Proteomic investigations are limited, in that, they typically yield information regarding static gene expression profiles. We demonstrate how to obtain a functional image of gene action by measuring protein dynamics in response to stimuli in vivo, i.e. one can determine the synthesis rate of virtually any protein by administering 2H2O (a safe, non-radioactive isotope) and then measuring the 2H-labeling of its peptide(s). Since 2H2O is given orally and the sensitivity of the proteomic assays allows measurements on ?l volumes of plasma, our method offers a minimally-invasive means of measuring the response to a dietary, pharmacological and/or surgical intervention. The objective of this proposal is to enable the routine application of our innovative technology for studying the development of proteome expression profiles. Two pivotal issues must be tested before one can envision wide-spread application.
Our first aim i s to determine the equilibration of 2H between body water and free amino acids.
Our second aim i s to test the applicability of 2H2O -proteomics by determining the influence of an obese insulin resistant phenotype and insulin resistant diabetes on protein synthesis in vivo. Experiments will focus on two proteins that lie at extreme ends of the plasma proteome, albumin and insulin, e.g. albumin has a relatively long t1/2 (~ 10 days in a human) and is present in high concentration (~ 600 umol per 1000 ml) whereas insulin has relatively short t1/2 (~ 30 min in a human) and is present in low concentration (~ 100 pmol per 1000 ml). Studies will contrast rodent models of extreme physiological homeostasis, e.g. fed vs. fasted states and normal vs. insulin resistant ? diabetes. These model experiments will draw out the main factors that challenge the use of the method in vivo. Significance. First, although we use conventional mass spectrometers (as compared to isotope ratio mass spectrometers), we have devised novel data integration routines to that yield """"""""high precision"""""""" measurements of isotope labeling. Second, given the existing proteomics infrastructure in the world, and the potential of using """"""""dynamic markers"""""""" of health/disease, one can envision the expansion of numerous areas of research. Last, since 2H2O can be administered in humans we plan to apply this technology for monitoring pancreatic islet function pre- and post-transplant, the technology development and model systems to be used herein will facilitate those future investigations. Public Health Relevance Statement: We have developed a new technology that is useful in diagnosing and monitoring patient health status, this relies on measurements of protein metabolism. We have outlined the essential studies that are required before one can envision wide-spread application.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRR1-BT-B (01))
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Friedman, Fred K
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Case Western Reserve University
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McCullough, Arthur; Previs, Stephen; Kasumov, Takhar (2018) Stable isotope-based flux studies in nonalcoholic fatty liver disease. Pharmacol Ther 181:22-33
Li, Ling; Zhang, Guo-Fang; Lee, Kwangwon et al. (2016) A Western diet induced NAFLD in LDLR(-/)(-) mice is associated with reduced hepatic glutathione synthesis. Free Radic Biol Med 96:13-21
Kasumov, Takhar; Li, Ling; Li, Min et al. (2015) Ceramide as a mediator of non-alcoholic Fatty liver disease and associated atherosclerosis. PLoS One 10:e0126910
Berisha, Stela Z; Brubaker, Greg; Kasumov, Takhar et al. (2015) HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function. J Lipid Res 56:653-64
Kasumov, Takhar; Dabkowski, Erinne R; Shekar, Kadambari Chandra et al. (2013) Assessment of cardiac proteome dynamics with heavy water: slower protein synthesis rates in interfibrillar than subsarcolemmal mitochondria. Am J Physiol Heart Circ Physiol 304:H1201-14
Heneghan, Helen M; Huang, Hazel; Kashyap, Sangeeta R et al. (2013) Reduced cardiovascular risk after bariatric surgery is linked to plasma ceramides, apolipoprotein-B100, and ApoB100/A1 ratio. Surg Obes Relat Dis 9:100-7
Kasumov, Takhar; Willard, Belinda; Li, Ling et al. (2013) 2H2O-based high-density lipoprotein turnover method for the assessment of dynamic high-density lipoprotein function in mice. Arterioscler Thromb Vasc Biol 33:1994-2003
Ilchenko, Serguei; Previs, Stephen F; Rachdaoui, Nadia et al. (2013) An improved measurement of isotopic ratios by high resolution mass spectrometry. J Am Soc Mass Spectrom 24:309-12
Li, Ling; Willard, Belinda; Rachdaoui, Nadia et al. (2012) Plasma proteome dynamics: analysis of lipoproteins and acute phase response proteins with 2H2O metabolic labeling. Mol Cell Proteomics 11:M111.014209
Kasumov, Takhar; Ilchenko, Serguey; Li, Ling et al. (2011) Measuring protein synthesis using metabolic ýýH labeling, high-resolution mass spectrometry, and an algorithm. Anal Biochem 412:47-55

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