Improved proteome analysis of <b><i>Saccharomyces cerevisiae</i></b> mitochondria by free‐flow electrophoresis

Zischka, Hans; Weber, Gerhard; Weber, Philipp; Posch, Anton; Braun, Ralf J.; Bühringer, Dietmute; Schneider, Ulrich; Nissum, Mikkel; Meitinger, Thomas; Ueffing, Marius; Eckerskorn, Christoph

doi:10.1002/pmic.200300376

Cited by 145 publications

(97 citation statements)

References 30 publications

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“…A second possibility is that Fd-GOGAT is present in a subset of a heterogeneous mitochondrial population and that this Fd-GOGAT-containing subset is lost during FFE purification. For example, distinct yeast mitochondrial populations originating from different biological conditions or experimental manipulations can be resolved by FFE and as little as deletion of a single outer-membrane protein can yield a mitochondrial fraction of distinct mobility in FFE (Zischka et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

et al. 2009

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The dual affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase for O 2 and CO 2 results in the net loss of fixed carbon and energy in a process termed photorespiration. The photorespiratory cycle is complex and occurs in three organelles, chloroplasts, peroxisomes, and mitochondria, which necessitates multiple steps to transport metabolic intermediates. Genetic analysis has identified a number of mutants exhibiting photorespiratory chlorosis at ambient CO 2 , including several with defects in mitochondrial serine hydroxymethyltransferase (SHMT) activity. One class of mutants deficient in SHMT1 activity affects SHM1, which encodes the mitochondrial SHMT required for photorespiration. In this work, we describe a second class of SHMT1-deficient mutants defective in a distinct gene, GLU1, which encodes Ferredoxin-dependent Glutamate Synthase (Fd-GOGAT). Fd-GOGAT is a chloroplastic enzyme responsible for the reassimilation of photorespiratory ammonia as well as for primary nitrogen assimilation. We show that Fd-GOGAT is dual targeted to the mitochondria and the chloroplasts. In the mitochondria, Fd-GOGAT interacts physically with SHMT1, and this interaction is necessary for photorespiratory SHMT activity. The requirement of protein-protein interactions and complex formation for photorespiratory SHMT activity demonstrates more complicated regulation of this crucial high flux pathway than anticipated.

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Section: Discussionmentioning

confidence: 99%

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

et al. 2009

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“…The group of Lubman [23,24] developed a method for the fractionation of complex protein samples by a combination of IEF or chromatofocusing and RP-LC. Other groups have used alternative methods for prefractionation, such as CE, affinity chromatography, and ion-exchange chromatography [4,21,22].…”

Section: Introductionmentioning

confidence: 99%

“…The purity of organelles and other protein complexes is crucial to subcellular proteome research [13][14][15][16][17]. For the isolation of subcellular structures and organelles several methods, such as differential-and density-gradient centrifugation [18][19][20], immunoisolation [8], affinity purification, [21], and freeflow electrophoresis [22], have been applied. It has been shown that such fractionations improved identification of proteins from targeted subcellular structures [8,10,[12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

et al. 2005

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Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteinsA model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion-and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

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Section: Introductionmentioning

confidence: 99%

“…3,7 μ-FFE's larger-scale, preparative counterpart has proven useful in separating a range of analytes, including cells, 8,9 cellular components, 10-14 and proteins. [15][16][17] In the near future, μ-FFE could be useful in analysis or micropreparative separations of the same analytes.Recently several researchers have investigated a variety of fabrication methods for μ-FFE devices to improve their performance. [4][5][6][18][19][20] In early designs two major problems were encountered.…”

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confidence: 99%

Fast Electrophoretic Separation Optimization Using Gradient Micro Free-Flow Electrophoresis

Fonslow

Bowser

2008

Anal. Chem.

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The continuous nature of micro free-flow electrophoresis (μ-FFE) was used to monitor the effect of a gradient of buffer conditions on the separation. This unique application has great potential for fast optimization of separation conditions and estimation of equilibrium constants. COMSOL was used to model pressure profiles in the development of a new μ-FFE design that allowed even application of a buffer gradient across the separation channel. The new design was fabricated in an all glass device using our previously published multiple-depth etch method (Fonslow, B. R.; Barocas, V. H.; Bowser, M. T. Anal. Chem. 2006, 78, 5369-5374, ref 1 ). Fluorescein solutions were used to characterize the applied gradients in the separation channel. Linear gradients were observed when buffer conditions were varied over a period of 5-10 min. The effect of a gradient of 0-50 mM hydroxypropyl-β-cyclodextrin (HP-β-CD) on the separation of a group of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) labeled primary amines was monitored as a proof of concept experiment. Direct comparisons to capillary electrophoresis (CE) separations performed under the same conditions were made. Gradient μ-FFE recorded 60 separations during a 5 min gradient allowing nearly complete coverage across a range of HP-β-CD concentrations. In comparison, 4 h were required to assess 15 sets of conditions across the same range of HP-β-CD concentrations using CE. Qualitatively, μ-FFE separations were predictive of the migration order and spacing of peaks in CE electropherograms measured under the same conditions. Data were fit to equations describing 1:1 analyte-additive binding to allow a more quantitative comparison between gradient μ-FFE and CE.Micro free-flow electrophoresis (μ-FFE) is an analytical separation technique used to separate a continuously flowing stream of charged analytes. 2,3 A thin sample stream is introduced into a planar separation channel with buffer running in parallel. An electric field is applied perpendicularly across the separation channel, and charged analytes are deflected laterally based on their electrophoretic mobility. Thus far, μ-FFE separations have been limited to simple separations of fluorescent dyes, 4-6 fluorescently labeled amino acids, 2 and fluorescently labeled proteins. 3,7 μ-FFE's larger-scale, preparative counterpart has proven useful in separating a range of analytes, including cells, 8,9 cellular components, 10-14 and proteins. [15][16][17] In the near future, μ-FFE could be useful in analysis or micropreparative separations of the same analytes.Recently several researchers have investigated a variety of fabrication methods for μ-FFE devices to improve their performance. [4][5][6][18][19][20] In early designs two major problems were encountered. Inefficient removal of electrolysis products, manifested as bubbles, from the electrode channels degraded separations. 4 Also, channel designs used to minimize the effects of the bubbles reduced the electric field applied in the separation channel. 5,6 These probl...

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Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free‐flow electrophoresis

Cited by 145 publications

References 30 publications

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

Arabidopsis Photorespiratory Serine Hydroxymethyltransferase Activity Requires the Mitochondrial Accumulation of Ferredoxin-Dependent Glutamate Synthase

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

Fast Electrophoretic Separation Optimization Using Gradient Micro Free-Flow Electrophoresis

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