure, affecting tandem MS results. For example, when native-like protein complexes undergo surface-induced dissociation (SID), the protein complexes dissociate along the weakest subunit interfaces, forming oligomeric product ions with various stoichiometries. The SID fragmentation pattern provides information regarding the protein’s quaternary structure, including how subunits were connected prior to dissociation. If the quaternary structure was rearranged due to high amounts of in-source activation, the SID fragmentation pattern can change, providing an inaccurate view of subunit connectivity. This study explores how in-source activation in the Waters SELECT Series Cyclic Ion Mobility Mass Spectrometer affects protein quaternary structure by adjusting the ion source’s cone voltage across a range of values, followed by SID at a fixed voltage. In agreement with previous results from our lab, high cone voltages caused pentameric C-reactive protein (115 kDa) to collapse from its native-like ring structure, which was confirmed by a decrease in the ion mobility arrival time. As SID was tracked across the cone voltage range, monomer formation decreased in favor of dimer and trimer formation at high cone voltages, indicative of the collapsed structure. When the cone voltage was varied for a mutant of 14mer 800 kDa GroEL (D398A) the ion mobility arrival time was minimally affected, suggesting that the larger protein structure was minimally disturbed by in-source activation. Additionally, the SID fragmentation pattern appeared qualitatively similar across the cone voltage range. These results suggest that larger protein complexes can sustain higher amounts of in-source activation without rearrangement, compared to smaller protein complexes, as expected. Overall, this study will provide guidance regarding appropriate amounts of in-source activation to balance adduct removal while maintaining native-like quaternary structures.
Variable-Temperature Electrospray Ionization Coupled with Electron Capture Dissociation to Study Temperature Induced Solution Phase Structural Changes
Kristie L. Baker, Phillip C. Lacey, Vicki H. Wysocki
The Ohio State University, Department of Chemistry and Biochemistry, Columbus, OH; Resource for Native Mass Spectrometry Guided Structural Biology, Columbus, OH
Native mass spectrometry (nMS) allows for analysis of gas-phase ions that retain native-like solution phase structure. Variable temperature electrospray ionization (vT-ESI) provides specific control over the solution temperature of the ESI nano-emitter. As temperature increases, temperature-induced solution phase changes can be monitored by observing a shift in the protein’s average charge state, due to an increase in solvent accessible surface area available for protonation during ionization. However, monitoring charge state shifts only gives a global view of the change in structure; it does not provide any information about what structural elements are changing. The addition of electron capture dissociation (ECD) provides key information on these structural changes because ECD cleaves the peptide backbone with greater efficiency at surface exposed terminal regions. Thus, as the structure expands and the exposed surface area increases, ECD sequence coverage can increase as well. Using vT-ESI followed by ECD (e-MSion) on a Waters SELECT Series Cyclic Ion Mobility Mass Spectrometer, the temperature-induced structural changes of carbonic anhydrase (CA, 29 kDa) were monitored. Our data show that at 20 °C the dominant charge states are 10+ and 9+, with the 9+ showing greater intensity. As temperature increases, charge states begin to shift to a dominant charge state of 10+ with some 11+ charge state. ECD of the higher charge state at elevated temperature shows an increase in fragmentation from the C-terminus of the protein, while fragmentation from the N-terminus appears to remain unchanged. This indicates that the expansion of the structure at elevated temperatures arises from the C-terminus. Additional work is being done to expand the size and oligomeric state of the proteins studied. This combination of the solution phase technique, vT-ESI, with the gas-phase technique, ECD, allows for residue specific information of temperature induced changes by monitoring change in fragmentation.
A 2D Microfluidic Paper-Based Analytical Device For Diagnosis of Canine Visceral Leishmaniasis in Developing Countries
Hianka Jasmyne Costa de Carvalho1,2, Ayesha Seth2, Maria Angelica Miglino1, Abraham Kwame Badu-Tawiah2*
1 Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
2 Department of Chemistry and Biochemistry, The Ohio State University, Columbus-OH, United States
This work relies on the development of a new tool based on paper-spray mass spectrometry (PS-MS) for diagnosing visceral leishmaniasis (VL) in dogs. VL is a protozoan, vector-bone, and life-threatening condition, only behind malaria in number of cases. Furthermore, VL can affect both human and dogs, and it is considered a neglected disease. Additionally, canine VL cases directly influences the disease in humans. Public measures for controlling VL include 1) implementing health education, 2) control of canine population and responsible ownership, 3) providing environmental management for vector control, and 4) screening and detection of infected dogs. Besides its importance, diagnostic tools used for screening dogs often have limitation in terms of sensitivity and specificity, also presenting high cost and being inaccessible to populations in developing countries. Herein, we propose a new canine VL diagnostic test applying cleavable ionic probe as targeting molecules instead of instable enzymes. We aim to achieve a highly stable test, enabling storage in ambient air, simple enough to permit dogs screening at any place, and sensitive enough to provide early detection in asymptomatic dogs through centralized analysis. Our proposed method consists of paper-based indirect immunoassay, analyzed through PS-MS/MS, providing a qualitative and quantitative result. A protein specific for canine VL biomarker was immobilized on hydrophilic zones of the paper surface. Unbound sites on paper were blocked and serum samples from VL positive and negative dogs were added to the spots and incubated. After washing, detection antibody previously conjugated to the cleavable ionic probe was added. After the final washing steps, each spot was analyzed by PS-MS and signals were obtained from negative and positive serum samples. Our method represents the first immunoassay analysis for VL on a paper substrate. The application of the target-mass probes successfully enabled us to distinguish between positive and negative results.
Dynamic Electrospray Ionization Mass Spectrometry
Purva S. Damale, Dmytro S. Kulyk, and Abraham K. Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United State
Mass spectrometry is an analytical technique characterized by its excellent sensitivity, robust selectivity, and rapid response. Nanoelectrospray ionization (nESI) is the preferred method for mass spectrometry (MS) when dealing with small sample volumes, but it is particularly effective for more polar analytes only. To tackle this, a versatile dual noncontact nESI/nAPCI (nanoatmospheric pressure chemical ionization) source was developed in our lab that enabled the concurrent detection of polar and nonpolar analytes in nanoliter/picoliter sample volumes while operating in ambient conditions and without sample pretreatment. However, this method requires laborious manual voltage optimizations for switching between ESI and APCI ionization modes, which makes it challenging for some compounds to get detected in complex mixtures/biofluids. Moreover, the robustness and clogging of glass tips is still a problem. To address these problems and make mass spectrometry more universal, we are proposing a novel dynamic high voltage power supply with automatic voltage ramping capabilities for optimizations of ionization and detection. For instance, automatic voltage tuning was effective to optimize ionization efficiency of highly non-polar molecules like cholesterol and vitamin D2 in APCI mode. Detection of such a wide range of molecules (polar, non-polar, proteins, and DNAs) in a complex mixture without any manual voltage tuning has been accomplished. Interestingly, this set-up has shown excellent stability of glass tips and lower flow rates, which allow longer analysis time. Our dynamic electrospray approach has potential to become a versatile ion source for the analysis of complex mixtures with inherent in-capillary separation abilities due to the temporal nature of ionization process in which different analytes will be ionized at different times. The robustness, simplicity, and longevity of this method render it appealing for significant applications in translational and biomedical research.
Paper-based immunoassay for highly sensitive detection of SARS-COV 2 using Miniature Mass Spectrometry
Monica Duarte da Silva1; Maria Angelica Miglino1; Abraham Badu Tawiah*2
1Department of Surgery, School of Veterinary Medicine and Animal Science, University of São Paulo-SP, Brazil. Prof. Dr. Orlando Marques Paiva Avenue, 87, 05508-270, São Paulo, Brazil, 2Department of Chemistry and Biochemistry, The Ohio State University. 100 W. 18th Avenue, Columbus, Ohio 43210, United States.
Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients. Though the real-time reverse transcription polymerase chain reaction (RT-PCR) acting as a gold-standard method has been widely used for COVID-19 diagnostics, it can hardly support rapid on-site applications or monitor the stage of disease development as well as to identify the infection and immune status of rehabilitation patients. Paper-based analytical devices (PADs) have shown great promise for point-of-care testing and on-site detection of analytes owing to their low cost, convenience, scalability, portability, and biocompatibility. In the present work to suit rapid on-site COVID-19 diagnostics under various application scenarios, we proposed the development a diagnostic strategy based on a paper-based mass spectrometry immunoassay platform that adopts stable and cleavable ionic probes as mass reporter. We synthesized pH-sensitive ionic probes and coupled them with monoclonal antibodies specific to the Spike protein antigen. We then used the antibody-ionic probe conjugates in a paper-based immunoassay to capture (Spike protein). After the immunoassay, the bound ionic probes were cleaved, and the released mass tags were analyzed through paper spray mass spectrometry strategy. The platform can simultaneously detect multiple samples at the same time thus supporting high-throughput sensing; and all these detecting operations can be implemented on-site using a portable mass spectrometer. Such a high-throughput multimodal immunoassay platform can provide a new all-in-one solution for rapid on-site diagnostics of COVID-19 for different detecting purposes.
The Proteomic Changes in HCT 116 Colon Cancer Spheroids During Growth
Catherine B. Edgington, Nicole C. Beller, Brian D. Fries, \ Amanda B. Hummon
The Ohio State University
Spheroids are a three-dimensional cell culture model that have steadily gained popularity because they mimic in vivo conditions better than monolayer cultures. Our lab uses HCT 116 cells to model avascularized colon cancer tumors. We harvest spheroids after two weeks of growth because previous data showed that the chemical gradients for oxygen and pH at this timepoint best mimic the tumor microenvironment. In the literature, spheroids are harvested after varying periods of growth. Some research groups allow spheroids to grow for only a few days before they begin experiments. However, little is known about the biological consequences of the time of spheroid harvest. To address this lack of information, we profiled the proteome of HCT 116 colon spheroids from Day 2 to Day 18, every two days of growth. Proteomic data was collected on a Thermo Orbitrap QE-HF in positive mode with a reserve-phase column. To elucidate the subtle proteomic changes, data independent acquisition and gas-phase fractionation was used. The proteins identified suggest that the time of harvest dictates clustering in initial principal component analysis (PCA) and hierarchical clustering analysis. In the PCA, day two samples cluster closest to the 2D control and the oldest sample was the most different from the 2D sample. Interestingly, the later harvest samples cluster closer to one another than the earlier time points. Further, Ingenuity Pathway Analysis showed that Senescence and DNA Damage Induced Signaling were significantly downregulated on the second day of growth, but significantly upregulated on the eighteenth day. These preliminary results suggest that there are clear proteomic differences in HCT 116 spheroids during growth which could be used as biomarkers for maturity.
Mass spectral properties of ubiquitylated peptides from non-tryptic proteolysis support alternative informatic strategies for probing the dark ubiquitylome
Regina Edgington1, Damien Beau Wilburn1
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210, USA
Ubiquitylation is a versatile post translational modification where the C-terminus of the small protein ubiquitin (Ub) is covalently attached to larger protein substrates. Proteins can be Ub-marked with monomers or chains of Ub on several different side chains as well as the N-terminus, and changes in proteome ubiquitylation have important consequences for protein homeostasis, DNA damage repair, and endocytosis. Bottom-up proteomics with LC/MS-MS is the most common analytical technique used to identify substrates for lysine ubiquitylation. After enzymatic digestion with trypsin, modified sites are identified by a characteristic “diGly” amino acid scar. Although this method is widely used, it fails to distinguish between ubiquitylated peptides and other peptides modified with ubiquitin-like proteins (NEDD8 and ISG15). Longer Ub scars provided by alternative proteases (LysC, GluC) that produce more Ub-specific fragment ions are usually avoided due to the complexity of mass spectra produced by these peptides. In a first proof of concept, we aimed to systematically evaluate the mass spectrometric behavior of such peptides. Using data from Akimov et al. (2018), tandem mass spectra were compared for 1941 matched pairs of tryptic and LysC peptides that vary in their Ub scar length (2 vs 13 amino acids). From this analysis, we found that LysC Ub peptides have a +1 or +2 higher charge state than their trypsin counterpart. Systematic fragmentation of the LysC scar itself is observed, which could serve as possible spectral signatures for detection of ubiquitylated peptides. Improved bioinformatic detection of peptides with longer Ub scars would also enable the use of other proteases (e.g. GluC) to quantify ubiquitylation in highly basic rich regions of the proteome that is invisible to trypsin-like proteases (i.e. the dark ubiquitylome).
Programmable Carbon-Carbon Bond Formation via Uncatalyzed Michael Addition in a Plasma-Water Microdroplet Fusing Green Synthetic Platform
Alexander J. Grooms, Isabella Marcelo, Abraham K. Badu-Tawiah
The Ohio State University
Charged water microdroplets fused with in situ generated non-thermal plasma discharge are found to facilitate carbon-carbon bond formation via a catalyst free Michael Addition reaction pathway without the use of strong base or organic solvent. A plasma-microdroplet fusing platform is utilized for the coaxial introduction of electron rich Michael donor carbon nucleophiles – formed in situ via reactive oxygen species (ROS) in plasma discharge from a chemically etched silica capillary – and electron poor Michael acceptors from a deactivated fused silica capillary. Water microdroplet-plasma fusion occurs online in a highly programmable reaction platform for direct process optimization and product evaluation via mass spectrometry. The platform is utilized herein with a variety of Michael donors for: i) straightforward 1,4-Michael addition of α,β-unsaturated carbonyls, ii) C-C bond formation with a series of Michael acceptors formed online via platform operation in an enhanced timescale and reactivity mode, and iii) selective product formation in an asymmetric Hantzch cascade reaction. Controlled ROS generation via plasma discharge during charged water microdroplet evolution establishes a green synthetic method for catalyst-free and organic solvent-free C-C bond formation.
Probabilistic Validation for Targeted Proteomics Using Parallel Reaction Monitoring
Alex W. Joyce,1,2 Yameng Wu,1 and Brian C. Searle1,2,3
1 Pelotonia Institute for Immuno-Oncology, Comprehensive Cancer Center The Ohio State University
2 Department of Biomedical Informatics, The Ohio State University Medical Center
3 Department of Chemistry and Biochemistry, The Ohio State University
Mass spectrometry is a powerful method for conducting high-throughput proteomic experiments. While global methods such as data-dependent acquisition (DDA) are effective for large-scale peptide identification, they are often unreliable for analyzing specific proteins of interest due to reproducibility issues caused by stochastic sampling. Targeted methods such as Selected Reaction Monitoring (SRM) and Parallel Reaction Monitoring (PRM) serve as alternatives that are more suitable for such tasks because they regularly sample targeted peptides regardless of the measured signal. A commonly used metric for validating proteomics experiments is the false discovery rate (FDR), which estimates the number of false positive matches in a dataset. While FDR thresholding is effective for evaluating results globally, targeted proteomics experiments could benefit from estimating a posterior error probability for each targeted peptide. SRM measurements are performed only on selected transitions that are tailored to targeted peptides, which limits the measurement size. On the other hand, PRM experiments computationally extract transition ions from full scan MS/MS spectra, which allows for other peptides that fall in the same retention time and precursor isolation window to also be measured. These additional “bonus” peptides can be used to derive distributions and statistics that help determine posterior error probabilities for individual peptides. Here, we introduce a method for determining these probabilities from the scoring distributions of “bonus” peptides identified in PRM. We expect that this will be a valuable method for evaluating and validating the accuracy of peptides measured by PRM.
Muricholic Bile Acid Enantiomers Separation by C18 RPLC-MS as a Function of Polarity Values of the Mobile Phase Components
Mst Ummul Khair, Eric Kipruto, David J Anderson
Department of Chemistry, Cleveland State University, Cleveland, OH, 44115.
The importance of drug enantiomers in clinical pharmacology is well recognized, where often only one of the enantiomer pair effectively binds to the target protein site to exert its therapeutic effect. It is essential for the analytical method to separately determine the two similar chemical structured compounds of the pair. The objective of this research is to optimize the reversed phase HPLC technique for the separation of alpha-muricholic acid and beta-muricholic bile acid enantiomers, by investigating the effects of the mobile phase’s three polarity components [hydrogen bond donor acidity (α), hydrogen bond donor basicity (β) and dipolarity/polarizability (π)] on the separation of the enantiomers (selectivity factor). In this work, steroid isomers are separated on a C18 column under column oven temperature at 40 oC using two different organic modifiers in water at a flow rate of 0.2 mL/min, with ESI-LC/MS detection and the separation was controlled by mobile phase (MP) component polarity adjusted with different proportions of binary organic modifiers added to the MP. When the selectivity factor versus the values of the individual MP polarity component was plotted for six different organic modifier pair combinations, the dipolarity(π) polarity component showed better correlation between the organic modifier pairs with the selectivity factor than basicity (β) and acidity(α) polarity components. And when the two individual polarity component values were summed and plotted for six different organic modifier pair combinations on the same plot, the summed dipolarity/polarizability (and basicity polarity (β) components of the MP showed reasonable correlation (R2=0.72), displaying mostly a linear superimposed trend for all the organic modifier pairs. This study shows that the separation of these bile acid enantiomers significantly depends on the summed π and β polarities of the MP, with poor correlation for total polarity (P) and acidity (α).
Utilizing HPLC-DAD and HPLC-high resolution mass spectrometry to identify iron chlorophyll derivatives synthesized from spinach
Agnes Josylyn Komey,1 Ziqi Li,2 Alireza Abbaspourrad,3 Michael B. Zimmermann,4,5 Rachel E. Kopec1,6
1Interdisciplinary Nutrition Graduate Program, Department of Human Sciences, The Ohio State University; 2 Department of Food Science & Technology, The Ohio State University: 3 Department of Food Science, Cornell University: 4University of Oxford, U.K.; 5Laboratory of Human Nutrition, ETH Zürich, Switzerland; 6Foods for Health Discovery Theme, The Ohio State University
Introduction: Heme iron is 2-10x more bioavailable (i.e. better-absorbed) than inorganic iron, partially due to the porphyrin ring in which it is chelated. Iron chlorophyllin derivatives (ICDs), which also have a porphyrin backbone, may provide a more bioavailable source of iron compared to inorganic iron, but remain understudied. However, the sole commercial source of ICDs contains only ~40% ICDs. The objective of this work was to identify a cost-effective synthesis method which also produced ICDs with higher yield.
Method: Spinach (containing ~100 mg chlorophyll/100 g F.W.) was homogenized with water and soybean oil (1:3, v/v). The puree was refluxed with aq. acetic acid for 20 minutes. The solids precipitated, the liquid phase was decanted and replaced with aq. acetic acid saturated with ferrous chloride (FeCl2). The mix was then refluxed for 20 min and cooled. A bi-phasic extraction was performed, and extracts analyzed via HPLC-DAD and HPLC-MS (Thermo Exploris 480). Separations were performed on a C30 column (for the lipid extract), and C18 column (for the polar extract). Eluent was interfaced with an ESI source operated in positive ion mode, with masses scanned over 100-1500 m/z. Selected masses of interest were fragmented via MS2.
Result: Polar extract: four compounds with primary m/z, isotope envelopes, and MS2 fragmentation consistent with iron-pheophorbide b and iron-pyropheophorbide a were observed. Lipid extract: two compounds with primary m/z, and isotope envelopes consistent with iron-pheophytin a and an iron-pheophytin a allomer were observed. All tentative ICDs had UV-Vis spectral shifts consistent with those previously reported for zinc and copper-containing derivatives. The polar and lipid extract yielded ~95% and ~40% ICDs, respectively.
Continuing research: Experiments are ongoing to increase ICDs in the lipid phase, and to concentrate the products (via crystallization and/or preparative HPLC). The higher ICD yield will permit testing in pigs and humans.
Acknowledgement: This study will be supported by the Bill and Melinda Gate Foundation (BMGF)
Plasma-in-Droplet Ionization Mass Spectrometry
Dmytro S. Kulyk, Purva S. Damale, and Abraham K. Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United State
Mass spectrometry is one of the most powerful modern analytical techniques in terms of speed and sensitivity. Nevertheless, it is still unable to ionize samples with different physicochemical properties by one single ionization source as well as to analyze them by one mass analyzer. Unfortunately, this has caused an unending production of different types of ion sources that are often analyte or sample dependent. To make mass spectrometry more universal and unified, we are proposing a new hybrid ion source as an online combination of charged microdroplets and nonthermal plasma discharge. This is achieved by merging the two most common ionization mechanisms: electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) into a single source. Interestingly, this new hybrid source does not require typical usage of nebulizing gas (unlike ESI) and external heating (unlike APCI). The new plasma-in-droplet ionization (PIDI) source has several modes of operation: ESI mode (pure droplets) and hybrid ESI/APCI mode (plasma is fused in charged microdroplet). While the ESI mode allows us to perform all conventional ESI MS detections, ESI/APCI mode opens opportunity to analyze polar/nonpolar and small/big molecules simultaneously during complex mixtures analysis. Moreover, we were able to detect proteins in the presence of nonthermal plasma that can be explained by the unique soft nature of our plasma-droplet fusion system, compared with conventional APCI MS. It is worth mentioning that ESI and APCI are distinct and incompatible processes. Our approach enabled these processes to co-exist efficiently, which improves analytical performance such as ionization efficiency, sensitivity, signal stability, and compatibility with HPLC. The hybrid ESI/APCI mode showed surprisingly higher sensitivity than conventional ESI MS that is not typical for any compromised dual ionization conditions. It was achieved by optimized physical parameters of the setup to instantaneously fuse and activate reactive plasma species to interact with charged microdroplet surface and gaseous ion species originating from the same source.
Characterizing Caspase-Lipopolysaccharide Oligomeric States with Electron Capture Charge Reduction MS
Philip Lacey1,2, Chengliang Wang3, Jianbin Ruan3, Vicki Wysocki1,2
1) Chemistry and Biochemistry Department, The Ohio State University, OH. 2) National Resource for Native MS-Guided Structural Biology, The Ohio State University, OH. 3) Department of Immunology, University of Connecticut Health, CT.
Understanding inflammatory responses can potentially help prevent or manage many inflammation-based conditions like septic shock, which has been found to be mediated by non-canonical inflammasome signaling. This process is initiated when cytosolic caspase-11 binds to a heterogenous class of lipopolysaccharides (LPS) from the outer membrane of gram-negative bacteria and begins to oligomerize. The oligomeric state or states themselves and mechanisms of interaction with their targets are still unknown; understanding the oligomerization of caspase-11 by LPS binding will help elucidate its role in non-canonical inflammasome signaling.
Native mass spectrometry (nMS) is a technique capable of measuring the mass-to-charge (m/z) of large proteins and protein complexes while maintaining their noncovalent interactions. We apply this technique to assess the oligomeric state or states of purified LPS-bound caspase-11. Preliminary data show an unresolvable mass spectrum which may have multiple caspase-11 oligomers. To probe these heterogeneous distributions in more detail, quadrupole selection and electron capture charge reduction (ECCR) was used, which selects a small m/z slice of a heterogeneous distribution in MS1 and uses electrons generated from a combined ExD-SID cell to charge reduce the ions, generating lower charge states that allow for individual species to be observed. This technique was used to more accurately measure the caspase-11 oligomeric masses, which were observed to range from 7-mer to 18-mer in varying abundances. Further work will include investigating the caspase-11 LPS-bound oligomer with dissociation techniques such as collision-induced dissociation (CID) to confirm stoichiometry and surface-induced dissociation (SID) to examine subunit connectivity. Combining quadrupole selection and ECCR with dissociation techniques will provide the first in depth examination of the LPS-bound caspase-11 oligomer with mass spectrometry and how it may affect activity in non-canonical inflammasome signaling.
nMS and ECD characterize interactions in RAS-SOS complexes
Ila A. Marathe1,2, Sangho (Davie) Yun3, Chen Du1,2, Arthur Laganowsky3, and Vicki H. Wysocki1,2
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210
2Resource for Native Mass Spectrometry-Guided Structural Biology, The Ohio State University, Columbus, OH 43210
Department of Chemistry, Texas A&M University, College Station, TX 77843
RAS proteins, molecular switches involved in many cellular processes, toggle between active (GTP-bound) and inactive (GDP-bound) states, with assistance from nucleotide exchange factors like Son of Sevenless (SOS)1. RAS allosterically binds SOS, resulting in increased nucleotide exchange rate. Mutations causing increased RAS activation are found in many forms of human cancers, thus making RAS an attractive target for anticancer therapeutics. However, little progress has been made in RAS targeting due to the ‘undruggable’ surface of RAS. Therefore, disrupting the RAS-SOS interaction provides an alternative approach. We employed native mass spectrometry (nMS) coupled with electron capture dissociation (ECD) and collision induced dissociation (CID) to establish a technique to gain better understanding of the RAS-SOS interaction. We used HRAS-GTP and the catalytic domain of SOS (SOScat) for preliminary studies. A mixture of HRAS-GTP and SOScat in ammonium acetate was sprayed by nano-electrospray ionization on a Thermo Q-Exactive UHMR, modified with a custom ECD-SID device (e-MSion). As reported previously2, free proteins, [HRAS-GTP + SOScat] (binary) and [HRAS-GTP + SOScat + HRAS] (ternary) complexes were observed. Each complex and SOScat alone was individually isolated using the quadrupole and then subjected to ECD, causing covalent fragmentation, while retaining inter-subunit interactions. CID was used to dissociate the complex-down and top-down fragments, which were then mapped using ExDviewer3. While free SOS fragmentated in two distinct regions corresponding to the active and allosteric sites, the binary complex only fragmented in the active site region. No fragmentation in the allosteric site region is likely due to RAS-GTP binding. The ternary complex, where both sites are occupied, did not show any distinct fragmentation patterns. Encouraged by our ability to localize RAS binding to SOS, we aim to further characterize RAS-SOS interactions using different RAS variants and their oncogenic mutants in the presence of various nucleotides, thus aiding anticancer therapeutic development.
- Wennerberg K, Rossman KL, and Der CJ. (2005) The Ras superfamily at a glance. J Cell Sci, 118: 843-846.
- Moghadamchargari Z, Shirzadeh M, Liu C, Schrecke S, Packianathan C, Russell DH, Zhao M, and Laganowsky A. (2021) Molecular assemblies of the catalytic domain of SOS with KRas and oncogenic mutants. Proc Natl Acad Sci, 118: e2022403118.
- Beckman JS, Voinov VG, Hare M, Sturgeon D, Vasil’ev Y, Oppenheimer D, Shaw JB, Wu S, Glaskin R, Klein C, Schwarzer C, and Stafford G (2021). Improved protein and ptm characterization with a practical electron-based fragmentation on Q-TOF instruments. J Am Soc Mass Spectrom, 32, 2081-2091. doi: 10.1021/jasms.0c00482.
- A Proteomic Analysis of Processed Proteins and Endogenous Peptides in Human Urine
A Proteomic Analysis of Processed Proteins and Endogenous Peptides in Human Urine
Madalyn G. Moore,1,2 Katelyn B. Brusach,3 Ariana E. Shannon,1,2,4 Vicki H. Wysocki,1 Brian C. Searle1,2,4
1Department of Chemistry and Biochemistry, The Ohio State University
2Pelotonia Institute for Immuno-Oncology, The Ohio State University Comprehensive Cancer Center
3Department of Veterinary Clinical Services, The Ohio State University
4Department of Biomedical Informatics, The Ohio State University Medical Center
Small proteins and endogenous peptides are excreted in human urine, consisting mainly of modified forms of proteins resulting from biochemical steps that occur in the body before their eventual removal as a waste product. Proteases are a source of post-translational sequence modifications and play essential roles in cell activity, including cellular localization, creation of biomolecules for signal amplification, and proliferation and cell fate. As a result, monitoring proteolytic activity could reveal information about how proteins are differentially utilized under specific pathological conditions. For example, the increase of fibrinogen degradation products in plasma has long been associated with cancer. Because urine is a near direct output from blood, analyzing these molecules allows disease states to be studied for potential biomarkers via a noninvasive route.
To properly study the processed urine proteome and peptidome, samples are first isolated from other components of urine, including small molecules and salts. Protein precipitation can be affected by different variables, so it is important that methodology is optimized. Secondly, endogenously processed proteins and peptides are separated from larger proteins that could originate from other sources. Suspension trapping using a protein filter common in proteomic sample preparation could help isolate shorter, processed proteins and endogenous peptides. We demonstrate a workflow using liquid-chromatography tandem mass spectrometry (LC-MS/MS) to compare tryptic peptides obtained from a standard bottom-up proteomics experiment with isolated proteins and peptides that could originate from alternative processing. Preliminary data reveals 29 endogenously digested peptides, 25 of which are tryptic in nature, from a sample isolated from the urine peptidome. Most of these endogenous peptides are from urobilin and albumin, which are highly abundant urinary proteins. The significance of these natural tryptic digestion events needs to be investigated further. This method used with urine samples from healthy and diseased patients has the potential to reveal peptide-based biomarkers.
Collision-Induced and Surface-Induced Unfolding of Protein Complexes
Martha Ortega Zepeda1, Yu-Fu Lin1, Dalton T. Snyder2, Vicki H. Wysocki1,2
The Ohio State University Department of Chemistry & Biochemistry1 Resource for Native MS Guided Structural Biology, The Ohio State University, Columbus, Ohio2
Native mass spectrometry (nMS) enables the study of intact non-covalent complexes in the gas phase, providing information such as the mass and stoichiometry of the complex analyzed. Analytes are introduced into the gas phase through positive or negative mode nanoESI, which allows non-covalent interactions to remain intact. Structural information is then elucidated by influencing the ions’ conditions prior to detection. Ion mobility (IM) is a complementary technique that separates ions based on their mobilities in a carrier buffer gas under the influence of an electric field. IM can be used to observe structural variations caused by pre-IM activation, providing information on fragmentation patterns and protein complex restructuring. Unfolding of three model protein complexes, streptavidin (SA), cholera toxin B (CTB), C-reactive protein (CRP), and IgG1 mAb in the gas phase was monitored using IM through collision induced unfolding (CIU) and surface-induced unfolding (SIU). Activation energy was varied by changing the collision voltage in 5 V increments prior to mobility measurements (voltage drop times charge state equals collision energy). Over a range of collision energies, more precursor remains with CIU than with SIU, and that remaining CIU precursor illustrates a range of unfolded forms. The collision against a surface deposits a large amount of energy at once, causing kinetically favored native-like fragmentation pathways. This is consistent with energy-resolved mass spectrum plots illustrating the appearance of fragments at low SID energies where only small amounts of precursor remain. Further work will include negative mode IMS measurements to compare the unfolding and dissociation behavior of the protein complexes as a function of positive vs. negative charge.
Separation and quantification of isomeric lipids of different classes via high flow rate plasma-droplet fusing using LC-Contained-Coaxial ESI-MS
Niraj Panday, Alexander J. Grooms, Abraham K. Badu-Tawiah
Department of Chemistry and Biochemistry, The Ohio State University, Columbus OH
Lipids encompass a wide array of organic compounds, including fats, sterols, and soluble vitamins exhibiting varying polarities and ionizable groups. Lipids exhibit different isomeric forms including the position of C=C bonds in fatty acyl side chains, along with sn-1 and sn-2 positional isomers. Various techniques such as C=C derivatization via Paterno-Buchi reaction, ozone-induced dissociation of the double bond, and epoxidation techniques via plasma generation are employed for the characterization of double bond positions. But still, the separation of positional isomers of different lipid classes poses an ongoing analytical challenge in the field of mass spectrometry-based lipidomics. To address this challenge, we couple – for the first time – a plasma-droplet fusing contained-electrospray source to liquid chromatography-mass spectrometry (LC-MS). Herein we optimize different source parameters for the formation of plasma at a higher flow rate and utilize the same parameters for the separation of various lipid isomers via LC-MS. For this, we optimize a common mobile phase composition for the separation of lipids of different polarities and varying ionizable groups. In addition, we characterize the separated lipid isomers via online epoxidation of the C=C bond. Furthermore, quantification of the lipid’s isomers will be considered using internal standards based on different classes of lipids.
Untargeted metabolomics analysis of LPS-stimulated mast cells by UHPLC-QTOF-MS
Akshay S Patil, Yan Xu*
Department of Chemistry, Cleveland State University, OH 44115
Metabolites play a crucial role in biological functions, which serve as building blocks, energy sources, and signaling molecules in cellular functions. The imbalance of metabolites in the biological systems leads to disease conditions. The UHPLC-QTOF-MS-based untargeted metabolomics is a powerful analytical technique for the global identification and quantification of metabolites in biological samples and offers invaluable insights into cellular and molecular changes under various experimental conditions. Hence, it can be used to study potential therapeutic agents with cell models.
In this work, we developed a UHPLC-QTOF-MS-based untargeted metabolomics method for the profiling, identification, semiquantitative analysis, and cross-comparison of key metabolites in mast cells under various conditions, including (a) control, (b) lipopolysaccharide (LPS)-stimulated, (c) triprolidine treated, and (d) zileuton treated. The cell lysates were normalized by protein contents and prepared by protein precipitation using a mixed solution of water, methanol, and acetonitrile. An exogenous stable isotope-labeled internal standard was used to correct the inter-run deviations and semiquantitative analysis. The chromatographic separation was performed using a high-strength silica (HSS) T3 column with gradient elution using a mobile phase consisting of (A) 5 mM ammonium acetate and 0.1% acetic acid, and (B) 5 mM ammonium acetate and 0.1 acetic acid in methanol/acetonitrile (80:20). The data acquired were processed with Agilent software and databases (i.e., MH Qualitative, MH Quantitative, Profinder, MPP, and METLIN AM) and MetaboAnalyst for metabolite identification, statistical and semiquantitative analysis, and cross-comparison. We demonstrated the measurable changes in metabolomic profiles under various experimental conditions and identified 21 key metabolites affected by LPS stimulation, triprolidine, and zileuton treatment. The method developed can be used to study inflammatory responses of mast cells.
Implementing Surface Collision in native MS for Essential DNA Repair Biomolecules
Zihao Qi1, 2; Charles E. Bell1, 3; Vicki H. Wysocki1, 2
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH; 2Resource for Native Mass Spectrometry Guided Structural Biology, Columbus, OH; 3Department of Biological Chemistry and Pharmacology, Columbus, OH
DNA double-strand breaks (DSB) severely damage genomic integrity, and if left unrepaired, can cause cancer. Single-strand annealing (SSA) repairs DSB and is promoted by single strand annealing proteins (SSAPs). SSAPs typically have disordered C-terminal domains, making them challenging to characterize with classical structural biology methods. In our study, we seek to understand the self-assembly and DNA binding behavior of the SSAPs, specifically RedB, a Rad52 homolog in bacteriophage, Rad52-DNA binding domain (Rad52-DBD), and Mgm101, a Rad52 related SSAP in yeast mitochondria, using native mass spectrometry (nMS) and surface induced dissociation (SID). nMS uses nanoelectrospray ionization (nESI) to transport biomolecules into the gas phase while retaining their non-covalent interactions, and SID breaks non-covalent interactions to measure oligomeric assembly and stability. With SID-nMS, we found RedB shows a dynamic range of oligomeric states, Rad52-DBD is a single 11-mer which corresponds to the known crystal structure, and Mgm101 forms a mixture of 18-mer and 19-mer. SID fragmentation for all SSAPs is consistent with a ring-shaped structure because monomer through n-1mer oligomers are produced due to similar interfacial strength, and the oligomeric state corresponding to half of the ring is the dominant species. We also investigated Rad52-DBD binding to DNA of different lengths, and we observed that Rad52-DBD will bind with two copies of the same ssDNA when the length is shorter than 44nt. We also collected energy-resolved mass spectra (ERMS) on the Rad52-DBD ssDNA complexes to understand the subcomplex connectivity. ERMS suggests the 83-mer ssDNA covers about 9 subunits of Rad52-DBD. This 83-mer ssDNA covers fewer subunits than we expected, presumably due to self-complementarity. As a result, we used dA38, which does not have self-complementary region and dA38 is expected to bind about 9 subunits; with SID, we observed a dominant species of 8-mer with ssDNA, indicating that dA38 covers 8 subunits.
Universal Mass Exclusion List for Organism Independent Modification Mapping of RNA
Asif Rayhan, Balasubrahmanyam Addepalli and 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.
Thermal Proteome Profiling for Characterizing Biophysical Properties of Chaperone Proteins
Chase P. Renzelmann1,2,3, Ariana E. Shannon1,2,3, Brian C. Searle1,2,3
1 Pelotonia Institute for Immuno-Oncology, Comprehensive Cancer Center The Ohio State University
2 Department of Biomedical Informatics, The Ohio State University Medical Center
3 Department of Chemistry and Biochemistry, The Ohio State University
Thermal Proteome Profiling (TPP) has emerged as a formidable method for investigating protein-ligand interactions, stabilization and destabilization of those interactions, and cellular responses to temperature shifts. Chaperones play a vital role in maintaining protein homeostasis and the facilitation of proper protein folding. In this work, the TPP approach is utilized to demonstrate the melting curves of proteins of interest, and in future studies, to evaluate changes in melting curves of proteins of interest in the presence and absence of a target chaperone to generate potential clientele candidates of the targeted chaperone. Chaperone thermal stability, in addition to interactions with partners, remains a crucial, yet challenging aspect to characterize.
A systematic approach was employed by treating cell lysates to a range of temperatures (37 – 64°C), in which proteins that are stable remain in solution, while destabilized proteins can crash out of solution. Proteins are then denatured followed by protein quantification to determine the levels of remaining protein concentration at the selected temperature intervals. Mass spectrometry analysis will characterize proteins that remain in-solution. By monitoring the abundance of protein concentration remaining in solution, comprehensive melting curve profiles will be generated. Notably at this point, we have observed confirmation in the widely understood direct inverse relationship between protein concentration and temperature.
We intend to generate a prospective data-driven workflow in which aims to present a detailed view of the chaperone protein’s behavior and clientele, offering a deeper understanding of its functional relevance. By capturing the temperature-dependent changes in protein concentration, and soon to follow changes in interactions and stability, this method contributes to identifying further intricacies within chaperone mediated folding and cellular proteostasis.
Self-Aspirating Contained Electrospray Ionization (SAC-ESI) for Microsampling and Sensitive Diagnosis.
Ayesha Seth, Dmytro S. Kulyk, and Abraham Badu-Tawiah*
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United State
Analyzing biological microsamples provides valuable information about complex biosystems. Proteomics and metabolomics analysis pose challenges due to the lack of uncomplicated, efficient, and reproducible microsampling techniques. Conventional methods like dried matrix spots have been used extensively for biomedical and diagnostic applications. However, quantitative assays are ambiguous with these platforms due to hematocrit/chromatographic effects and sample stability. We introduce a straightforward microsampling/electrospray device that can use thermal assistance for sample extraction and self-aspiration avoiding the need of any nebulizer gas. The device incorporates a pre-weighed light-protective amber glass container(vial) with sorbent to contain and facilitate the extraction of analyte from the bio-sample with determined exact mass, simplifying the process for both patients and medical laboratory experts. This device addresses multiple challenges related to the stability of samples in home kits, ensuring biohazard safety during transportation, and enabling high throughput processing, including the potential for use with an autosampler. It allows for the direct collection of samples from a finger prick, placing them in vial, and sealing securely with a snap cap. At the time of analysis, the extraction solvent with internal standard is injected, and silica capillary is pierced through the snap top and directed towards the mass spectrometer. Due to capillary action, the silica capillary gets filled with sample and heat assistance helps achieve a continuous flow when potential is applied between the sample vial and entrance orifice of a mass spectrometer. As proof of concept, we have used Ubiquitin, to detect charged states of protein within 2000Da mass range. We have also detected different amines and cocaine. Self-aspirating contained electrospray ionization (SAC-ESI) setup with its simple structure, aspiration capabilities and low sample consumption benefits can easily be used as a microsampling platform for direct analysis with mass spectrometers and will be applied for illicit drugs detection in different biofluids.
Modifaction of Commercial Capillary Electrophoresis Instrument to use Vibrational Sharp-Edge Spray Ionization
Tyler A. Shaffer1, Yousef Elshamy1, Lisa A Holland1, John A. Lucas1
1 C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26505, United States
Vibrational Sharp-Edge Spray Ionization (VSSI) is a recent development in the field of MS ionization. This innovative method allows for a voltage free method to ionize a wide range of molecules relatively independent of the effects of pH and without the use of organic solvents as well as the ability to analyze anionic compound more easily. So far, the coupling of Capillary Electrophoresis (CE) instrumentation to MS via VSSI has been done with the use of lab-built instruments. This work uses a commercial instrument (Beckman P/ACE) instrument that has been modified to use the Ionization method VSSI. By modifying the instrument for use with VSSI, more variables and processes will be under operator control than with the current lab-built systems. Temperature control over the capillary and integrated sample storage allow for the analysis of temperature sensitive analytes as well as negate the risk of joule heating. Temperature control also allows for the use of temperature sensitive capillary coatings used to suppress electroosmotic flow. The enhanced control over injection and pressure pushing also allow for better and safer injections and the ability to do operations like stacking and capillary flushing more easily and efficiently. This method also uses a sheathed VSSI probe with a small tip diameter in order to provide makeup flow as well as achieve a focused plume of droplets for analysis at the MS. To test this modified instrument, it is tested against three other CE instruments. Firstly, the modified instrument is compared to a standard Beckman CE-UV instrument to prove repeatability and reliability of separations. The instrument is then compared to a lab-built system currently in use to compare both ease of use and repeatability of measurements. And finally, the instrument is compared to a commercial CE-ESI instrument to compare performance and show the strengths of VSSI.
The effects of a lycopene-rich tomato juice enhanced with soy isoflavones on inflammation in individuals with obesity
Maria J. Sholola1, Jenna L. Miller1, Emma A. Bilbrey2, Molly A. Torok3, Janet A. Novotny4, David M. Francis5, Thomas A. Mace3, Jessica L. Cooperstone1,2
1Department of Food Science and Technology, The Ohio State University, Columbus, OH
2Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH
3Division of Gastroenterology, Hepatology, and Nutrition, The Ohio State University Wexner Medical Center, Columbus, OH
4Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD
5Department of Horticulture and Crop Science, The Ohio State University, Wooster, OH
Tomatoes and soy, both combined and separately, are linked to anti-inflammatory outcomes. Lycopene from tomatoes and soy isoflavones are often rationalized to produce these effects. In this randomized, crossover clinical trial, the objective is to test the effects of lycopene and soy isoflavones on markers of inflammation in obesity.
Participants (n=12) consumed daily servings of a high-lycopene tomato-soy juice, or control yellow tomato juice devoid of lycopene, both for 4 weeks each. A washout period of 2 weeks preceded the study and another for 4 weeks in between interventions. Plasma and 24-hour urine samples were collected at baseline, before and following each intervention.
Fifteen inflammatory cytokines and 40 immune cell types were profiled in plasma using high-throughput bioplex-based assays and mass cytometry, respectively. Plasma carotenoids were quantified using high performance liquid chromatography. With mass spectrometry-based metabolomics, assessment of urinary soy isoflavones along with the urine metabolome before and after each intervention for metabolite levels and global effects are underway. Unsupervised analyses will be used to visualize natural clustering within the data. Paired t-tests and mixed linear modeling will be conducted to determine significant metabolites and their effects on inflammatory markers.
Plasma lycopene levels increased significantly (P = 0.001) for all patients while on the tomato-soy juice intervention as compared to both baseline and control. Interleukin-5 levels decreased (P = 0.048), while mature and naïve B cell levels increased (P < 0.01) post tomato-soy, but were not different between tomato-soy and control. Linear modeling did not reveal any relationships between lycopene and inflammatory markers.
Preliminary data shows a lack of effect for lycopene and isoflavones on inflammatory markers, suggesting that other compounds from tomato-soy might be at play; however, other significant markers may not have been observed due to small sample size. Germplasms specifically designed for whole food interventions can aide in understanding the roles that phytochemicals play in human health.
A Multi-Modal Approach for the On-Line Modification of Lipids for Efficient Analysis of Complex Mixtures
Octavion Spears, Alex Grooms, Ben Burris, Abraham Badu-Tawiah
The Ohio State University, Columbus, OH
This project aims to develop a miniaturized orthogonal analytical system that is based on the coupling of a custom-made solid-phase extraction (SPE) cartridge with a reactive contained-electrospray (ES) source for online fractionation and ionization of lipids followed by their in-situ analysis by a portable mass spectrometer. With the increasing interest to apply mass spectrometry (MS) in middle- and third-world countries, a multi-functional ion source that is widely applicable can transform simple standalone mass spectrometers, including portable instruments, into a powerful tool for applications in clinical chemistry, environmental analysis, agricultural, and biomedical research.1 This PhD dissertation focuses on metabolomics, a field that is growing rapidly because of its importance in disease diagnosis.3 The metabolome represents the most downstream stage in molecular biology and thus has higher significance in early disease detection.3 Lipidomics is a subcategory focusing on the analysis of lipids, which is now prevalent in routine blood tests. Human serum contains many thousands of distinct lipids, and the physiological contributions of these diverse lipids are now beginning to emerge.1 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 metabolomics, including lipidomics, rely on the enhanced resolution provided by high-field NMR and some mass spectrometers.3 I hypothesize that the ability to effectively perform metabolomics on a low-resolution mass spectrometer will bring a significant advancement to the field, including implementation in low- and middle-income countries. We will use a custom-made solid phase extraction (SPE) cartridge coupled to a contained-electrospray ionization (ESI) source for online fractionation of several lipid classes based on differences in lipid polarity and interaction with select mobile and stationary phases. Further characterization will be enabled via online reaction of the SPE eluents with lipase enzymes – catalyzing lipid hydrolysis.
Paper-Based Microfluidic Device for Early Diagnosis of Severe Acute Pancreatitis using Ambient Mass Spectrometry Detection
Ruth Speidel1, Peter Lee2, and Abraham Badu-Tawiah1*
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210
2Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH
A paper-substrate device for the early detection of severe acute pancreatitis is being developed using paper-based immunoassay followed by 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. 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. Method optimization will include bioconjugation chemistry to attach the ionic probe to reporter antibodies, capture antibody immobilization and blocking, and direct detection of captured species by MS. We expect the optimized method will enable detection limits in the tenths of ng/mL range for most of the biomarkers. Associated analytical merits for repeatability and reproducibility will be reported.
Thread-Based Microfluidic Device for Real-Time Reaction Monitoring by Thread Spray Mass Spectrometry
Salmika G. Wairegi, Abraham K. Badu-Tawiah
Department of Chemistry, The Ohio State University
Organic reaction screening is foundational to clinical and pharmaceutical studies. Microfluidic devices are of special interest as they offer advantages such as small reagent volumes and short mixing times. Unfortunately, mixing in microfluidic devices is nontrivial due to laminar flow. Herein, we use thread-based microfluidic device to facilitate mixing, where reagents are passively pumped by capillary action. The device was integrated with thread spray mass spectrometry for real-time monitoring of reaction products. Thread is naturally composed of intertwined sub-fibers that can serve as effective mixing medium. We fabricated this thread-based microfluidic platform on a microscope glass, with reagent reservoirs created from adhesive tape with defined holes. The tip of the combined threads was placed parallel to the mass inlet for real-time reaction monitoring. For ionization, 20 μL of methanol/water (1:1, v/v) was used as spray solvent, with an application 5 kV DC voltage. The analytical performance of the device was investigated by monitoring the Katrizky and Aza-Michael addition reactions. In the Katrizky reaction, a triphenylpyrylium (TPP) cation (MW 309 Da) reacts with a primary amine. The TPP cation reacted with each amine to produce the corresponding pyridinium cation. In the Aza-Michael addition reaction, the formation of a C-N bond was observed. All products were analyzed in positive-ion mode and MS2 was utilized to fragment and structurally characterize each product. The preliminary results suggest that the combination of thread-based microfluidic device with thread spray MS is efficient and useful for monitoring the Katrizky and Aza-Michael addition reactions in real-time. Future work will involve validation of the thread-based microfluidic device by performing each reaction using nanoelectrospray (nESI) and theta-capillaries to explore each reaction via bulk-phase and microdroplet fusion. We also plan to optimize the platform to monitor a three-component reaction.
Probing Protein Structures Using Covalent Labeling Mass Spectrometry
Evan Whitford, Philip Lacey, Vicki Wysocki
Ohio State University Department of Chemistry and Biochemistry
Covalent labeling methods involve modifying solvent accessible protein side chains with a covalent mass tag that minimally perturbs a protein’s native structure and provides information on a protein’s surface structure. This empirical information can be used in protein modeling workflows, providing valuable constraints to limit the number of possible structures predicted by computational means. Covalent labeling and constrained computational modeling can arrive at reasonable native-like protein structures. Diethylpyrocarbonate (DEPC) is an easy-to-use, commercially available reagent that, in solution, directly labels nucleophilic residues of proteins, which can compose up to 30% of the average protein sequence. Previous work done by the Vachet lab of the University of Massachusetts demonstrated that limiting the amount of labeling to 1-1.2 labels per protein helps preserve the structural integrity of the sample protein. A typical bottom-up proteomics workflow is used to analyze labeled samples. Samples are enzymatically digested and then analyzed by a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method. This allows for identifying the labeled residues and gives the relative extent of labeling. Labeled sites can provide site-specific information about the degree of their reactivity. The long-term goal of this work is to compare covalent labeling data to a protein structure prediction software for proteins whose structures contain an intrinsically disordered region and are not predicted well by Alphafold 2, although the current focus is on collecting the experimental data. Work has been done confirming the labeling reaction with the well-characterized proteins beta-lactoglobulin and lysozyme. Isotopic resolution and a top-down approach were utilized to support that labels could be seen on samples compared to a control. Work is being done with a bottom-up proteomics approach to identify the specific residues being labeled and to quantify the extent of the modification occurring for each residue.
Resolving the Challenges of Overlapping Charge States in Native MS and SID through Electron Capture Charge Reduction and Charge Detection Mass Spectrometry
Shixiang Xu, Marius M. Kostelic, Vicki H.Wysocki
Department of Chemistry and Biochemistry. Ohio State University, Columbus, OH. nMS guided structural biology resource.
Native mass spectrometry (nMS) has become a popular tool for studying protein structure because the non-covalent interfaces within the protein are retained in the gas phase. To gain deeper insights into protein structure, MS/MS analysis with collision induced dissociation (CID) is typically performed. CID is useful for accurate monomer mass measurement and stoichiometry information. As an alternative ion activation technique, surface induced dissociation (SID) yields a symmetrical charge distribution and preferentially cleaves the weakest noncovalent interfaces, providing information about subunit connectivity within the protein complex. A notable challenge in native MS with SID is the occurrence of overlapping charge states among different SID fragment oligomers, which can lead into inaccurate mass deconvolution. To address this issue, electron capture charge reduction (ECCR) and charge detection mass spectrometry (CDMS) have been employed. ECCR increases the spacing between the adjacent charge states by extending them into a higher m/z range, and CDMS measures charge, which separates peaks overlapping in m/z. Experiments were conducted using an Orbitrap UHMR (Ultra-High Mass Range) MS with a custom ExD cell coupled with a 3 mm SID device. For glutamate dehydrogenase (GDH) hexamer precursor, the trimer subunit fragments from SID dissociation typically overlap in m/z with precursor, complicating interpretation/deconvolution of the SID spectrum. By employing ECCR and CDMS, precursors and products are well separated. The potential of CDMS to yield more accurate relative abundances for product and precursor ions in energy-resolved mass spectra will be illustrated.
Electron Capture Charge Reduction (ECCR) for Heterogeneous Glycoproteins
Zhixin Xu1,2, Regina M. Edgington1, Chen Du1,2, Vicki H. Wysocki1,2
Department of Chemistry & Biochemistry, The Ohio State University, Columbus, Ohio1
Resource for Native MS Guided Structural Biology, The Ohio State University, Columbus, Ohio2
Glycosylation is a common post-translational modification, affecting a protein’s conformation, stability, and solubility. Glycosylated proteins are called glycoproteins. Glycoproteins contain diverse oligosaccharide chains called glycans, creating sample heterogeneity. Due to this heterogeneity, characterization of glycoproteins can be challenging because different glycosylation patterns on a protein or protein complex lead to different charge state distributions that can overlap in m/z space.
One native mass spectrometric strategy that we are developing to characterize glycoprotein heterogeneity uses narrow-window quadrupole mass-to-charge (m/z) selection followed by electron capture charge reduction (ECCR) to detangle the multiple glycoforms hiding in the selected m/z range. Electron capture charge reduction helps to reduce the charge states of proteins/protein complexes, shifting the original m/z peaks to the right and spreading them across a larger m/z space. This strategy helps to improve our spectral resolution, allowing easier mass spectral deconvolution to identify the glycoform masses.
Using these techniques, we focused on determining the mass distributions of several SARS-COV-2 VFLIP stabilized spike glycoprotein variants of concern, VOC (e.g., Wuhan, Delta, Brazil, Omicro BA.x). We determined masses of the intact spike proteins cover a range from 494 kDa to 524 kDa. We then reduced the glycan heterogeneity by using the PNGase F enzyme to remove N-glycans. A typical spectrum for spike protein has only one broad peak; after applying ECCR, the charge state peaks resolve, and the mass of the spike protein can be calculated. The masses for incubated samples reduced to around 470 KDa. We employed computational methods to complement our empirical mass measurements to determine the possible glycan composition. Using our measured masses and published glycomics data, we produced a potential mass distribution for intact spike glycoprotein with clearly identified glycan compositions, which reflect the microheterogeneity of our samples. Differences are apparent between different VOC.
Defining the Developmental Dynamics of Ubiquitylation Proteomes Regulated by ASK1-Containing Ubiquitin Ligases
Peifeng Yua,b, Yang Lia, and Zhihua Huaa,b
aEnviromental and Plant Biology Department, Ohio University, Athens, Ohio 45701
bInterdisciplinary Program in Molecular and Cellular Biology, Ohio University, Athens, Ohio 45701
The ubiquitin-26S proteasome system (UPS) and autophagy comprise the two large protein degradation systems in plants for managing a healthy proteome that is extremely important for maintaining the homeostasis of cellular metabolism and conducting signaling transduction. It is commonly agreed that the UPS targets the turnover of many individual proteins that are specifically determined through ubiquitylation by a large group of ubiquitin ligases. In addition, many substrates, including organelles, targeted by autophagy are also ubiquitylated prior to proteolysis. Therefore, understanding the ubiquitylation processes plays a key role in deciphering the molecular mechanisms underlying the two degradation systems. Consistent with the sessile lifestyle, the genomes of land plants, particularly flowering plants, have evolved to encode an extremely larger group of ubiquitin ligase genes than any other eukaryotic organisms. Among the plant ubiquitin ligases, the Skp1-Cul1-F-box (SCF) complexes constitute the largest group, in which the Skp1 and F-box proteins assemble the substrate receptor module to determine the specificity of each SCF complex. Through functional genomic studies, we demonstrated that the Arabidopsis Skp1-Like (ASK) 1 protein plays a predominant Skp1 function in Arabidopsis, which allowed us to develop an affinity purification-mass spectrometry approach to identify the active ASK1-F-box SRM complexes in vivo. In combining RNA-Seq transcriptomics analysis in the ask1 null mutant, our multi-omics studies uncovered a dynamic yet evolutionary conserved regulatory proteome by the Skp1-Cul1-F-box ubiquitin ligases across the key developmental transition stages of Arabidopsis.
Development of Isobaric Peptide Probes for Multiplex Disease Detection using Paper Spray Mass Spectrometry
Stephanee Zerrudo1 and Abraham Badu-Tawiah1*
1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210
Mass spectrometry (MS) is a sensitive and accurate method, but for detection of large molecular protein biomarkers this process can be expensive and sophisticated to allow implementation in resource-limited settings. We have developed a diagnostic platform for disease detection based on inexpensive paper-based microfluidic devices that can detect large molecular weight protein biomarkers on a miniature mass spectrometer via the application of cleavable reporter probes. In this work, isobaric peptide probes are proposed for multiplex detection in a single scan tandem MS (MS/MS). The peptide probes will be attached to specific monoclonal antibodies, which will be subsequently used for immuno-extraction of desired biomarkers from complex biofluids. After the immunoassay, the peptide is cleaved and directly analyzed by an on-chip paper spray MS/MS. This presentation will focus on the design and characterization of the peptide sequence with potential multiplex capability. We aim for isobaric peptides in which all peptide probes will have the same molecular weight but dissociate to give distinct fragment ions.
We observed that a set of peptide sequences with cleavage site at aspartic acid (Asp) and proline (Pro) residue gave only two fragment peaks dictated by the position of these two amino acids. The required fragments can be observed if arginine (Arg) is placed at the C-terminus, with controllable proton sites. Based on this sequence structure, multiple isobaric peptide probes were designed by moving Asp-Pro position along the peptide chain. Through Steglich esterification, the peptides were attached to a cleavable conjugate unit, which enabled subsequent attachment to monoclonal antibodies. For LRNPTIDPLNR (MW 1307.73 Da), the +2-charge state (m/z 655) produced expected fragment ions at m/z 499 and 810 by breaking Asp-Pro bond. Conditions for generating abundance +2 charge state will be discussed. The designed peptide probes will be used for the detection of malaria biomarkers in paper-based immunoassays.