Course-based undergraduate research experiences (CUREs) integrate authentic research into undergraduate chemistry laboratories, introducing students to research while simultaneously reinforcing fundamental concepts. Despite their ubiquitous nature in bioanalytical research, few CUREs have been published applying the fundamental techniques of separations, spectroscopy, quantification, and mass spectrometry. To engage students in learning these increasingly essential bioanalytical techniques, we designed and implemented a semester-long project-based course centered around the purification, quantification, and identification of heterologously expressed proteins in five succinct and adaptable modules. Instructors can use these modules to form the foundation of a CURE specific to their research interests. The extensive commercial availability of plasmids for transformation combined with the modular approach to laboratory experiments enables convenient customization to accommodate diverse research goals. Instructors can tailor the modules to meet the curricular requirements and instrumentation capabilities of their institutions and can easily extend the research goals to incorporate more specialized analytical techniques, as needed. Through the implementation of the five modules, students apply the fundaments of acid−base chemistry, statistics, quantification strategies, spectrophotometry, separations, and mass spectrometry, thus covering the material required in most undergraduate introductory analytical courses. Instructors can then use these modules as a backbone to support student-led discovery-based investigations for the remainder of the course. Students demonstrate their understanding through the completion of a comprehensive, publication-style laboratory report as well as a poster presentation at a university-wide undergraduate research symposium. Since first offering this course in 2016, student evaluations have been exceedingly positive, with over 75% of students indicating that the course both increased their scientific skills as well as their confidence in their ability to succeed in further science courses. Furthermore, 22% of students reported they were "much more" or "extremely more" likely to enroll in a Ph.D. program in science, math, or engineering following the courses, emphasizing the impact that project-based laboratories can have on undergraduate chemists' career trajectories.
Melanoma, a type of cancer that develops in melanocytes, is usually caused by direct exposure of skin to ultraviolet (UV) radiation resulting in cellular damage. In this study, a procedure to determine the effects of various commercial sunscreens with SPF values ranging from 15 to 100 was developed using pig skin to mimic human skin. These sunscreens contain inorganic filters, such as zinc oxide and titanium dioxide; active organic ingredients, such as octocrylene and oxybenzone; or both. As a model for human skin, pig skin was analyzed before and after UV exposure, and the presence of free radicals was measured using electron paramagnetic resonance (EPR) spectroscopy. Using this method, students were able to quantify radical formation following irradiation and use this as a basis to compare the efficacies of sunscreens against UVA. This experiment allowed undergraduate students to characterize a complex chemical process (light-induced radical formation) and relate it to something they experience every day (sun damage). Interestingly, students found higher levels of postillumination radical formation in sunscreen-treated samples, perhaps indicating sunscreen-induced stabilization of these species. Student outcomes included learning how to collect and interpret EPR data, statistical analysis of these data, and the preparation of reproducible biological samples. Students also consulted literature sources to properly display their measurements.
Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a mediator against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of cysteine oxidation in understanding biological stress response, cysteine sites most reactive toward ROS remain largely unknown and are a major analytical challenge. Herein, a chemical proteomic method to quantify site-specific cysteine reactivity using a maleimide-activated, thiol-reactive probe (N-propargylmaleimide, NPM) is described. Implementation of a gel-based approach via conjugation of rhodamine-azide to NPM-labeled cysteine residues by copper-catalyzed azide−alkyne cycloaddition (CuAAC) click chemistry allowed simple and highly sensitive fluorescence profiling. Relative quantification of >1500 unique cysteine sites from greater than 800 proteins was achieved by conjugating dialkoxydiphenylsilane (DADPS) biotin-azide by the CuAAC reaction and subsequently performing biotin−streptavidin affinity purification and mass-spectrometry-based proteomics. Taken together, this work defines a novel role for the NPM probe in chemical proteomics and presents a robust method for determination of cysteine reactivity during oxidative stress response.
The target of rapamycin (TOR) kinase is a master metabolic regulator with roles in nutritional sensing, protein translation, and autophagy. In Chlamydomonas reinhardtii, a unicellular green alga, TOR has been linked to the regulation of increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target(s) is responsible for the elusive “lipid switch” in control of increasing TAG accumulation under nutrient limitation. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems, and little work has been done to show the role of oxidative signaling in TOR regulation. In this study, the TOR inhibitor AZD8055 was used to relate reversible thiol oxidation to the physiological changes seen under TOR inhibition, including increased TAG content. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 401 proteins were determined to have significant changes in oxidation following TOR inhibition. These oxidative changes mirrored characterized physiological modifications, supporting the role of reversible thiol oxidation in TOR regulation of TAG production, protein translation, carbohydrate catabolism, and photosynthesis through the use of reversible thiol oxidation. The delineation of redox-controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.
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