Proteomic survey of copper-binding proteins inArabidopsis roots by immobilized metal affinity chromatography and mass spectrometry

PROTEOMICS ◽  
2006 ◽  
Vol 6 (9) ◽  
pp. 2746-2758 ◽  
Author(s):  
Cheng-Che S. Kung ◽  
Wei-Ning Huang ◽  
Yan-Chen Huang ◽  
Kuo-Chen Yeh
Keyword(s):  
Mass Spectrometry ◽  
Affinity Chromatography ◽  
Binding Proteins ◽  
Immobilized Metal Affinity Chromatography ◽  
Copper Binding ◽  
Metal Affinity ◽  
Copper Binding Proteins

scholarly journals Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC)

2005 ◽  
Vol 388 (1) ◽  
pp. 371-378 ◽  
Author(s):  
Henrik MÜLLER ◽  
Alexander STROM ◽  
Gerhard HUNSMANN ◽  
Andreas W. STUKE
Keyword(s):  
Affinity Chromatography ◽  
Prion Protein ◽  
Cellular Prion Protein ◽  
Immobilized Metal Affinity Chromatography ◽  
Proteinase K ◽  
Transmissible Spongiform Encephalopathies ◽  
Copper Binding ◽  
Metal Affinity ◽  
Binding Forms ◽  
Ionic Detergent

The conformational conversion of the normal cellular prion protein (PrPC) into the pathology-associated PrPSc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrPC molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrPC, PrPSc is insoluble in non-ionic detergents and does not bind to Cu2+ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-β-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu2+-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrPC and denatured PrPSc were retained by a Cu2+-loaded resin, native PrPSc and PrPres [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrPC from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrPC from aggregated PrPSc in infected brains. Our results indicate that in contrast with PrPSc in uninfected as well as infected brains, PrPC is predominantly present in the glycosylated forms.

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