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Characterization of human plasma proteome dynamics using deuterium oxide.
Wang, D, Liem, DA, Lau, E, Ng, DC, Bleakley, BJ, Cadeiras, M, Deng, MC, Lam, MP, Ping, P
Proteomics. Clinical applications. 2014;(7-8):610-9
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Abstract
PURPOSE High-throughput quantification of human protein turnover via in vivo administration of deuterium oxide ((2) H2 O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of (2) H2 O to human subjects. EXPERIMENTAL DESIGN We recruited ten healthy human subjects with a broad demographic variety to evaluate the safety, feasibility, efficacy, and reproducibility of (2) H2 O intake for studying protein dynamics. We designed a protocol where each subject orally consumed weight-adjusted doses of 70% (2) H2 O daily for 14 days to enrich body water and proteins with deuterium. Plasma proteome dynamics was measured using a high-resolution MS method we recently developed. RESULTS This protocol was successfully applied in ten human subjects to characterize the endogenous turnover rates of 542 human plasma proteins, the largest such human dataset to-date. Throughout the study, we did not detect physiological effects or signs of discomfort from (2) H2 O consumption. CONCLUSIONS AND CLINICAL RELEVANCE Our investigation supports the utility of a (2) H2 O intake protocol that is safe, accessible, and effective for clinical investigations of large-scale human protein turnover dynamics. This workflow shows promising clinical translational value for examining plasma protein dynamics in human diseases.
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Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis.
Plum, S, Helling, S, Theiss, C, Leite, REP, May, C, Jacob-Filho, W, Eisenacher, M, Kuhlmann, K, Meyer, HE, Riederer, P, et al
Journal of proteomics. 2013;:202-206
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Abstract
UNLABELLED This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (≥0.15g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls. BIOLOGICAL SIGNIFICANCE Key features of Parkinson's disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta, an associated loss of the brain pigment neuromelanin and a resulting impairment of the neuronal network. The accumulation of iron binding neuromelanin granules is age- and disease-dependent and disease specific alterations could affect the neuronal iron homeostasis leading to oxidative stress induced cell death. The focus of the described method is the analysis of neuromelanin granules as well as axonal cell-endings of nerve cells (synaptosomes) of individual donors (control and diseased). It is the basis for the identification of disease-relevant changes in the iron homeostasis and the generation of new insight into altered protein compositions or regulations which might lead to disturbed communications between nerve cells resulting in pathogenic processes.
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Proteomic approach to the study of statin pleiotropy in kidney transplant patients.
Pérez, V, Navarro-Muñoz, M, Mas, S, Bayés, B, Pastor, MC, Martínez-Cáceres, E, Lauzurica, R, Egido, J, Romero, R
Pharmacology. 2011;(3-4):161-8
Abstract
BACKGROUND/AIMS: Statins are prescribed in kidney transplant recipients in order to manage dyslipidemia, a common complication in these patients. The efficacy of statins in reducing cholesterol levels has been accompanied by pleiotropic effects. Fifty-four kidney transplant patients were included in the present study, the objective of which was to ascertain the effect of 12 weeks of atorvastatin therapy (10 mg/day) on the patients' lipid profile, renal function, markers of inflammation and plasma peptide profile. METHODS Biochemical variables were determined with a routine clinical laboratory analyzer, and the proteomic approach was based on magnetic particle-assisted sample processing coupled to mass spectrometry readout. RESULTS Atorvastatin therapy improved the lipid profile of patients and caused significant changes in their plasma peptide profile; peptides with m/z 1063 and 1898 decreased after treatment and were identified as fragments derived from molecules involved in vascular inflammation, i.e. high-molecular-weight kininogen and complement factor C4, respectively. CONCLUSION These findings may contribute to the growing body of evidence of the anti-inflammatory actions attributed to statins, by which these drugs could improve these patients' clinical status.
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Antibiotics and probiotics in chronic pouchitis: a comparative proteomic approach.
Turroni, S, Vitali, B, Candela, M, Gionchetti, P, Rizzello, F, Campieri, M, Brigidi, P
World journal of gastroenterology. 2010;(1):30-41
Abstract
AIM: To profile protein expression in mucosal biopsies from patients with chronic refractory pouchitis following antibiotic or probiotic treatment, using a comparative proteomic approach. METHODS Two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry were used to characterize the changes related to antibiotic therapy in the protein expression profiles of biopsy samples from patients with chronic refractory pouchitis. The same proteomic approach was applied to identify differentially expressed proteins in the non-inflamed pouch before and after probiotic administration. RESULTS In the first set of 2D gels, 26 different proteins with at least 2-fold changes in their expression levels between the pouchitis condition and antibiotic-induced remission were identified. In the second set of analysis, the comparison between mucosal biopsy proteomes in the normal and probiotic-treated pouch resulted in 17 significantly differently expressed proteins. Of these, 8 exhibited the same pattern of deregulation as in the pouchitis/pouch remission group. CONCLUSION For the first time, 2D protein maps of mucosal biopsies from patients with ileal pouch-anal anastomosis were provided, and differentially expressed proteins following antibiotic/probiotic treatment were identified.