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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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In-depth proteomic analysis of the human cerumen-a potential novel diagnostically relevant biofluid.
Feig, MA, Hammer, E, Völker, U, Jehmlich, N
Journal of proteomics. 2013;:119-29
Abstract
UNLABELLED Human cerumen, also called earwax, is a substance secreted by various glands in the outer ear canal. Although the variation of texture and color during otorhinolaryngological diseases is a generally known phenomenon, cerumen as biofluid remains relatively unexplored. However, there is an emerging interest for protein biomarkers which are easily accessible and predictive for diagnostics and therapy outcome. Here we provide a thorough investigation of human cerumen applying two different prefractionation techniques: i) 1D-PAGE prefractionation with subsequent LC-MS/MS, and ii) online SCX-fractionation coupled to LC-MS/MS. Additionally, individual variation was addressed by shotgun LC-MS/MS of specimens from 5 subjects. In total, we identified 11,562 distinct peptides representing 2013 proteins in human cerumen. The in-depth characterization revealed a high complexity of cerumen comparable with other human biofluids such as urine, plasma, or saliva. A probiotic or antibiotic character of cerumen has previously been discussed. In this study we provide further evidence for the important role of cerumen as an antimicrobial barrier and in local immune response, e.g. by assessing high amounts of zinc-alpha-2-glycoprotein. PRACTICAL IMPLICATIONS Cerumen analysis might have promising potential as diagnostic body fluid for biomarker characterization and disease specific objectives. Disease-associated or infection-specific changes may support diagnostics in otorhinolaryngology and may lead to a better understanding of human cerumen's function in immune response. An easy-to-handle and standardized sample collection and preparation of cerumen can further improve individualized medicine strategies.
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Identification of proteins implicated in the development of pancreatic cancer-associated diabetes mellitus by iTRAQ-based quantitative proteomics.
Wang, WS, Liu, XH, Liu, LX, Jin, DY, Yang, PY, Wang, XL
Journal of proteomics. 2013;:52-60
Abstract
UNLABELLED Studies have revealed that pancreatic cancer (PC) may lead to diabetes mellitus (DM). We aimed to identify the proteins implicated in the development of PC-associated DM in PC tissues with DM. We used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography-tandem mass spectrometry to compare protein expression in PC tissues with DM with that in PC tissues without DM and in adjacent nontumor tissues with or without DM. A total of 80 surgically resected fresh tissues from 40 PC patients were included to identify differential protein expression. Western blotting and immunohistochemistry were then applied to evaluate the differential expression of selected proteins. A total of 1611 proteins were repeatedly identified and quantified by performing the iTRAQ-based experiments twice. Of these, 23 proteins were differentially expressed according to our defined criteria (12 upregulated and 11 downregulated). The S100 calcium binding protein A9 and aldehyde dehydrogenase 2 family were selected to validate the proteomic results by western blotting and immunohistochemistry. The identification of key proteins implicated in the development of PC-associated DM could serve as a foundation to better understand and further explore the etiology and pathogenesis of PC-associated DM. BIOLOGICAL SIGNIFICANCE The identification of key proteins implicated in the development of PC-associated DM could serve as a foundation to better understand and further explore the etiology and pathogenesis of PC-associated DM.
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"Cheek-to-cheek" urinary proteome profiling via combinatorial peptide ligand libraries: A novel, unexpected elution system.
Candiano, G, Santucci, L, Bruschi, M, Petretto, A, D'Ambrosio, C, Scaloni, A, Righetti, PG, Ghiggeri, GM
Journal of proteomics. 2012;(3):796-805
Abstract
A new method is here reported for facile elution of the human urinary proteome after being captured with combinatorial peptide ligand libraries (CPLL, ProteoMiner). It consists in challenging the beads with 100mM Tris, pH 7.4, or with 100mM Lys, pH 7.4 or even better with a mixture of Lys, Arg, Asp and Glu (150mM final concentration). These elutions permit recovery of species in a native form, for monitoring any biological activity of the eluted species, while avoiding the noxious presence of sodium dodecyl sulphate (SDS), reported as the best eluant so far from CPLL beads. SDS, albeit permitting quantitative recovery from the beads, has to be removed from the sample prior to mass spectrometry analysis. This unorthodox elution, which most likely will work only for urine samples, seems to be due to the fact that bile salts and urinary pigments are massively adsorbed by the beads, thus masking the hydrophobic binding sites of aromatic and non-aromatic amino acids. The binding thus occurs mostly via ionic and hydrogen bond interactions via the "Grand Catchers" Arg, Lys, His, which can then be easily challenged by positively charged species, such a Tris, free Lys and free Arg in the eluant as well as by negatively charged compounds, such as Glu and Asp. When eluting with the four-amino acid mix, at least 3300 spots can be visualized in a 2D map.
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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.