Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability.
To further our knowledge of intracellular copper transport, we used a proteomics strategy to search for hepatic proteins with copper-binding ability. Hep G2 cytosolic and microsomal fractions were applied to a copper(II)-loaded immobilized metal-affinity chromatography (IMAC) column. Protein identification was performed with 2-D gel electrophoresis and mass spectrometry. We identified 48 cytosolic proteins and 19 microsomal proteins displaying copper-binding ability. These proteins are diverse in function. Fifty-two of the 67 proteins contain putative metal-binding domains. We have identified many components of the Hep G2 copper metalloproteome including a large number of proteins not previously known to bind copper.
Smith
Scott D
SD
Department of Structural Biology and Biochemistry, The Hospital for Sick Children Research Institute, Toronto, Canada.
She
Yi-Min
YM
Roberts
Eve A
EA
Sarkar
Bibudhendra
B
D002352:Carrier Proteins / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D022781:Hepatocytes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008667:Metalloproteins / Q000032:analysis*; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D008667:Metalloproteins; D020543:Proteome; C023548:copper-binding protein; D003300:Copper