We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. The use of label-free approaches for quantitative proteomics studies has been implemented. This methodology involves both data dependent (DDA) and data-independent analyses (Mse). The data obtained from depleted serum (depletion of the 14 most abundant proteins) followed by six different sample treatments has been acquired. The results obtained by data-independent analyses and by data-dependent analyses are being evaluated. Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions and thus travel more slowly than those ions that comprise a smaller cross-section. Thus, ion mobility serves as a useful means of orthogonal separation in unrelated molecules as well. This is a real benefit because the ability to identify and quantify proteins is directly linked to the power of chromatographic separations. 2. Myositis Study. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein or a suite of proteins can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and digested, data has been acquired, and the results are being processed. 3. Isoprostane Study. Isoprostanes are a family of compounds that are identical to their corresponding prostaglandins except for differences in stereochemistry. Because isoprostanes can be formed nonenzymatically, they are often studied as markers of oxidative stress. The generation of the most often measured isoprostane, 8-iso-PGF2alpha, by prostaglandin-endoperoxide synthase and arachidonic acid even with low enzymatic activity was investigated. From our studies, an increase in the concentration of 8-iso-PGF2alpha does not necessarily reflect an increase in chemical lipid peroxidation. We recommend using the 8-iso-PGF2alpha/PGF2alpha ratio to quantitatively distinguish between increases in 8-iso-PGF2alpha by enzymatic and/or chemical lipid peroxidation. We recently analyzed plasma from smokers and compared the 8-iso-PGF2alpha levels to nonsmokers. An increase in the 8-iso-PGF2 levels were observed in chronic smokers and were determined to come primarily from the inflammation-induced, PGHS-dependent lipid peroxidation pathways and not directly from free radicals. 4. Fatty Acid Study. We performed a comparison of short chain fatty acids and amino acids in feces to determine if obesity has an effect on the metabolism of cells. It was shown previously that obesity can result in some epigenetic changes in colon epithelial cells and make them resemble tumor cells. 5. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 71 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. In addition, we are developing an untargeted approach for these analytes on another instrument. 6. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. Steroids are often present at low concentrations and exhibit low response in electrospray ionization. We are evaluating chemical derivatization procedures that will increase the signal intensity across the spectrum of hydroxy steroids and keto steroids. 7. Clozapine-N-oxide Studies. Over the last decade, a novel chemogenetic tool, DREADDs (designer receptors exclusively activated by designer drugs), has enabled researchers to manipulate cellular activity with increased spatial and temporal specificity. This tool relies on the use of an inert ligand clozapine-N-oxide (CNO), which upon injection can activate or inhibit a mutated receptor. While this method is ideal for studying early neural development in rodents, as manipulation of the embryo may be achieved by CNO injection to the pregnant dams, little is known about the dynamics or mechanisms when CNO is transmitted during this process. Using mass spectrometry, we previously detected CNO and its metabolites in sera and are now looking for these molecules in embryonic brain tissue.

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