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Journal RefereesPROTEOMICS, Journal of Proteomics, Journal of Proteome Research, Microbiology, Proteome Sc
Publications (41) View all
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Article: A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria.
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ABSTRACT: The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 10(6) cells of S. aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.Methods 04/2013; · 4.01 Impact Factor -
Article: Comparative immunoproteome analysis of the response of susceptible A.BY/SnJ and resistant C57BL/6 mice to Coxsackievirus B3-infection
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ABSTRACT: Both, innate and cell-mediated immunity contribute to prevention of chronic myocarditis and consecutively, cardiomyopathy. 4us, in resistant C57BL/6 mice myocarditis induced by Coxsackievirus B3 (CVB3)-infection is abrogated by immune-mediated mechanisms. However, susceptible A.BY/SnJ mice develop dilated cardiomyopathy (DCM) due to chronic myocarditis. Cardiac auto-antibodies have been shown to play a pivotal role in the initiation and/or progression of inWammatory DCM. In order to investigate diXerences in the autoimmune response of susceptible and resistant mice to infection with CVB3, the patterns of autoantibodies reacting with heart proteins in A.BY/SnJ and C57BL/6 mice were proYled by 2-D Western blot analysis during the acute and chronic phases of myocarditis up to three months, when the pathophysiological phenotype in the susceptible mice has progressed to DCM. In the early phase of infection both mouse strains displayed similar autoantibody patterns. In contrast, at later time points compared to the resistant C57BL/6 strain susceptible A.BY/SnJ mice displayed a much stronger autoimmune response against proteins associated with cell structure, protein transport as well as primary metabolic processes such as energy production. During chronic myocarditis strong antibody responses against myosin heavy chain 6, mitochondrial and heat shock proteins were observed in A.BY/SnJ mice. Antibodies directed against alpha-enolase, serotransferrin, radixin and two processed myosin protein species accumulated late and only in A.BY/SnJ mice suXering from inWammatory DCM. Functional assignment of the target proteins of cardiac autoantibodies indicates that these might be directly involved in cardiac dysfunction.Journal of Integrated OMICS. 12/2012; 2(2):54-63. -
Article: Quantitative analysis of the intra- and inter-subject variability of the whole salivary proteome.
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ABSTRACT: BACKGROUND AND OBJECTIVE: Interest in human saliva is increasing for disease-specific biomarker discovery studies. However, protein composition of whole saliva can grossly vary with physiological and environmental factors over time and it comprises human as well as bacterial proteins. MATERIAL AND METHODS: We compared intra- and inter-subject variabilities using complementary gel-based (two-dimensional difference gel electrophoresis, 2-D DIGE) and gel-free (liquid chromatography tandem mass spectrometry, LC-MS/MS) proteomics profiling of saliva. Unstimulated whole saliva of four subjects was examined at three different time-points (08.00 h, 12.00 h and 17.00 h) and variability of the saliva proteome was analyzed on two successive days by LC-MS/MS. RESULTS: In the 2-D DIGE experiment, the median coefficient of variation (CV) for intra-subject variability was significantly lower (CV of 0.39) than that for inter-subject variability (CV of 0.57; CV of technical replicates 0.17). LC-MS/MS data confirmed the significantly lower variation within subjects over time (CV of 0.37) than the inter-subject variability (CV of 0.53; CV of technical replicates 0.11), and that the inter-subject variability was not time-dependent. CONCLUSION: Both techniques revealed similar trends of variations on technical, intra- and inter-subject level but provided peptide and protein focused information and should thus be used as complementary approaches. The data presented indicate that 2-D DIGE as well as LC-MS/MS approaches are suitable for biomarker screening in saliva.Journal of Periodontal Research 11/2012; · 1.69 Impact Factor -
Article: Myocardial gene expression profiles and cardiodepressant autoantibodies predict response of patients with dilated cardiomyopathy to immunoadsorption therapy.
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ABSTRACT: AimsImmunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) represents a novel therapeutic approach in the treatment of dilated cardiomyopathy (DCM) which leads to the improvement of left ventricular ejection fraction (LVEF). However, response to this therapeutic intervention shows wide inter-individual variability. In this pilot study, we tested the value of clinical, biochemical, and molecular parameters for the prediction of the response of patients with DCM to IA/IgG.Methods and resultsForty DCM patients underwent endomyocardial biopsies (EMBs) before IA/IgG. In eight patients with normal LVEF (controls), EMBs were obtained for clinical reasons. Clinical parameters, negative inotropic activity (NIA) of antibodies on isolated rat cardiomyocytes, and gene expression profiles of EMBs were analysed. Dilated cardiomyopathy patients displaying improvement of LVEF (≥20 relative and ≥5% absolute) 6 months after IA/IgG were considered responders. Compared with non-responders (n = 16), responders (n = 24) displayed shorter disease duration (P = 0.006), smaller LV internal diameter in diastole (P = 0.019), and stronger NIA of antibodies. Antibodies obtained from controls were devoid of NIA. Myocardial gene expression patterns were different in responders and non-responders for genes of oxidative phosphorylation, mitochondrial dysfunction, hypertrophy, and ubiquitin-proteasome pathway. The integration of scores of NIA and expression levels of four genes allowed robust discrimination of responders from non-responders at baseline (BL) [sensitivity of 100% (95% CI 85.8-100%); specificity up to 100% (95% CI 79.4-100%); cut-off value: -0.28] and was superior to scores derived from antibodies, gene expression, or clinical parameters only.Conclusion Combined assessment of NIA of antibodies and gene expression patterns of DCM patients at BL predicts response to IA/IgG therapy and may enable appropriate selection of patients who benefit from this therapeutic intervention.European Heart Journal 10/2012; · 10.48 Impact Factor -
Article: Profiling alterations in platelets induced by Amotosalen/UVA pathogen reduction and gamma irradiation--a LC-ESI-MS/MS-based proteomics approach.
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ABSTRACT: Pathogen reduction in platelet concentrates (PC) using Amotosalen/UVA-light reduces the risk of transfusion transmitted infections but also decreases the post-transfusion platelet count increment. Little is known about potential platelet lesions caused by Amotosalen/UVA-light, which may reduce therapeutic efficacy of PCs. Platelets from buffy coat (n=15) derived PCs were pooled and split into three equal PC-units. PC-1 was left untreated (control). PC-2 was Amotosalen/UVA treated using the INTERCEPT Blood System(™), PC-3 was gamma irradiated (30 Gy). Samples were prepared one and five days after PC-production for LC-ESI-MS/MS analysis. Proteins displaying treatment-dependent changes in intensity were classified according to Gene Ontology. In total, 948 proteins were identified, 721 with ≥2 peptides. At day 1, Amotosalen/UVA-treatment triggered alteration of 23 proteins, and gamma irradiation of 49 proteins (overlap: 11 proteins). Five days storage revealed 58 (Amotosalen/UVA treated), 50 (gamma irradiated), and 36 (controls) changes in the platelet proteome compared to control platelets at day 1. Gene Ontology analysis revealed that many affected proteins were displaying specific catalytic activities and/or protein/nucleic acid binding capacity. We identified platelet endothelial aggregation receptor 1 precursor, chloride intracellular channel protein 4, and protein-tyrosine sulfotransferase 2 as proteins uniquely and consistently altered after treatment and storage of Amotosalen/UVA treated platelets. While Amotosalen/UVA-treatment causes less pronounced proteome changes than gamma irradiation at day 1, our data indicate an increase in storage lesions at day 5 caused by this pathogen reduction treatment.Blood transfusion = Trasfusione del sangue 05/2012; 10 Suppl 2:s63-70. · 2.10 Impact Factor
