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Pietsch J.,Otto Von Guericke University of Magdeburg | Sickmann A.,Leibniz Institute for Analytical Sciences | Weber G.,FFE Service GmbH | Bauer J.,Max Planck Institute of Biochemistry | And 5 more authors.
Proteomics | Year: 2012

Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Ma X.,University of Aarhus | Sickmann A.,Leibniz Institute for Analytical Sciences | Pietsch J.,Otto Von Guericke University of Magdeburg | Wildgruber R.,FFE Service GmbH | And 5 more authors.
Proteomics | Year: 2014

Proteomic changes of two types of human endothelial cells (ECs) were determined and compared to morphological alterations occurring during the scaffold-free in vitro formation of 3D structures resembling vascular intimas. The EA.hy926 cell line and human microvascular ECs (HMVECs) were cultured on a random positioning machine or static on ground (normal gravity) for 5 and 7 days, before their morphology was examined and their protein content was analysed by MS after free-flow electrophoretic separation. A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVEC forming 3D structures faster than the EA.hy926 cells. Five hundred and eighty-four of these kinds of proteins were present in both types of cells. They included a number of metabolic enzymes, of structure-related and stress proteins. Comparing proteins of EA.hy926 cells growing either adherently on ground or in 3D aggregates on the random positioning machine revealed that ribosomal proteins were enhanced, while tubes are formed and various components of 26S proteasomes remained prevalent in static normal gravity control cells only. The fast developing tube-like 3D structures of HMVEC suggested a transient augmentation of ribosomal proteins during the 3D assembling of ECs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Ma X.,University of Aarhus | Wildgruber R.,FFE Service GmbH | Bauer J.,Max Planck Institute of Biochemistry | Weber G.,FFE Service GmbH | And 4 more authors.
Electrophoresis | Year: 2012

Prefractionation of proteins enhances the resolution of proteome analysis of whole cells. Free-flow electrophoresis (FFE) provides a useful step in various prefractionation protocols, since matrix-free isoelectric focusing (FF-IEF) performed in this machine enables the enrichment of large, easily absorbable, sensitive proteins. The impact of the FFE on the success of a proteome analysis depends on the quality of the FF-IEF separation procedure. Therefore, attempts are continuously being made to improve FF-IEF. Here, we applied sigmoid pH gradients to the prefractionation of endothelial cell proteins. Small steps of pH incline between neighboring FFE fractions were established in pH ranges, in which the proteins of interest have their pIs. With the help of this advanced technology, we separated vimentin and cytoplasmic actin as well as triosephosphate isomerase and glyceraldehyde phosphate dehydrogenase preparatively, and found a pI of 5.9 ± 0.2 for nonmuscle myosin. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Pietsch J.,Free University of Berlin | Pietsch J.,Institute of Clinical Pharmacology and Toxicology | Sickmann A.,Leibniz Institute for Analytical Sciences | Weber G.,FFE Service GmbH | And 6 more authors.
Proteomics | Year: 2011

The human cell lines FTC-133 and CGTH W-1, both derived from patients with thyroid cancer, assemble to form different types of spheroids when cultured on a random positioning machine. In order to obtain a possible explanation for their distinguishable aggregation behaviour under equal culturing conditions, we evaluated a proteomic analysis emphasising cytoskeletal and membrane-associated proteins. For this analysis, we treated the cells by ultrasound, which freed up some of the proteins into the supernatant but left some attached to the cell fragments. Both types of proteins were further separated by free-flow IEF and SDS gel electrophoresis until their identity was determined by MS. The MS data revealed differences between the two cell lines with regard to various structural proteins such as vimentin, tubulins and actin. Interestingly, integrin α-5 chains, myosin-10 and filamin B were only found in FTC-133 cells, while collagen was only detected in CGTH W-1 cells. These analyses suggest that FTC-133 cells express surface proteins that bind fibronectin, strengthening the three-dimensional cell cohesion. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Wildgruber R.,FFE Service GmbH | Weber G.,FFE Service GmbH | Wise P.,University of Southern California | Grimm D.,University of Aarhus | Bauer J.,Max Planck Institute of Biochemistry
Proteomics | Year: 2014

An aim of proteome research is to identify the entire complement of proteins expressed in defined cell types of humans, animals, plants, and microorganisms. The approach requires searching for low abundant or even rarely expressed proteins in many cell types, as well as the determination of the protein expression levels in subcellular compartments and organelles. In recent years, rather powerful MS technologies have been developed. At this stage of MS device development, it is of highest interest to purify intact cell types or isolate subcellular compartments, where the proteins of interest are originating from, which determine the final composition of a peptide mixture. Free-flow electrophoresis proved to be useful to prepare meaningful peptide mixtures because of its improved capabilities in particle electrophoresis and the enhanced resolution in protein separation. Sample preparation by free-flow electrophoresis mediated particle separation was preferentially performed for purification of either organelles and their subspecies or major protein complexes. Especially, the introduction of isotachophoresis and interval zone electrophoresis improved the purity of the gained analytes of interest. In addition, free-flow IEF proved to be helpful, when proteins of low solubility, obtained, e.g. from cell membranes, were investigated. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Pietsch J.,Otto Von Guericke University of Magdeburg | Riwaldt S.,Otto Von Guericke University of Magdeburg | Bauer J.,Max Planck Institute of Biochemistry | Sickmann A.,Leibniz Institute for Analytical Sciences | And 6 more authors.
International Journal of Molecular Sciences | Year: 2013

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Justesen B.H.,Copenhagen University | Laursen T.,Copenhagen University | Weber G.,FFE Service GmbH | Fuglsang A.T.,Copenhagen University | And 2 more authors.
Analytical Chemistry | Year: 2013

Free flow electrophoresis is used for rapid and high-recovery isolation of homogeneous preparations of functionally active membrane proteins inserted into nanodiscs. The approach enables isolation of integral and membrane anchored proteins and is also applicable following introduction of, e.g., fluorescent tags. Preparative separation of membrane protein loaded nanodiscs from empty nanodiscs and protein aggregates results in monodisperse nanodisc preparations ideal for structural and functional characterization using biophysical methods. © 2013 American Chemical Society.


Patent
FFE Service GmbH | Date: 2015-01-07

A free-flow electrophoresis method for separating at least one analyte from a mixture of analytes is disclosed. The method comprises- flowing a separation medium through a separation chamber in a flow direction;- applying an electric field in the separation medium across an anode and a cathode, wherein the anode and the cathode are located at a distance from each other and the separation medium flows between the anode and the cathode - applying the mixture of analytes to the separation medium, whereupon the at least one analyte of interest is separated from the mixture of analytes,- collecting fractions of the separation medium with at least one fraction comprising the analyte of interest. The separation medium comprises two or more individual separation media, which differ in their pH value and form a pH step gradient between the anode and the cathode.


Patent
Ffe Service Gmbh | Date: 2014-07-04

The present invention is related to a free-flow electrophoresis method for separating at least one analyte of interest from a mixture of analytes, wherein the method uses a separation medium comprising two or more individual separation media, wherein the two or more individual separation media differ in their pH value, and wherein each of the two or more individual separation media comprise at least one anion of at least one acid and at least one cation of at least one base, wherein the at least one acid is the same in each of the two or more individual separation media and the at least one base is the same in each of the two or more individual separation media.


PubMed | FFE Service GmbH
Type: Journal Article | Journal: Proteomics | Year: 2014

An aim of proteome research is to identify the entire complement of proteins expressed in defined cell types of humans, animals, plants, and microorganisms. The approach requires searching for low abundant or even rarely expressed proteins in many cell types, as well as the determination of the protein expression levels in subcellular compartments and organelles. In recent years, rather powerful MS technologies have been developed. At this stage of MS device development, it is of highest interest to purify intact cell types or isolate subcellular compartments, where the proteins of interest are originating from, which determine the final composition of a peptide mixture. Free-flow electrophoresis proved to be useful to prepare meaningful peptide mixtures because of its improved capabilities in particle electrophoresis and the enhanced resolution in protein separation. Sample preparation by free-flow electrophoresis mediated particle separation was preferentially performed for purification of either organelles and their subspecies or major protein complexes. Especially, the introduction of isotachophoresis and interval zone electrophoresis improved the purity of the gained analytes of interest. In addition, free-flow IEF proved to be helpful, when proteins of low solubility, obtained, e.g. from cell membranes, were investigated.

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