TY - JOUR AB - The ascidian Boytryllus schlosseri is a marine chordate that thrives under conditions of anthropogenic climate change. The B. schlosseri expressed proteome contains unusually high levels of proteins adducted with 4-hydroxy-2-nonenal (HNE). HNE represents a prominent posttranslational modification resulting from oxidative stress. Prior to this study, which identified 1052 HNE adducted proteins in B. schlosseri by LCMS, HNE protein modification has not been determined in any marine species. Adducted residues were ascertained for 1849 HNE modifications, 1195 of which had a maximum amino acid localization score. Most HNE modifications were at less reactive lysines (rather than more reactive cysteines). HNE prevalence on most sites was high, suggesting that B. schlosseri experiences and tolerates high intracellular reactive oxygen species levels, resulting in substantial lipid peroxidation. HNE adducted B. schlosseri proteins show enrichment in mitochondrial, proteostasis, and cytoskeletal functions. We propose that redox signaling contributes to regulating energy metabolism, the blastogenic cycle, oxidative burst defenses, and cytoskeleton dynamics in B. schlosseri. DIA-LCMS quantification of 72 HNE-adducted sites across 60 proteins revealed significant population-specific differences. We conclude that the vast amount of HNE protein adduction in this circumpolar tunicate is indicative of high oxidative stress tolerance contributing to its range expansion into diverse environments. SUMMARY: Oxidative stress results from environmental challenges that increase in frequency and severity during the Anthropocene. Oxygen radical attack causes lipid peroxidation, leading to HNE production. Proteome-wide HNE adduction is highly prevalent in Botryllus schlosseri, a widely distributed, highly invasive, and economically important biofouling ascidian, and the first marine species to be analyzed for proteome HNE modification. HNE adduction of specific proteins may physiologically sequester reactive oxygen species, which could enhance fitness and resilience during environmental change. AU - Kültz, D.* AU - Gardell, A.M.* AU - DeTomaso, A.* AU - Stoney, G.* AU - Rinkevich, B.* AU - Qarri, A. AU - Hamar, J.* C1 - 75422 C2 - 58238 CY - 111 River St, Hoboken 07030-5774, Nj Usa SP - 12-25 TI - Proteome-wide 4-hydroxy-2-nonenal signature of oxidative stress in the marine invasive tunicate Botryllus schlosseri. JO - Proteomics VL - 25 IS - 19 PB - Wiley PY - 2025 SN - 1615-9853 ER - TY - JOUR AB - INSC94Y transgenic pigs represent a model for mutant insulin gene-induced diabetes of youth, with impaired insulin secretion and beta cell loss, leading to elevated fasting blood glucose levels. A key complication of diabetes mellitus is diabetic retinopathy (DR), characterized by hyperglycemia-induced abnormalities in the retina. Adjacent to the retina lies the vitreous, a gelatinous matrix vital for ocular function. It harbors proteins and signaling molecules, offering insights into vitreous biology and ocular health. Moreover, as a reservoir for secreted molecules, the vitreous illuminates molecular processes within intraocular structures, especially under pathological conditions. To uncover the proteomic profile of porcine vitreous and explore its relevance to DR, we employed discovery proteomics to compare vitreous samples from INSC94Y transgenic pigs and wild-type controls. Our analysis identified 1404 proteins, with 266 showing differential abundance in INSC94Y vitreous. Notably, the abundances of ITGB1, COX2, and GRIFIN were significantly elevated in INSC94Y vitreous. Gene Set Enrichment Analysis unveiled heightened MYC and mTORC1 signaling in INSC94Y vitreous, shedding light on its biological significance in diabetes-associated ocular pathophysiology. These findings deepen our understanding of vitreous involvement in DR and provide valuable insights into potential therapeutic targets. Raw data are accessible via ProteomeXchange (PXD038198). AU - Degroote, R.L.* AU - Schmalen, A.* AU - Renner, S.* AU - Wolf, E.* AU - Hauck, S.M. AU - Deeg, C.A.* C1 - 71450 C2 - 56180 CY - 111 River St, Hoboken 07030-5774, Nj Usa TI - Diabetic retinopathy from the vitreous proteome perspective: The INSC94Y transgenic pig model study. JO - Proteomics PB - Wiley PY - 2024 SN - 1615-9853 ER - TY - JOUR AB - Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions. AU - Kültz, D.* AU - Gardell, A.M.* AU - DeTomaso, A.* AU - Stoney, G.* AU - Rinkevich, B.* AU - Rinkevich, Y. AU - Qarri, A. AU - Dong, W.* AU - Luu, B.* AU - Lin, M.* C1 - 70033 C2 - 55369 CY - 111 River St, Hoboken 07030-5774, Nj Usa TI - Deep quantitative proteomics of North American Pacific coast star tunicate (Botryllus schlosseri). JO - Proteomics PB - Wiley PY - 2024 SN - 1615-9853 ER - TY - JOUR AB - Data-independent acquisition (DIA) of tandem mass spectrometry spectra has emerged as a promising technology to improve coverage and quantification of proteins in complex mixtures. The success of DIA experiments is dependent on the quality of spectral libraries used for data base searching. Frequently, these libraries need to be generated by labor and time intensive data dependent acquisition (DDA) experiments. Recently, several algorithms have been published that allow the generation of theoretical libraries by an efficient prediction of retention time and intensity of the fragment ions. Sequential windowed acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) is a DIA method that can be applied at an unprecedented speed, but the fragmentation spectra suffer from a lower quality than data acquired on Orbitrap instruments. To reliably generate theoretical libraries that can be used in SWATH experiments, we developed deep-learning for SWATH analysis (dpSWATH), to improve the sensitivity and specificity of data generated by Q-TOF mass spectrometers. The theoretical library built by dpSWATH allowed us to increase the identification rate of proteins compared to traditional or library-free methods. Based on our analysis we conclude that dpSWATH is a superior prediction framework for SWATH-MS measurements than other algorithms based on Orbitrap data. AU - Sun, B.* AU - Smialowski, P. AU - Aftab, W.* AU - Schmidt, A.* AU - Forne, I.* AU - Straub, T.* AU - Imhof, A.* C1 - 67129 C2 - 53487 CY - 111 River St, Hoboken 07030-5774, Nj Usa TI - Improving SWATH-MS analysis by deep-learning. JO - Proteomics VL - 23 IS - 9 PB - Wiley PY - 2023 SN - 1615-9853 ER - TY - JOUR AB - Metabolomics, the systematic measurement of small molecules (<1000 Da) in a given biological sample, is a fast-growing field with many different applications. In contrast to transcriptomics and proteomics, sharing of data is not as widespread in metabolomics, though more scientists are sharing their data nowadays. However, to improve data analysis tools and develop new data analytical approaches and to improve metabolite annotation and identification, sharing of reference data is crucial. Here, different possibilities to share (metabolomics) data are reviewed and some recent approaches and applications regarding the (re-)use and (re-)analysis are highlighted. AU - Witting, M. C1 - 68321 C2 - 54737 CY - 111 River St, Hoboken 07030-5774, Nj Usa TI - (Re-)use and (re-)analysis of publicly available metabolomics data. JO - Proteomics VL - 23 IS - (23-24) PB - Wiley PY - 2023 SN - 1615-9853 ER - TY - JOUR AB - Skin affections after sulfur mustard (SM) exposure include erythema, blister formation and severe inflammation. An antidote or specific therapy does not exist. Anti-inflammatory compounds as well as substances counteracting SM-induced cell death are under investigation. In this study, we investigated the benzylisoquinoline alkaloide berberine (BER), a metabolite in plants like berberis vulgaris, which is used as herbal pharmaceutical in Asian countries, against SM toxicity using a well-established in vitro approach. Keratinocyte (HaCaT) mono-cultures (MoC) or HaCaT/THP-1 co-cultures (CoC) were challenged with 100, 200 or 300 mM SM for 1 h. Post-exposure, both MoC and CoC were treated with 10, 30 or 50 mu M BER for 24 h. At that time, supernatants were collected and analyzed both for interleukine (IL) 6 and 8 levels and for content of adenylate-kinase (AK) as surrogate marker for cell necrosis. Cells were lysed and nucleosome formation as marker for late apoptosis was assessed. In parallel, AK in cells was determined for normalization purposes. BER treatment did not influence necrosis, but significantly decreased apoptosis. Anti-inflammatory effects were moderate, but also significant, primarily in CoC. Overall, BER has protective effects against SM toxicity in vitro. Whether this holds true should be evaluated in future in vivo studies. AU - Ruzafa, N.* AU - Pereiro, X.* AU - Lepper, M.F. AU - Hauck, S.M. AU - Vecino, E.* C1 - 53395 C2 - 44845 CY - Elsevier House, Brookvale Plaza, East Park Shannon, Co, Clare, 00000, Ireland TI - A proteomics approach to identify candidate proteins secreted by Müller glia that protect ganglion cells in the retina. JO - Proteomics VL - 11 IS - 11 PB - Elsevier Ireland Ltd PY - 2018 SN - 1615-9853 ER - TY - JOUR AB - Equine recurrent uveitis (ERU) is the only spontaneous model for recurrent autoimmune uveitis in humans, where T cells target retinal proteins. Differences between normal and autoaggressive lymphocytes were identified in this study by analyzing peripheral blood derived lymphocytes (PBL) proteomes from the same case with IRBP-induced uveitis sampled before (day 0), during (day 15) and after uveitic attack (day 23). Relative protein abundances of PBL were investigated in a quantitative, label-free differential proteome analysis in cells that were kept frozen for 14 years since the initial experiment. Quantitative data could be acquired for 2632 proteins at all three time-points. Profound changes (≥2 fold change) in PBL protein abundance were observed when comparing day 0 to 15, representing acute inflammation (1070 regulated proteins) and day 0 to 23 (cessation; 1571 regulated). Significant differences applied to proteins with functions in integrin signaling during active uveitis, involving "Erk and pi-3 kinase are necessary for collagen binding in corneal epithelia", "Integrins in angiogenesis" and "Integrin-linked kinase signaling" pathways. In contrast, at cessation of uveitic attack, significantly changed proteins belonged to pathways of "nongenotropic androgen signaling", "classical complement pathway" and "Amb2 integrin signaling". Several members of respective pathways were earlier shown to be changed in naturally occurring uveitis, underscoring the significance of these findings here and proofing the value of the induced model in mimicking spontaneous autoimmune uveitis. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository 2 (dataset identifier PXD005580). AU - Hauck, S.M. AU - Lepper, M.F. AU - Hertl, M.* AU - Sekundo, W.* AU - Deeg, C.A.* C1 - 51762 C2 - 43513 CY - Hoboken TI - Proteome dynamics in biobanked horse peripheral blood derived lymphocytes (PBL) with induced autoimmune uveitis. JO - Proteomics VL - 17 IS - 19 PB - Wiley PY - 2017 SN - 1615-9853 ER - TY - JOUR AB - Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease (COPD). Many of its more than 4500 chemicals are highly reactive thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative mass spectrometry to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar-derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix (ECM) organization. In particular, secretion of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in ECM organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and COPD. AU - Mossina, A. AU - Lukas, C. AU - Merl-Pham, J. AU - Uhl, F.E.* AU - Mutze, K. AU - Schamberger, A.C. AU - Staab-Weijnitz, C.A. AU - Jia, J. AU - Yildirim, A.Ö. AU - Königshoff, M. AU - Hauck, S.M. AU - Eickelberg, O. AU - Meiners, S. C1 - 50091 C2 - 42017 CY - Hoboken TI - Cigarette smoke alters the secretome of lung epithelial cells. JO - Proteomics VL - 17 IS - 1-2 PB - Wiley-blackwell PY - 2017 SN - 1615-9853 ER - TY - JOUR AB - Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity and availability of anti-histone antibodies. Here, we describe a novel MALDI imaging mass spectrometry (MALDI-IMS) based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available. AU - Lahiri, S.* AU - Sun, N. AU - Solis-Mezarino, V.* AU - Fedisch, A.* AU - Ninkovic, J. AU - Feuchtinger, A. AU - Götz, M. AU - Walch, A.K. AU - Imhof, A.* C1 - 47408 C2 - 39318 CY - Hoboken SP - 437-447 TI - In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry. JO - Proteomics VL - 16 IS - 3 PB - Wiley-blackwell PY - 2016 SN - 1615-9853 ER - TY - JOUR AB - Iron is an essential micronutrient for plants, and iron deficiency requires a variety of physiological adaptations. FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR) is essential for the regulation of iron uptake in Arabidopsis thaliana roots. FIT is transcriptionally as well as post-transcriptionally regulated in response to iron supply. To investigate to which extent post-transcriptional regulation upon iron deficiency applies to proteins and to determine the dependency on FIT, we performed a parallel proteomic and transcriptomic study with wild-type, a fit knock-out mutant and a FIT over-expressing Arabidopsis line. Among 92 proteins differentially regulated by iron and/or FIT, we identified 30 proteins, which displayed differential regulation at the transcriptional level. 11 protein spots were regulated in at least one of the data points even contrary to the respective genes dependent on FIT. We found 10 proteins in at least two forms. The analysis of functional classification showed enriched Gene Ontology (GO) terms among the post-transcriptionally regulated genes and of proteins, that were down-regulated or absent in the fit knock-out mutant. Taken together, we provide evidence for iron and FIT-dependent post-transcriptional regulation in iron homeostasis in A. thaliana. AU - Mai, H.J.* AU - Lindermayr, C. AU - von Toerne, C. AU - Fink-Straube, C.* AU - Durner, J. AU - Bauer, P.* C1 - 45085 C2 - 37204 SP - 3030-3047 TI - Iron and FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)-dependent regulation of proteins and genes in Arabidopsis thaliana roots. JO - Proteomics VL - 15 IS - 17 PY - 2015 SN - 1615-9853 ER - TY - JOUR AB - Molecular interaction databases are essential resources that enable access to a wealth of information on associations between proteins and other biomolecules. Network graphs generated from these data provide an understanding of the relationships between different proteins in the cell, and network analysis has become a widespread tool supporting -omics analysis. Meaningfully representing this information remains far from trivial and different databases strive to provide users with detailed records capturing the experimental details behind each piece of interaction evidence. A targeted curation approach is necessary to transfer published data generated by primarily low-throughput techniques into interaction databases. In this review we present an example highlighting the value of both targeted curation and the subsequent effective visualization of detailed features of manually curated interaction information. We have curated interactions involving LRRK2, a protein of largely unknown function linked to familial forms of Parkinson's disease, and hosted the data in the IntAct database. This LRRK2-specific dataset was then used to produce different visualization examples highlighting different aspects of the data: the level of confidence in the interaction based on orthogonal evidence, those interactions found under close-to-native conditions, and the enzyme-substrate relationships in different in vitro enzymatic assays. Finally, pathway annotation taken from the Reactome database was overlaid on top of interaction networks to bring biological functional context to interaction maps. AU - Porras, P.* AU - Duesbury, M.* AU - Fabregat, A.* AU - Ueffing, M. AU - Orchard, S.* AU - Gloeckner, C.J. AU - Hermjakob, H.* C1 - 44016 C2 - 36698 CY - Hoboken SP - 1390-1404 TI - A visual review of the interactome of LRRK2: Using deep-curated molecular interaction data to represent biology. JO - Proteomics VL - 15 IS - 8 PB - Wiley-blackwell PY - 2015 SN - 1615-9853 ER - TY - JOUR AB - Mass spectrometers equipped with matrix-assisted laser desorption/ionization (MALDI-MS) require frequent multi-point calibration to obtain good mass accuracy over a wide mass range and across large numbers of samples. In this study, we introduce a new synthetic peptide mass calibration standard termed PAS-cal tailored for MALDI-MS based bottom-up proteomics. This standard consists of 30 peptides between 8 and 37 amino acids long and each constructed to contain repetitive sequences of Pro, Ala and Ser as well as one C-terminal Arg residue. MALDI spectra thus cover a mass range between 750 and 3200 m/z in MS mode and between 100 and 3200 m/z in MS/MS mode. Our results show that multi-point calibration of MS spectra using PAS-cal peptides compares well to current commercial reagents for protein identification by peptide mass finger printing. Calibration of tandem mass spectra from LC-MALDI experiments using the longest peptide, PAS-cal37, resulted in smaller fragment ion mass errors, more matching fragment ions and more protein and peptide identifications compared to commercial standards, making the PAS-cal standard generically useful for bottom-up proteomics. AU - Maier, S.K. AU - Bashkueva, K.* AU - Rösli, C.* AU - Skerra, A.* AU - Kuster, B.* C1 - 31964 C2 - 34914 SP - 2427-2431 TI - PAS-cal: A repetitive peptide sequence calibration standard for MALDI mass spectrometry. JO - Proteomics VL - 14 IS - 21-22 PY - 2014 SN - 1615-9853 ER - TY - JOUR AB - Mass spectrometry imaging (MSI) is a valuable tool for diagnostics and systems biology studies, being a highly sensitive, label-free technique capable of providing comprehensive spatial distribution of different classes of biomolecules. The application of MSI to the study of endogenous compounds has received considerable attention because metabolites are the result of the interactions of a biosystem with its environment. MSI can therefore enhance understanding of disease mechanisms and elucidate mechanisms for biological variation. Here we present the in situ comparative metabolomics imaging data for analyses of light- and dark-treated retina. A wide variety of tissue metabolites were imaged at a high spatial resolution. These include nucleotides, central carbon metabolism pathway intermediates, 2-oxocarboxylic acid metabolism, oxidative phosphorylation, glycerophospholipid metabolism, and cysteine and methionine metabolites. The high lateral resolution enabled the differentiation of retinal layers, allowing determination of the spatial distributions of different endogenous compounds. A number of metabolites demonstrated differences between light and dark conditions. These findings add to the understanding of metabolic activity in the retina. This article is protected by copyright. All rights reserved. AU - Sun, N. AU - Ly, A. AU - Meding, S. AU - Witting, M. AU - Hauck, S.M. AU - Ueffing, M. AU - Schmitt-Kopplin, P. AU - Aichler, M. AU - Walch, A.K. C1 - 29077 C2 - 33621 CY - Hoboken SP - 913-923 TI - High-resolution metabolite imaging of light and dark treated retina using MALDI-FTICR mass spectrometry. JO - Proteomics VL - 14 IS - 7-8 PB - Wiley-Blackwell PY - 2014 SN - 1615-9853 ER - TY - JOUR AU - Zahedi, R.P.* AU - Ueffing, M. AU - Sickmann, A.* C1 - 34378 C2 - 35240 SP - 1953 TI - Proteomics - moving from inventory to personalized medicine? JO - Proteomics VL - 14 IS - 17-18 PY - 2014 SN - 1615-9853 ER - TY - JOUR AB - Chronic low-dose ionizing radiation induces cardiovascular disease in human populations but the mechanism is largely unknown. We suggested that chronic radiation exposure may induce endothelial cell senescence that is associated with vascular damage in vivo. We investigated whether chronic radiation exposure is causing a change in the onset of senescence in endothelial cells in vitro. Indeed, when exposed to continuous low-dose rate gamma radiation (4.1 mGy/h), primary human umbilical vein endothelial cells (HUVECs) initiated senescence much earlier than the nonirradiated control cells. We investigated the changes in the protein expression of HUVECs before and during the onset of radiation-induced senescence. Cellular proteins were quantified using isotope-coded protein label technology after 1, 3, and 6 weeks of radiation exposure. Several senescence-related biological pathways were influenced by radiation, including cytoskeletal organization, cellcell communication and adhesion, and inflammation. Immunoblot analysis showed an activation of the p53/p21 pathway corresponding to the progressing senescence. Our data suggest that chronic radiation-induced DNA damage and oxidative stress result in induction of p53/p21 pathway that inhibits the replicative potential of HUVECs and leads to premature senescence. This study contributes to the understanding of the increased risk of cardiovascular diseases seen in populations exposed to chronic low-dose irradiation. AU - Yentrapalli, R. AU - Azimzadeh, O. AU - Barjaktarovic, Z. AU - Sarioglu, H. AU - Wojcik, A.* AU - Harms-Ringdahl, M.* AU - Atkinson, M.J. AU - Haghdoost, S.* AU - Tapio, S. C1 - 24330 C2 - 31522 SP - 1096-1107 TI - Quantitative proteomic analysis reveals induction of premature senescence in human umbilical vein endothelial cells exposed to chronic low-dose rate gamma radiation. JO - Proteomics VL - 13 IS - 7 PB - Wiley-Blackwell PY - 2013 SN - 1615-9853 ER - TY - JOUR AB - Protein interactions mediate essentially all biological processes and analysis of proteinprotein interactions using both large-scale and small-scale approaches has contributed fundamental insights to the understanding of biological systems. In recent years, interactome network maps have emerged as an important tool for analyzing and interpreting genetic data of complex phenotypes. Complementary experimental approaches to test for binary, direct interactions, and for membership in protein complexes are used to explore the interactome. The two approaches are not redundant but yield orthogonal perspectives onto the complex network of physical interactions by which proteins mediate biological processes. In recent years, several publications have demonstrated that interactions from high-throughput experiments can be equally reliable as the high quality subset of interactions identified in small-scale studies. Critical for this insight was the introduction of standardized experimental benchmarking of interaction and validation assays using reference sets. The data obtained in these benchmarking experiments have resulted in greater appreciation of the limitations and the complementary strengths of different assays. Moreover, benchmarking is a central element of a conceptual framework to estimate interactome sizes and thereby measure progress toward near complete network maps. These estimates have revealed that current large-scale data sets, although often of high quality, cover only a small fraction of a given interactome. Here, I review the findings of assay benchmarking and discuss implications for quality control, and for strategies toward obtaining a near-complete map of the interactome of an organism. AU - Braun, P. C1 - 7688 C2 - 29888 SP - 1499-1518 TI - Interactome mapping for analysis of complex phenotypes: Insights from benchmarking binary interaction assays. JO - Proteomics VL - 12 IS - 10 PB - Wiley-VCH PY - 2012 SN - 1615-9853 ER - TY - JOUR AB - Today, it is widely appreciated that proteinprotein interactions play a fundamental role in biological processes. This was not always the case. The study of protein interactions started slowly and evolved considerably, together with conceptual and technological progress in different areas of research through the late 19th and the 20th centuries. In this review, we present some of the key experiments that have introduced major conceptual advances in biochemistry and molecular biology, and review technological breakthroughs that have paved the way for today's systems-wide approaches to proteinprotein interaction analysis. AU - Braun, P. AU - Gingras, A.C.* C1 - 7687 C2 - 29889 SP - 1478-1498 TI - History of protein-protein interactions: From egg-white to complex networks. JO - Proteomics VL - 12 IS - 10 PB - Wiley-VCH PY - 2012 SN - 1615-9853 ER - TY - JOUR AB - Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder. AU - Dong, G.F.* AU - Callegari, E.* AU - Gloeckner, C.J. AU - Ueffing, M. AU - Wang, H.M.* C1 - 7759 C2 - 29878 SP - 2060-2064 TI - Mass spectrometric identification of novel posttranslational modification sites in huntingtin. JO - Proteomics VL - 12 IS - 12 PB - Wiley-VCH PY - 2012 SN - 1615-9853 ER - TY - JOUR AB - To better understand the involvement of retinal Muller glial (RMG) cells in retinal diseases, we phenotyped primary porcine RMGs in dependence of cultivation time using different quantitative proteomic strategies. A well-established LC-MS/MS-based quantification method was employed: stable isotope labeling by amino acids in cell culture (SILAC) and directly compared to label-free (LF) quantifications, based on total peak intensities using two different programs (MaxQuant and Progenesis LC-MS). The overall numbers of detected proteins were largely similar (overlap of 1324 proteins), only a total of 173 proteins were significantly altered between the different culture conditions. However, among these, only 21 proteins were shared between the three analytical strategies. Hence, the majority of altered proteins only reached significance thresholds in one of the applied analyses with a larger overlap between the two LF approaches. Among the shared, differentially abundant proteins were known RMG markers as well as new proteins associated with glial cell transition. However, proteins correlated to cellular transitions and dedifferentiation were also found among the proteins only significant in one or two of the applied strategies. Consequently, the application of different quantification and analytical strategies could increase the analytical depths of proteomic phenotyping. AU - Merl, J. AU - Ueffing, M. AU - Hauck, S.M. AU - von Toerne, C. C1 - 7763 C2 - 29876 SP - 1902-1911 TI - Direct comparison of MS-based label-free and SILAC quantitative proteome profiling strategies in primary retinal Müller cells. JO - Proteomics VL - 12 IS - 12 PB - Wiley-VCH PY - 2012 SN - 1615-9853 ER - TY - JOUR AB - Accidental nuclear scenarios lead to environmental contamination of unknown level. Immediate radiation-induced biological responses that trigger processes leading to adverse health effects decades later are not well understood. A comprehensive proteomic analysis provides a promising means to identify and quantify the initial damage after radiation exposure. Early changes in the cardiac tissue of C57BL/6 mice exposed to total body irradiation were studied, using a dose relevant to both intentional and accidental exposure (3 Gy gamma ray). Heart tissue protein lysates were analyzed 5 and 24 h after the exposure using isotope-coded protein labeling (ICPL) and 2-dimensional difference-in-gel-electrophoresis (2-D DIGE) proteomics approaches. The differentially expressed proteins were identified by LC-ESI-MS-MS. Both techniques showed similar functional groups of proteins to be involved in the initial injury. Pathway analyses indicated that total body irradiation immediately induced biological responses such as inflammation, antioxidative defense, and reorganization of structural proteins. Mitochondrial proteins represented the protein class most sensitive to ionizing radiation. The proteins involved in the initial damage processes map to several functional categories involving cardiotoxicity. This prompts us to propose that these early changes are indicative of the processes that lead to an increased risk of cardiovascular disease after radiation exposure. AU - Azimzadeh, O. AU - Scherthan, H.* AU - Sarioglu, H. AU - Barjaktarovic, Z. AU - Conrad, M. AU - Vogt, A.* AU - Calzada-Wack, J. AU - Neff, F. AU - Aubele, M. AU - Buske, C. AU - Atkinson, M.J. AU - Tapio, S. C1 - 5023 C2 - 28708 CY - Weinheim SP - 3299-3311 TI - Rapid proteomic remodeling of cardiac tissue caused by total body ionizing radiation. JO - Proteomics VL - 11 IS - 6 PB - Wiley-VCH PY - 2011 SN - 1615-9853 ER - TY - JOUR AB - In recent years, nitric oxide (NO) has been recognized as a signalling molecule of plants, being involved in diverse processes like germination, root growth, stomatal closing, and responses to various stresses. A mechanism of how NO can regulate physiological processes is the modulation of cysteine residues of proteins (S-nitrosylation) by S-nitrosoglutathione (GSNO), a physiological NO donor. The concentration of GSNO and the level of S-nitrosylated proteins are regulated by GSNO reductase, which seems to play a major role in NO signalling. To investigate the importance of NO in plant defense response, we performed a proteomic analysis of Arabidopsis wildtype and GSNO-reductase knock-out plants infected with both the avirulent and virulent pathogen strains of Pseudomonas syringae. Using 2-D DIGE technology in combination with MS, we identified proteins, which are differentially accumulated during the infection process. We observed that both lines were more resistant to avirulent infections than to virulent infections mainly due to the accumulation of stress-, redox-, and defense-related proteins. Interestingly, after virulent infections, we also observed accumulation of defense-related proteins, but no or low accumulation of stress- and redox-related proteins, respectively. In summary, we present here the first detailed proteomic analysis of plant defense response. AU - Holzmeister, C.F. AU - Fröhlich, A. AU - Sarioglu, H. AU - Bauer, N. AU - Durner, J. AU - Lindermayr, C. C1 - 4270 C2 - 28827 SP - 1664-1683 TI - Proteomic analysis of defense response of wildtype Arabidopsis thaliana and plants with impaired NO-homeostasis. JO - Proteomics VL - 11 IS - 9 PB - Wiley-VCH Verl. PY - 2011 SN - 1615-9853 ER - TY - JOUR AB - Recent advances in experimental technologies allow for the detection of a complete cell proteome. Proteins that are expressed at a particular cell state or in a particular compartment as well as proteins with differential expression between various cells states are commonly delivered by many proteomics studies. Once a list of proteins is derived, a major challenge is to interpret the identified set of proteins in the biological context. Protein-protein interaction (PPI) data represents abundant information that can be employed for this purpose. However, these data have not yet been fully exploited due to the absence of a methodological framework that can integrate this type of information. Here, we propose to infer a network model from an experimentally identified protein list based on the available information about the topology of the global PPI network. We propose to use a Monte Carlo simulation procedure to compute the statistical significance of the inferred models. The method has been implemented as a freely available web-based tool, PPI spider (http://mips.helmholtz-muenchen.de/proj/ppispider). To support the practical significance of PPI spider, we collected several hundreds of recently published experimental proteomics studies that reported lists of proteins in various biological contexts. We reanalyzed them using PPI spider and demonstrated that in most cases PPI spider could provide statistically significant hypotheses that are helpful for understanding of the protein list. AU - Antonov, A.V. AU - Dietmann, S. AU - Rodchenkov, I. AU - Mewes, H.-W. C1 - 77 C2 - 27007 SP - 2740-2749 TI - PPI spider: A tool for the interpretation of proteomics data in the context of protein-protein interaction networks. JO - Proteomics VL - 9 IS - 10 PB - Wiley-VCH PY - 2009 SN - 1615-9853 ER - TY - JOUR AB - Members of the transforming growth factor (TGF)-beta superfamily are key regulators of lung development and homeostasis, in particular by controlling alveolar/bronchial epithelial cell function. TGF-beta signaling involves ligand-dependent activation of receptor serine/threonine kinases, activation and subsequent nuclear translocation of pathway-specific transcription factors (Smads), and ultimately, modulation of gene expression. While Smad-dependent responses represent the primary signaling components activated by TGF-beta receptors, their function is controlled by a variety of cofactors. In addition, alternative signaling systems mediating TGF-beta-induced effects have recently been described such as MAP kinase pathways. To uncover novel proteins that participate in TGF-beta signaling via nuclear/cytoplasmic shuttling in lung epithelial cells, we have analyzed A549 human lung epithelial cells, using subcellular fractionation combined with 2-D PAGE, tryptic digestion, and MS. We identified a rapid increase in the cytosolic localization of KH-type splicing regulatory protein (KHSRP), far upstream element-binding protein (FUBP1), hnRNP-L, and hnRNP-H1, concomitant with a decrease in their nuclear localization in response to TGF-beta 1. Proteomic data were confirmed by immunofluorescence and immunoblot analyses. In summary, we represent a powerful novel technology for the identification of previously unknown signaling intermediates. AU - Milosevic, J.* AU - Bulau, P.* AU - Mortz, E.* AU - Eickelberg, O. C1 - 1497 C2 - 26817 SP - 1230-1240 TI - Subcellular fractionation of TGF-β1-stimulated lung epithelial cells: A novel proteomic approach for identifying signaling intermediates. JO - Proteomics VL - 9 IS - 5 PB - Wiley-VCH PY - 2009 SN - 1615-9853 ER - TY - JOUR AB - We have correlated transcriptomics, proteomics and toponomics analyses of hippocampus tissue of inbred C57BL/6 mice to analyse the interrelationship of expressed genes and proteins at different levels of organization. We find that transcriptome and proteome levels of function as well as the topological organization of synaptic protein clusters, detected by toponomics at physiological sites of hippocampus CA3 region, are all largely conserved between different mice. While the number of different synaptic states, characterized by distinct synaptic protein clusters, is enormous (>155,000), these states together form synaptic networks defining distinct and mutually exclusive territories in the hippocampus tissue. The findings provide insight in the systems biology of gene expression on transcriptome, proteome and toponome levels of function in the same brain subregion. The approach will lay the ground for designing studies of neurodegeneration in mouse models and human brains. AU - Bode, M.* AU - Irmler, M. AU - Friedenberger, M.* AU - May, C.* AU - Jung, K.* AU - Stephan, C.* AU - Meyer, H.E.* AU - Lach, C. AU - Hillert, R.* AU - Krusche, A.* AU - Beckers, J. AU - Marcus, K.* AU - Schubert, W.* C1 - 3622 C2 - 25171 SP - 1170-1178 TI - Interlocking transcriptomics, proteomics and toponomics technologies for brain tissue analysis in murine hippocampus. JO - Proteomics VL - 8 IS - 6 PB - Wiley-VCH PY - 2008 SN - 1615-9853 ER - TY - JOUR AU - Hamacher, M.* AU - Herberg, F.* AU - Ueffing, M. AU - Meyer, H.E. C1 - 2303 C2 - 25771 SP - 1116-1117 TI - Seven successful years of Omics research: The Human Brain Proteome Project within the National German Research Network (NGFN). JO - Proteomics VL - 8 IS - 6 PB - Wiley-VCH PY - 2008 SN - 1615-9853 ER - TY - JOUR AB - Mouse embryonic brain development involves sequential differentiation of multipotent progenitors into neurons and glia cells. Using microarrays and large 2-DE, we investigated the mouse brain transcriptome and proteome of embryonic days 9.5, 11.5, and 13.5. During this developmental period, neural progenitor cells shift from proliferation to neuronal differentiation. As expected, we detected numerous expression changes between all time points investigated, but interestingly, the rate of alteration remained in a similar range within 2 days of development. Furthermore, up- and down-regulation of gene products was balanced at each time point which was also seen at embryonic days 16-18. We hypothesize that during embryonic development, the rate of gene expression alteration is rather constant due to limited cellular resources such as energy, space, and free water. A similar complexity in terms of expressed genes and proteins suggests that changes in relative concentrations rather than an increase in the number of gene products dominate cellular differentiation. In general, expression of metabolism and cell cycle related gene products was down-regulated when precursor cells switched from proliferation to neuronal differentiation (days 9.5-11.5), whereas neuron specific gene products were up-regulated. A detailed functional analysis revealed their implication in differentiation related processes such as rearrangement of the actin cytoskeleton as well as Notch- and Wnt-signaling pathways. AU - Hartl, D.* AU - Irmler, M. AU - Römer, I.* AU - Mader, M.T. AU - Mao, L.* AU - Zabel, C.* AU - Hrabě de Angelis, M. AU - Beckers, J. AU - Klose, J.* C1 - 2659 C2 - 25173 SP - 1257-1265 TI - Transcriptome and proteome analysis of early embryonic mouse brain development. JO - Proteomics VL - 8 IS - 6 PB - Wiley-VCH PY - 2008 SN - 1615-9853 ER - TY - JOUR AB - A major aim of the Human Brain Proteome Project (HBPP) is a better understanding of the molecular etiology and progression of neurodegenerative diseases. Transgenic and loss-of-function mouse mutant lines (MMLs) serve as experimental models. Transcriptome and proteome regulate each other in a complex and controlled way, and their comparative analysis is an essential aspect. As a fundamental study, we have assessed transcript profiles using a microarray containing 21 000 cDNA probes in a series of disease models within the German Mouse Clinic (GMC). Seventeen distinct organs of one adult stage were systematically collected for each submitted MML. Samples for gene expression profiling are individually selected based on conspicuous phenotypes in at least one of 14 GMC phenotype screens or on previous knowledge of the mutant phenotype. By microarray experiments expression patterns of 90 organs from 46 MMLs were analysed, identifying up to 232 differentially expressed genes in 45 organs. Here we present an overview of the results of all MMLs analysed and demonstrate the efficiency of systematic genome-wide expression profiling for the detection of molecular phenotypes in organs of a mammalian model organism. We identify the recurring regulation of particular genes and groups of coexpressed genes in apparently unrelated MMLs. AU - Horsch, M. AU - Schädler, S. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Meyer, H.* AU - Hrabě de Angelis, M. AU - Beckers, J. C1 - 2658 C2 - 25172 SP - 1248-1256 TI - Systematic gene expression profiling of mouse model series reveals coexpressed genes. JO - Proteomics VL - 8 IS - 6 PB - Wiley-VCH PY - 2008 SN - 1615-9853 ER - TY - JOUR AB - A major challenge towards a comprehensive analysis of biological systems is the integration of data from different omics sources and their interpretation at a functional level. Here we address this issue by analysing transcriptomic and proteomic datasets from mouse brain tissue at embryonic days 9.5 and 13.5. We observe a high concordance between transcripts and their corresponding proteins when they were compared at the level of expression ratios between embryonic stages. Absolute expression values show marginal correlation. We show in examples, that poor concordance between protein and transcript expression is in part explained by the fact, that single genes give rise to multiple transcripts and protein variants. The integration of transcriptomic and proteomic data therefore requires proper handling of such ambiguities. A closer inspection of such cases in our datasets suggests, that comparing gene expression at exon level instead of gene level could improve the comparability. To address the biological relevance of differences in expression profiles, literature-data mining and analysis of gene ontology terms are widely used. We show here, that this can be complemented by the inspection of physical properties of genes, transcripts, and proteins. AU - Irmler, M. AU - Hartl, D.* AU - Schmidt, T.* AU - Schuchhardt, J.* AU - Lach, C. AU - Meyer, H.E.* AU - Hrabě de Angelis, M. AU - Klose, J.* AU - Beckers, J. C1 - 3621 C2 - 25170 SP - 1165-1169 TI - An approach to handling and interpretation of ambiguous data in transcriptome and proteome comparisons. JO - Proteomics VL - 8 IS - 6 PB - Wiley-VCH PY - 2008 SN - 1615-9853 ER - TY - JOUR AB - BrainProfileDB is a database system for integrating large sets of high throughput functional genomics data of the Human Brain Proteome Project (HBPP). Within HBPP (http://www.smp-proteomics.de/) the molecular pathology of neurodegenerative diseases is investigated, using complementary methods from transcriptomics, proteomics, toponomics and interaction measurements. Aim of the database system is to provide a broad spectrum of scientific users joined in the consortium with a practical integrated view on their data. Employing appropriate mapping techniques and levels of data representation the user is relieved from technical details of gene identification or experimental measurement technique. AU - Schuchhardt, J.* AU - Glintschert, A.* AU - Hartl, D.* AU - Irmler, M. AU - Beckers, J. AU - Stephan, C.* AU - Marcus, K.* AU - Klose., J.* AU - Meyer, H.E.* AU - Malik, A.* C1 - 3540 C2 - 25169 SP - 1162-1164 TI - BrainProfileDB - a platform for integration of functional genomics data. JO - Proteomics VL - 8 IS - 6 PB - Wiley-VCH PY - 2008 SN - 1615-9853 ER - TY - JOUR AB - Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease. AU - Deeg, C.A.* AU - Altmann, F.* AU - Hauck, S.M. AU - Schoeffmann, S. AU - Amann, B.* AU - Stangassinger, M.* AU - Ueffing, M. C1 - 5084 C2 - 24479 SP - 1540-1548 TI - Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier. JO - Proteomics VL - 7 IS - 9 PB - Wiley-VCH PY - 2007 SN - 1615-9853 ER - TY - JOUR AB - Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3. AU - Gloeckner, C.J. AU - Boldt, K. AU - Schumacher, A. AU - Roepman, R.* AU - Ueffing, M. C1 - 3057 C2 - 25055 SP - 4228-4234 TI - A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. JO - Proteomics VL - 7 IS - 23 PB - Wiley-VCH PY - 2007 SN - 1615-9853 ER - TY - JOUR AB - The Wellcome Trust Conference Centre at Hinxton, UK, was the meeting place of the 7th HUPO Brain Proteome Project Workshop entitled High Performance Proteomics. It started on Wednesday, March 7, 2007 with a steering committee meeting followed by a two days series of talks dealing with the standardization and handling of tissues, body fluids as well as of proteomics data. The presentation and accompanying vivid discussions created a picture of actual strategies and standards in recent proteomics. AU - Hamacher, M.* AU - Stephan, C.* AU - Eisenacher, M.* AU - Lewczuk, P.* AU - Wiltfang, J.* AU - Martens, L.* AU - Vizcaíno, J.A.* AU - Kwon, K.H.* AU - Yoo, J.S.* AU - Park, Y.M.* AU - Beckers, J. AU - Horsch, M. AU - Hrabě de Angelis, M. AU - Cho, Z.H.* AU - Apweiler, R.* AU - Meyer, H.E.* C1 - 5844 C2 - 24537 SP - 2490-2496 TI - High Performance Proteomics: 7th HUPO Brain Proteome Project Workshop. JO - Proteomics VL - 7 IS - 15 PB - Wiley-VCH PY - 2007 SN - 1615-9853 ER - TY - JOUR AB - How is the yeast proteome wired? This important question, central in yeast systems biology, remains unanswered in spite of the abundance of protein interaction data from high-throughput experiments. Unfortunately, these large-scale studies show striking discrepancies in their results and coverage such that biologists scrutinizing the "interactome" are often confounded by a mix of established physical interactions, functional associations, and experimental artifacts. This stimulated early attempts to integrate the available information and produce a list of protein interactions ranked according to an estimated functional reliability. The recent publication of the results of two large protein interaction experiments and the completion of a comprehensive literature curation effort has more than doubled the available information on the wiring of the yeast proteome. This motivates a fresh approach to the compilation of a yeast interactome based purely on evidence of physical interaction. We present a procedure exploiting both heuristic and probabilistic strategies to draft the yeast interactome taking advantage of various heterogeneous data sources: application of tandem affinity purification coupled to MS (TAP-MS), large-scale yeast two-hybrid studies, and results of small-scale experiments stored in dedicated databases. The end result is WI-PHI, a weighted network encompassing a large majority of yeast proteins. AU - Kiemer, L.* AU - Costa, S. AU - Ueffing, M. AU - Cesareni, G.* C1 - 3769 C2 - 24660 SP - 932-943 TI - WI-PHI: A weighted yeast interactome enriched for direct physical interactions. JO - Proteomics VL - 7 IS - 6 PB - Wiley-VCH PY - 2007 SN - 1615-9853 ER - TY - JOUR AU - Blazek, E. AU - Meisterernst, M. C1 - 832 C2 - 23963 SP - 2070-2078 TI - A functional proteomics approach for the detection of nuclear proteins based on derepressed importin alpha. JO - Proteomics VL - 6 PB - Wiley-VCH PY - 2006 SN - 1615-9853 ER - TY - JOUR AU - Islinger, M.* AU - Lüers, G.H.* AU - Zischka, H. AU - Ueffing, M. AU - Völkl, A.* C1 - 2176 C2 - 23625 CY - Weinheim SP - 804-816 TI - Insights into the membrane proteome of rat liver peroxisomes: Microsomal glutathione-S-transferase is shared by both subcellular compartments. JO - Proteomics VL - 6 PB - Wiley-VCH PY - 2006 SN - 1615-9853 ER - TY - JOUR AU - Sarioglu, H. AU - Brandner, S. AU - Jacobsen, C. AU - Meindl, T.* AU - Schmidt, A.* AU - Kellermann, J.* AU - Lottspeich, F.* AU - Andrae, U. C1 - 3285 C2 - 23861 SP - 2407-2421 TI - Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein lables. JO - Proteomics VL - 6 PB - Wiley-VCH PY - 2006 SN - 1615-9853 ER - TY - JOUR AU - Schmitt, S.* AU - Prokisch, H. AU - Schlunck, T.* AU - Camp II, D.G.* AU - Ahting, U. AU - Waizenegger, T.* AU - Scharfe, C.* AU - Meitinger, T. AU - Imhof, A.* AU - Neupert, W.* AU - Oefner, P.J.* C1 - 1913 C2 - 23452 SP - 72-80 TI - Proteome analysis of mitochondrial outer membrane from Neurospora crassa. JO - Proteomics VL - 6 PB - Wiley-VCH PY - 2006 SN - 1615-9853 ER - TY - JOUR AU - Facius, A. AU - Englbrecht, C.C. AU - Birzele, F. AU - Groscurth, A. AU - Benjamin, S. AU - Wanka, S. AU - Mewes, H.-W. C1 - 3129 C2 - 23420 SP - 76-80 TI - PRIME: A graphical interface for integrating genomic/proteomic databases. JO - Proteomics VL - 5 PB - Wiley-VCH PY - 2005 SN - 1615-9853 ER - TY - JOUR AU - Hauck, S.M. AU - Schoeffmann, S. AU - Deeg, C.A.* AU - Gloeckner, C.J. AU - Swiatek-de Lange, M. AU - Ueffing, M. C1 - 4478 C2 - 23183 SP - 3623-3636 TI - Proteomic analysis of the porcine interphotoreceptor matrix. JO - Proteomics VL - 5 PB - Wiley-VCH PY - 2005 SN - 1615-9853 ER - TY - JOUR AU - Klein, C.* AU - Garcia-Rizo, C.* AU - Bisle, B.* AU - Scheffer, B.* AU - Zischka, H. AU - Pfeiffer, F.* AU - Siedler, F.* AU - Oesterhelt, D.* C1 - 3799 C2 - 22913 SP - 180-197 TI - The membrane proteome of Halobacterium salinarum. JO - Proteomics VL - 5 PB - Wiley-VCH PY - 2005 SN - 1615-9853 ER - TY - JOUR AB - Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability. AU - Zischka, H. AU - Gloeckner, C.J. AU - Klein, C.* AU - Willmann, S. AU - Swiatek-de Lange, M. AU - Ueffing, M. C1 - 2800 C2 - 22267 SP - 3776-3782 TI - Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction. JO - Proteomics VL - 4 IS - 12 PB - Wiley-VCH PY - 2004 SN - 1615-9853 ER - TY - JOUR AB - The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles. AU - Zischka, H. AU - Weber, G.* AU - Weber, P.J.* AU - Posch, A.* AU - Braun, R.J. AU - Bühringer, D.* AU - Schneider, U.* AU - Nissum, M.* AU - Meitinger, T. AU - Ueffing, M. AU - Eckerskorn, C.* C1 - 22437 C2 - 31082 SP - 906-916 TI - Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. JO - Proteomics VL - 3 IS - 6 PB - Wiley-VCH PY - 2003 SN - 1615-9853 ER -