IU Indianapolis ScholarWorks :: Browsing by Subject "Mass Spectrometry"

Browsing by Subject "Mass Spectrometry"

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Advancing the Applicability of Fast Photochemical Oxidation of Proteins to Complex Systems
(2016-08) Rinas, Aimee Lynn; Jones, Lisa; Georgiadis, Millie M.; Long, Eric C.; Manicke, Nicholas
Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced increases the complexity of identifying and quantifying oxidation products. The current methods available for quantifying the extent of oxidation either involve manual analysis steps, or limit the number of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. In addition to the data complexity, the peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. The recently published in cell FPOP method exemplifies where this field is headed - larger and more complex systems. This dissertation describes two new methodologies and one new technology for hydroxyl radical-mediated protein footprinting, expanding the applicability of the method. First is development of a new footprinting analysis method for both peptide and residue level analysis, allowing for faster quantification of results. This method utilizes a customized multilevel search workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in, expediting the analysis process. Second is the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Last is the design and implementation of a flow system for in cell FPOP, which hydrodynamically focuses the cells, and when used yielded a 13-fold increase in oxidized proteins and 2 orders of magnitude increase in the dynamic range of the method.
Characterizing the Unfolded Protein Response by Changes in Protein Thermal Stability
(2023-09) McCracken, Neil Andrew; Mosley, Amber; Wek, Ron; Evans-Molina, Carmella; Georgiadis, Millie; Quinney, Sara
The Unfolded Protein Response (UPR) protects eukaryotic cells from the threat of excessive protein flux into the Endoplasmic Reticulum (ER). UPR sentries PERK, Ire1 and ATF6 detect unfolded protein in the ER and alert the cell of the condition. Downstream pathways increase translation of select responders while simultaneously decreasing the global protein load in order that toxic protein aggregates do not form in the cell. While this warning system has been characterized over several decades through extensive reporting of UPR impact on transcript and protein abundance, little is known about the biophysical changes that occur to proteins as part of the UPR in the context of the cellular environment. An understanding of how the UPR affects the folding, stability and protein oligomerization is vital for describing subtle but important changes that occur and contribute to maladaptive physiology in diseases including diabetes, cancer, and neurodegeneration. I propose that deficiencies in characterizing the UPR can be overcome by using thermal shifts assays (TSA) that quantify changes in protein stability post stimuli. Findings described herein show the utility of the biophysical thermal shift assay in characterizing the UPR. Thermal shift assays (TSA) measure susceptibility of proteins to denature upon heat treatment and consequently detect changes in protein structure, modification, and interactions in the cellular environment. Previously unobserved protein relationships related to the UPR were detected using TSA. These workflows were improved through more strategic upstream sampling and downstream data analysis through creation of the publicly available InflectSSP program. Observed UPR phenomena during N-linked glycosylation inhibition and UPR induction include protein degradation, changes in stability of N-linked glycosylation enzymes, and transcriptional targets canonical to the UPR. Stability changes in proteins downstream of PERK were also observed in experiments where PERK genetic ablation was combined with UPR induction. Finally, the thermal shift assay was used to develop a “signature” for the UPR that holistically describes the ER stress response. Results described in this dissertation provide an improved perspective of the UPR along with an approach that can be used to identify novel targets for therapeutic intervention of the UPR.
Deep Proteome Profiling in the Progression of Pancreatic Ductal Adenocarcinoma-Associated Cachexia
(2021-09) Umberger, Tara; Mosley, Amber L.; Zimmers, Teresa; Goebl, Mark; Doud, Emma
Cachexia is a devastating muscle wasting syndrome affecting multiple biochemical pathways and is a comorbidity of many diseases including pancreatic ductal adenocarcinoma (PDAC). PDAC patients with cachexia commonly experience systemic inflammation, progressive loss of lean muscle and adipose tissue, and cardiac dysfunction. The present workflow identifies proteins and their post-translational modifications extracted from both cardiac and skeletal muscle tissue isolated from a murine model of PDAC-associated cachexia. Reported here are differentially occurring post-translational modifications found on the most abundant contractile proteins. Tissue from mouse muscle samples were collected two weeks after either receiving a sham surgery or orthotopically implanted with PDAC tumor cells, with or without a follow-up chemotherapy treatment of the standard of care agent gemcitabine with nab-Paclitaxel. Whole tissue blocks of gastrocnemius or heart were either flash frozen and pulverized or homogenized in denaturing lysis buffer and then sonicated to facilitate protein extraction. After disulfide bond reduction, cysteine alkylation, and trypsin digestion, the resultant peptides were subjected to molecular barcoding using tandem mass tag isobaric labeling reagents to facilitate multiplexing. The first and second dimension of peptide separation in the multiplexed sample is accomplished with an offline, high pH, reverse phase (RP)-LC fractionation followed by an online RP-LC at lower pH. The use of high-field asymmetric-waveform ion mobility spectrometry provided a last dimension of separation before MSn analyses. This novel, proteomic workflow enables deep proteome profiling in the progression of cancer-induced cachexia. The use of multi-dimensional chromatographic separation and differential ion mobility technique have allowed us to identify almost 4,500 proteins groups of gastrocnemius muscle tissue and nearly 7,100 protein groups of myocardium taken from the murine PDAC model of pancreatic cancer. A comprehensive analysis of the data collected from this workflow was used to calculate differential post-translational modifications on major contractile proteins isolated from PDAC model muscle tissue, with or without chemotherapy, when compared to sham surgery controls. Differential post-translational modifications and protein expression changes found to contribute to cancer cachexia may elucidate novel molecular mechanisms and cellular signaling that drive cachexia progression.
Detection and Quantitation of Hazardous Chemicals in Environmental Matrices using Paper Spray Mass Spectrometry
(2019-08) Dowling, Sarah Naciye; Manicke, Nicholas; Goodpaster, John; Sardar, Rajesh
Paper spray mass spectrometry (PS-MS) is an ambient ionization technique that has been proven useful in many types of investigative analyses. However, the use of this technique with regards to environmental samples has been largely unexplored since the technique’s development. In this work, paper spray mass spectrometry was utilized to detect and quantify compounds for environmental, forensic and chemical defense applications. Due to the sensitive nature of some projects, the work was split into two volumes. Volume 1 focuses on the detection of pharmaceuticals in soil using paper spray (Chapter 2) and the detection of chemical warfare agent (CWA) simulants and CWA hydrolysis products (Chapter 3). Volume 2 focuses on the detection and quantitation of fentanyl analogs in environmental matrices. Chapter 5 focuses on the rapid analysis of fentanyl analogs in soil matrices. The following chapter evaluates the ability of PS-MS to detect low concentrations of fentanyl analogs in water (Chapter 6). Throughout this work, paper spray has proven to be an effective, rapid alternative to chromatography for the analysis of environmental samples.
Development and Application of a Mass Spectrometry-Based Quantitative Assay for Apolipoprotein M in Human and Mouse Serum
(2008-10-13T19:13:53Z) Copeland, Marci Lynn; Wang, Mu
Apolipoprotein M (apoM) is necessary for the formation of lipid-poor preβ-HDL particles, the initial precursor of HDL and acceptors of cholesterol efflux from peripheral cells. An assay to quantify apoM in serum is not widely-available, hampering the efforts to further understand apoM and to develop therapeutic methods to increase circulating levels of apoM. An antibody-free, high throughput mass spectrometry (MS)-based assay was developed to quantitatively measure apoM from a variety of species including human, mouse, and rat. Apolipoproteins were enriched by selectively binding to Liposorb, an affinity resin, followed by enzymatic digestion. This peptide mixture was separated by HPLC coupled in-line with tandem MS/ MS. Signal intensities from the MS/ MS fragmentation of apoM-specific peptides were measured simultaneously in a targeted method spanning many commonly used species. The same amount of purified human apolipoprotein A-IV uniformly labeled with 15N was spiked into all samples and was used as an internal standard to correct for any variation in sample handling and recovery. Assay variability and accuracy was statistically validated in a three-day spike recovery experiment to determine the working range of the assay. The concentration range for quantification of apoM using this assay was 11.2-500 nM, whereas average concentration of human apoM measured from a large sampling (n>100) was 370 nM. This assay was used to measure changes in apoM in mouse serum from a pre-clinical study that was designed to evaluate the effects of a microsomal triglyceride transfer protein (MTTP) inhibitor. All measured lipoproteins and apolipoproteins showed a dose-dependent decrease in concentration and the response of apoM closely followed the response of HDL. In a clinical application of the assay, apoM was measured in human serum to evaluate the effects of two cholesterol-lowering compounds, a statin drug and an experimental PPAR-α agonist. ApoM levels did not change with PPAR-α agonist or combination treatments, but significantly decreased with atorvastatin. The measurement of apoM provided additional information on the effects of these drug treatments that previously could not be measured. The availability of a quantitative assay for apoM provides a valuable tool in the development of cardio-protective therapeutics and understanding the mechanisms of these drugs.
Development of Mass Spectrometry-Based Analytical Assays for Environmental and Chemical Defense Applications
(2023-12) Dowling, Sarah Naciye; Manicke, Nicholas; Goodpaster, John; Laulhé, Sébastien; Sardar, Rajesh
Mass spectrometry (MS) is a powerful and versatile technique that is useful for addressing a wide range of complex analytical challenges. In this work, mass spectrometry-based assays were developed to address issues relating to environmental contamination and for detecting analytes of interest to the defense industry. Chapter one is an overview of the history of mass spectrometry, the fundamental operation of a mass spectrometer, as well as, advancements in chromatographic separation and ionization methods. Chapter two focuses on the development of an assay that uses blow flies as environmental sensors of chemical weapon release. In that work, a liquid chromatography – tandem mass spectrometry (LC-MS/MS) method was developed to detect chemical warfare agent simulants and chemical warfare agent hydrolysis products in flies exposed to the chemicals in controlled feeding experiments. The work in chapter three describes the development of a surface enhanced Raman spectroscopy assay coupled to paper spray mass spectrometry for a more fieldable and environmentally friendly approach to detect organophosphorus compounds. Chapter four describes the development of a paper spray mass spectrometry assay for the detection and semi-quantitation of per- and polyfluoroalkyl substances in whole blood without sample cleanup or chromatographic separations. This method would be useful in detecting high levels of these carcinogenic compounds in individuals highly exposed via their occupations. The final chapter (chapter five) returns to using blow flies as environmental sensors, but this time to detect insensitive munitions in the environment. The work focuses on the development of two different liquid chromatography mass spectrometry methods for the detection of insensitive munitions, which are less shock sensitive explosives, and their transformation products in the environment. Controlled feeding experiments were also performed where flies were exposed to contaminated soil and water sources to show the feasibility of this method in a more realistic scenario. The projects detailed herein show the extensive range with which mass spectrometry can be used for the detection of harmful chemistries of environmental concern.
Expanding the Capabilities of Paper Spray Mass Spectrometry: High-Throughput Drug Screening and On-Paper Electrokinetic Stacking
(2024-12) Rydberg, Magnus Per; Manicke, Nicholas; Deiss, Frédérique T.; Web, Ian K.; Naumann, Christoph T.
Paper spray mass spectrometry (PS-MS) offers advantages in simplicity and rapid analysis but faces challenges that have limited its widespread adoption. These include insufficient sensitivity for certain analytes, susceptibility to matrix effects in complex samples, and inconsistent quantitative performance. Such limitations have restricted the application of PS-MS in fields requiring high sensitivity in analysis of complex biological matrices. This dissertation explores approaches to enhance the capabilities and applications of PS-MS, with a particular focus on overcoming sensitivity limitations. Chapter 2 demonstrates the potential of PS-MS in forensic toxicology through an automated high-throughput urine drug screening method. Chapter 3 investigates the negative impact of laser cutting paper spray substrates on sensitivity and provides practical remedies. The core of this work revolves around the development and application of electrokinetic stacking coupled to PS-MS. Chapter 4 describes the integration of field-amplified sample stacking and faradaic ion concentration polarization into paper-based MS ionization cartridges. Chapter 5 presents mathematical modeling to complement the experimental work, offering a theoretical framework for understanding system behavior. Chapter 6 demonstrates practical applications of the technique, utilizing stacking devices to detect low levels of per- and polyfluoroalkyl substances (PFAS) in tap water and drugs of abuse in urine. These applications demonstrate substantial enhancements in sensitivity over conventional PS-MS, while preserving the technique's advantage of minimal sample preparation. Moreover, this work also identifies a potential path towards incorporating electrokinetic separations in PS-MS, addressing a longstanding limitation of the technique and potentially broadening its applicability in complex sample analysis.
The HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B
(Public Library of Science, 2018-05-09) Carmichael, Jillian C.; Yokota, Hiroki; Craven, Rebecca C.; Schmitt, Anthony; Wills, John W.; Biomedical Engineering, School of Engineering and Technology
All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1), direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor), we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B), and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4) blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease.
Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis
(Wiley-Blackwell, 2015-02-01) Micanovic, Radmila; Khan, Shehnaz; El-Achkar, Tarek M.; Department of Medicine, IU School of Medicine
Laser micro-dissection (LMD) is a very useful tool that allows the isolation of finite areas from tissue specimens for downstream analysis of RNA and protein. Although LMD has been adapted for use in kidney tissue, the use of this powerful tool has been limited by the diminished ability to identify specific tubular segments in the kidney. In this study, we describe a major improvement in the methodology to isolate specific cells in the mouse kidney using immunofluorescence LMD (IF-LMD). Using IF-LMD, we can reproducibly isolate not only glomeruli, but also S1-S2 proximal segments, S3 tubules, and thick ascending limbs. We also demonstrate the utility of a novel rapid immunofluorescence staining technique, and provide downstream applications for IF-LMD such as real-time PCR and cutting-edge proteomic studies. This technical breakthrough may become an invaluable tool for understanding cellular and molecular events in the heterogeneous kidney milieu.
Ion Mobility and Gas-Phase Covalent Labeling Study of the Structure and Reactivity of Gaseous Ubiquitin Ions Electrosprayed from Aqueous and Denaturing Solutions
(2021-12) Carvalho, Veronica Vale; Webb, Ian; Manicke, Nicholas; Laulhé, Sébastien
Gas-phase ion/ion covalent modification was coupled to ion mobility/mass spectrometry analysis to directly correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross-section (CCS) distributions were measured prior to ion/ion reactions to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Ubiquitin ions were electrosprayed from aqueous and methanolic solutions yielding a range of different charge states that were analyzed by ion mobility and time-of-flight mass spectrometry. Aqueous solutions stabilizing the native state of ubiquitin generated folded ubiquitin structures with CCS values consistent with the native state. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision-induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous solutions were covalently modified preferentially at the lysine 29 and arginine 54 residues, indicating that elements of secondary structure, as well as tertiary structure, were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase.

Browsing by Subject "Mass Spectrometry"

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