@article{mellinger_kibbe_rabbani_meritet_muddiman_gamcsik_2022, title={Mapping glycine uptake and its metabolic conversion to glutathione in mouse mammary tumors using functional mass spectrometry imaging}, volume={193}, ISSN={["1873-4596"]}, DOI={10.1016/j.freeradbiomed.2022.11.010}, abstractNote={Although glutathione plays a key role in cancer cell viability and therapy response there is no clear trend in relating the level of this antioxidant to clinical stage, histological grade, or therapy response in patient tumors. The likely reason is that static levels of glutathione are not a good indicator of how a tissue deals with oxidative stress. A better indicator is the functional capacity of the tissue to maintain glutathione levels in response to this stress. However, there are few methods to assess glutathione metabolic function in tissue. We have developed a novel functional mass spectrometry imaging (fMSI) method that can map the variations in the conversion of glycine to glutathione metabolic activity across tumor tissue sections by tracking the fate of three glycine isotopologues administered in a timed sequence to tumor-bearing anesthetized mice. This fMSI method generates multiple time point kinetic data for substrate uptake and glutathione production from each spatial location in the tissue. As expected, the fMSI data shows glutathione metabolic activity varies across the murine 4T1 mammary tumor. Although glutathione levels are highest at the tumor periphery there are regions of high content but low metabolic activity. The timed infusion method also detects variations in delivery of the glycine isotopologues thereby providing a measure of tissue perfusion, including evidence of intermittent perfusion, that contributes to the observed differences in metabolic activity. We believe this new approach will be an asset to linking molecular content to tissue function.}, journal={FREE RADICAL BIOLOGY AND MEDICINE}, author={Mellinger, Allyson L. and Kibbe, Russell R. and Rabbani, Zahid N. and Meritet, Danielle and Muddiman, David C. and Gamcsik, Michael P.}, year={2022}, month={Nov}, pages={677–684} }
 @article{mellinger_garrard_khodjaniyazova_rabbani_gamcsik_muddiman_2022, title={Multiple Infusion Start Time Mass Spectrometry Imaging of Dynamic SIL-Glutathione Biosynthesis Using Infrared Matrix-Assisted Laser Desorption Electrospray Ionization}, volume={21}, ISSN={["1535-3907"]}, DOI={10.1021/acs.jproteome.1c00636}, abstractNote={Due to the high association of glutathione metabolism perturbation with a variety of disease states, there is a dire need for analytical techniques to study glutathione kinetics. Additionally, the elucidation of microenvironmental effects on changes in glutathione metabolism would significantly improve our understanding of the role of glutathione in disease. We therefore present a study combining a multiple infusion start time protocol, stable isotope labeling technology, infrared matrix-assisted laser desorption electrospray ionization, and high-resolution accurate mass-mass spectrometry imaging to study spatial changes in glutathione kinetics across in sectioned mouse liver tissues. After injecting a mouse with the isotopologues [2-13C,15N]-glycine, [1,2-13C2]-glycine, and [1,2-13C2,15N]-glycine at three different time points, we were able to fully resolve and spatially map their metabolism into three isotopologues of glutathione and calculate their isotopic enrichment in glutathione. We created a tool in the open-source mass spectrometry imaging software MSiReader to accurately compute the percent isotope enrichment (PIE) of these labels in glutathione and visualize them in heat-maps of the tissue sections. In areas of high flux, we found that each label enriched an approximate median of 1.6%, 1.8%, and 1.5%, respectively, of the glutathione product pool measured in each voxel. This method may be adapted to study the heterogeneity of glutathione flux in diseased versus healthy tissues.}, number={3}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Mellinger, Allyson L. and Garrard, Kenneth P. and Khodjaniyazova, Sitora and Rabbani, Zahid N. and Gamcsik, Michael P. and Muddiman, David C.}, year={2022}, month={Mar}, pages={747–757} }
 @article{yao_rabbani_sattler_nguyen_zaharoff_walker_gamcsik_2019, title={Flow-Encoded Oxygen Control to Track the Time-Dependence of Molecular Changes Induced by Static or Cycling Hypoxia}, volume={91}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.9b03709}, abstractNote={Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.}, number={23}, journal={ANALYTICAL CHEMISTRY}, author={Yao, Ming and Rabbani, Zahid N. and Sattler, Tyler and Nguyen, Khue G. and Zaharoff, David A. and Walker, Glenn and Gamcsik, Michael P.}, year={2019}, month={Dec}, pages={15032–15039} }
 @article{yao_sattler_rabbani_pulliam_walker_gamcsik_2018, title={Mixing and delivery of multiple controlled oxygen environments to a single multiwell culture plate}, volume={315}, ISSN={["1522-1563"]}, DOI={10.1152/ajpcell.00276.2018}, abstractNote={ Precise oxygen control is critical to evaluating cell growth, molecular content, and stress response in cultured cells. We have designed, fabricated, and characterized a 96-well plate-based device that is capable of delivering eight static or dynamically changing oxygen environments to different rows on a single plate. The device incorporates a gas-mixing tree that combines two input gases to generate the eight gas mixtures that supply each row of the plate with a different gas atmosphere via a removable manifold. Using air and nitrogen as feed gases, a single 96-well plate can culture cells in applied gas atmospheres with Po2 levels ranging from 1 to 135 mmHg. Human cancer cell lines MCF-7, PANC-1, and Caco-2 were grown on a single plate under this range of oxygen levels. Only cells grown in wells exposed to Po2 ≤37 mmHg express the endogenous hypoxia markers hypoxia-inducible factor-1α and carbonic anhydrase IX. This design is amenable to multiwell plate-based molecular assays or drug dose-response studies in static or cycling hypoxia conditions. }, number={5}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY}, author={Yao, Ming and Sattler, Tyler and Rabbani, Zahid N. and Pulliam, Thomas and Walker, Glenn and Gamcsik, Michael P.}, year={2018}, month={Nov}, pages={C766–C775} }