Article

RCADiA: simple automation platform for comparative multidimensional protein identification technology.

Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, SR11, La Jolla, California 92037, USA.
Analytical Chemistry (impact factor: 5.86). 09/2007; 79(16):6410-8. DOI:10.1021/ac070585g pp.6410-8
Source: PubMed

ABSTRACT Multidimensional liquid chromatography in combination with tandem mass spectrometry has been used to analyze a variety of biological structures including protein complexes. Incorporating this approach with autosampling devices presents a number of problems including decreased sensitivity due to exposure to extra surfaces, carryover from run to run, and increased dead volume. We developed a device, termed Radial Column Array for Distribution and Automation (RCADiA), to automate multiple MuDPIT experiments while eliminating many of these problems and maintaining a high resolution and sensitive analysis. The design, which places each sample downstream of any common fluid path, presents a low risk of carryover between successive analyses. Beyond the convenience of automation, the RCADiA platform also produces data of similar quality to the standard method of performing individual MuDPIT experiments. We demonstrate this device by performing a comparative analysis of mitochondria enriched from rat liver and spinal cord.

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    Article: The influence of nickel and cobalt on putative members of the oxygen-sensing pathway of erythropoietin-producing HepG2 cells.
    T Porwol, W Ehleben, K Zierold, J Fandrey, H Acker
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    ABSTRACT: Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 microM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 microM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three-dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The .OH levels decreased in cobalt-treated (to 81%) as well as in nickel-treated (to 67%) HepG2 cells, accompanied by an increase of EPO expression to 167% and 150%, respectively. Our results underline the importance of .OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins.
    European Journal of Biochemistry 09/1998; 256(1):16-23. · 3.58 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

automate multiple MuDPIT experiments
 
Automation
 
autosampling devices presents
 
common fluid path
 
comparative analysis
 
convenience
 
individual MuDPIT experiments
 
mitochondria enriched
 
Multidimensional liquid chromatography
 
protein complexes
 
Radial Column Array
 
RCADiA
 
RCADiA platform
 
sample downstream
 
sensitive analysis
 
similar quality
 
spinal cord
 
standard method
 
successive analyses