The goal of the SC COBRE in Oxidants, Redox Balance and Signaling Proteomics Core is to provide state-of-the-art LC-MS/MS instrumentation, expertise, and training for comprehensive proteomic analyses to advance the research endeavors of investigators with interests in redox signaling. Dedicated core personnel assist with experimental design, method development, data acquisition, and computational analysis for protein characterization, interactions, and quantitative proteomic applications using metabolic labeling (SILAC), isobaric tandem mass tagging (TMT), and label free quantitation (MaxQuant LFQ). The core utilizes complementary peptide fragmentation modes (CID, HCD, ETD, EThcD, UVPD) as needed to characterize post-translational modifications of cysteine (sulfenic, sulfinic, sulfonic acid, S-glutathionylation, disulfide bonds); arginine (glycation by methylglyoxal, dihydroxyimidazolidine); tyrosine (nitrated and crosslinked), serine (O-GlcNAc and phosphorylation); lysine (acetylation, ubiquitylation, glycation, hydroxylation); and N- and O-linked glycosylation.

COBRE investigators have sequenced putative biomarker peptides discovered by MALDI-tissue imaging mass spectrometry and identified differentially expressed or post-translationally regulated proteins following genetic manipulation of anti-oxidant enzymes, drug treatment, or disease. Methodology has been established to determine the effects of altered redox-balance on differential protein abundance in FAC-sorted cell populations, primary cells, and exosomes. COBRE investigators have also utilized quantitative proteomics to elucidate the mechanism of drug action and identify the targets of drugs identified in phenotypic screens. We will continue to assist COBRE investigators and members of the COBRE Redox Center with customized method development as needed to advance their research endeavors.

Instrumentation available for quantitative LC-MS/MS-based proteomics include a ThermoScientific Orbitrap Fusion Lumos ETD/UVPD MS with EASY nanoUHPLC 1300 and an Orbitrap Elite ETD MS with Ultimate 3000 nanoLC acquired through the NIH Shared Instrumentation Program (S10 OD010731 and S10 OD025126, PI Ball). The core recently acquired a Waters Xevo TQ-S triple quadrupole MS with M class LC which will expand our capabilities to include absolute quantitation and multiple reaction monitoring (MRM) assays for targeted proteomics. Instrumentation is housed within the University supported MUSC Mass Spectrometry Facility and Proteomics Center.

Cell and Molecular Imaging (CMI) Core D provides COBRE investigators access and assistance for high end laser scanning confocal/multiphoton/super-resolution microscopy and related imaging techniques. Core D houses five confocal/multiphoton systems: 1) a state-of-the-art Zeiss LSM 880 NLO Quasar confocal/multiphoton microscope with a Fast Airyscan super-resolution detector; 2) an Olympus FV1200 silicone oil optics multiphoton microscope configured especially for intravital imaging; 3) an Olympus FV10i LIV confocal microscope with water immersion optics for live cell imaging; 4) a Zeiss LSM 510 META laser scanning confocal microscope for general purpose imaging of live and fixed specimens; and 5) a BD CARV II disk-scanning confocal microscope for video rate “real-time” confocal imaging. Major recent upgrades include Fast Airyscan for the Zeiss LSM 880 for super-resolution imaging, a near UV laser upgrade for the Zeiss LSM 510 to permit imaging of DAPI and other blue-emitting fluorophores, and the acquisition of Bitplane Imaris software for 3- and 4-D visualization of image data sets. The Core together with the Drug Discovery Core at MUSC is currently preparing a Shared Instrumentation Grant (SIG) S10 application to secure a high content automated imager for submission in May, 2019, which would greatly help the drug discovery efforts of several current and graduated COBRE investigators.

In 2018-2019, CMI held workshops on newly acquired and emerging technologies, including “Zeiss Celldiscoverer 7: A New Platform for Automated Live Cell Imaging for Drug Discovery” (March 14, 2018), “Simplifying High Content Analysis for Cell Biology and Drug Development” (April 16), Imaris Image Visualization & Analysis Workshop” (June 4-5), “Zeiss Fast Airyscan Super-Resolution Microscopy” (November 26), “Streamline Imaging and Analysis with a Single Platform” (December 20), and “Current Landscape of Biological Testing using High Content Analysis” (January 31, 2019).

To ensure that COBRE members are expertly trained in cell and molecular imaging, especially as it relates to oxidative stress and redox signaling biology, the Core also organizes a biennial Charleston Workshop on Light Microscopy for the Biosciences (LMB), which will next be held June 9-14, 2019. Participation of COBRE investigators and their personnel is given priority. The 7th LMB Workshop will provide a solid introduction to the concepts and practical applications of light microscopy relevant to modern cell and molecular biology. Students will have opportunities for extensive hands-on experience with state-of-the-art equipment for optical imaging, digital image processing, fluorescence, confocal/multiphoton microscopy and super-resolution microscopy guided by experienced academic and commercial faculty. Commercial faculty representing leading microscope manufacturers will make available for students use of the latest and most advanced instrumentation for light microscopy, image detection and computerized image analysis. The keynote speaker and invited faculty for the workshop will be Dr. Eeva-Liisa Eskelinen of the University of Turku, who will give presentations on “Correlative Light-Electron Microscopy (CLEM)”.

By providing sophisticated imaging technologies, expertise and training, CMI promotes the success of the individual COBRE projects and also provides training and assistance to junior investigators studying oxidative stress and stress signaling related to the overall theme of the COBRE.

Understanding the complexities of redox mediated signaling events requires a multidisciplinary approach. The SC COBRE in Oxidants, Redox Balance and Stress Signalling has assembled a cohort of promising junior faculty with expertise in relevant biomedical model systems. Analytical biochemistry specific to the detection and quantification of redox sensitive molecules and coordinate protein changes that drive homeostasis is a unique niche fulfilled by the Analytical Redox Biology Core (ARBC).

The primary objective of the Core is to provide comprehensive analytical redox biochemistry methods and mentoring support for the COBRE junior faculty with the goal to advance their research endeavors, publications and fundability. The specific aims of the ARBC are: 1) Provide ROS /RNS identification and quantification using state-of-the-art techniques; 2) Perform quantitative analysis of ROS/RNS (redox molecules and metabolites), including those associated with calcium mobilization and changes in energy metabolism; 3) Provide expertise and technology for in depth biochemical analysis of thiol-centered enzyme activities and define protein:protein interactions.

Since oxidative (nitrosative) stress often is associated with a conditional increase in antioxidant protection, the Core has established methods to detect and measure various antioxidant enzyme activities as a function of oxidant stress/antioxidant protection equilibrium. Comprehensive analysis of redox status also includes measurement of intracellular GSH, GSSG, protein surface and “buried” thiols utilizing both endpoint and/ or real-time kinetic measurements with millisecond resolution. In complex studies of redox signaling, certain protein:protein interactions appear to be redox dependent and attributed to post-translational modifications, including S-nitrosylation and S-glutathionylation. The ARBC has developed fluorescent labeling and FRET analysis to evaluate redox dependent protein:protein interactions with subsequent in silico molecular modeling using ZDOCK, GOLD Suite (v 5.2) software. Collectively, these technologies will provide a multidisciplinary approach to advance the understanding of redox mediated signaling events specific to the model systems presented by the junior faculty in their research.

Cellular redox species are produced directly or indirectly via bioenergetic metabolic reactions. Although the leak of electrons is often cited as the primary source of superoxide and hydroxy radicals, the leak is only a small contributor to the many different species involved in cellular redox reactions. Indeed, the fundamental basis of bioenergetics involves the oxidation (loss of electrons) of reduced nutrients and subsequent production of metabolites with a range of redox potentials (i.e., nicotinamides, reduced/oxidized metalloproteins, and thiol-containing species with varied redox potentials. Profiling the flux of these various metabolites through their attendant metabolic reactions is fundamental to any studies aimed at understanding cellular redox reactions.

The Bioenergetics Core provides several of the leading technologies that enable researchers to quantify the fluxes of these metabolic reactions in cells, tissues, organoids and small animal models such as zebrafish embryos and nematodes. The central technologies include high resolution respirometry using the XF technology from Seahorse Biosciences/Agilent. Dr. Beeson was involved in the original design of the XF technology profiles extracellular fluxes of oxygen, lactate and CO2 as the samples are interrogated with pharmacological and/or genetic manipulations. The instrumentation utilizes 96-well microplates to provide sufficient sample numbers to provide robust, statistically validated flux profiles of glycolysis, mitochondrial respiration, fatty acid oxidation, glutamine utilization and other related metabolic processes. Rapid, high-throughput imaging optimized to the XF plate architecture provides normalization of cell/tissue numbers, health, and other.

Isotopomer analyses of Krebs cycle intermediates and their byproducts utilizes LC-GC/MS. Typical 13C-labels at particular positions of, for example glucose, are introduced to cells or tissue small samples are periodically quenched, lysed and derivatized to volatile esters. The key advantage is that only the low molecular weight acids readily enter the gas phase for analyses that enable determination of enrichment of the 13C at positions that reveal the fluxes through specific pathways and/or the conversions to key species such as 2-hydroxy-glutamic acid – and oncometabolite produced by a mutated form of isocitrate dehydrogenase seen in many tumors that have escaped from therapeutic pressures. The oncometabolites affect epigenetic mechanisms that regulate tumor cell growth and proliferation.

A key feature of the core is that Dr. Beeson and his team also provide extensive training, data analyses support and aid in experimental design – it is fundamentally a collaborative unit.

Lab produces purified proteins on a large scale to support research activities at MUSC and outside customers. The lab also contains equipment for biophysical characterization of proteins, including ITC, circular dichroism and dynamic light scattering

The COBRE Mass Spectrometry Core is housed within the MUSC Mass Spectrometry Facility which provides expertise, services, education, and training to enhance biomedical research endeavors through mass spectrometry-based proteomics. Currently there are over 50 investigators which utilize the facility for protein identification and characterization. Protein analysis includes in-gel or in-solution protease digestion, chromatographic separation and tandem mass spectrometric analysis of the resulting peptides, and interpretation of MS/MS data using Sequest , Mascot, Protein Pilot, MaxQuant, and other search algorithms. The facility also assists in the development of customized applications for the isolation, detection and characterization of posttranslationally modified peptides (e.g. phosphorylation, glycosylation, oxidation, glutathionylation, and O-GlcNAc modification). With the recent acquisition of the Orbitrap Elite Mass Spectrometer we are expanding our services to couple quantitative approaches (SILAC, iTRAQ®, ICAT®, TMT®) to modification-specific experiments (eg., phosphoproteomics, redox proteomics). We are developing methodology to analyze alterations in posttranslational regulation that impact signal transduction, epigenetic modulation, and the response to therapeutics with the goal of enabling investigators to discover molecular mechanisms underlying disease progression and therapeutic responses that may not be revealed through genomic studies.