Identification of arsenic-binding proteins in human cells by affinity chromatography and mass spectrometry.
Exposure to high levels of arsenic can cause a wide range of health effects, including cancers of the bladder, lung, skin, and kidney. However, the mechanism(s) of action underlying these deleterious effects of arsenic remains unclear. Arsenic binding to cellular proteins is a possible mechanism of toxicity, and identifying such binding is analytically challenging because of the large concentration range and variety of proteins. We describe here an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or having cysteine blocked showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind to arsenic. The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurity in the commercial preparation of carbonic anhydrase II, demonstrating the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. Application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the nuclear fraction, and 24 proteins in the membrane/organelle fraction that could bind to arsenic, adding to the current list of only a few known arsenic-binding proteins.
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G3.
Huiming
H
Yan
Nan
N
Wang
Michael
M
Weinfeld
William R
WR
Cullen
X Chris
XC
Le
D000595:Amino Acid Sequence; D001151:Arsenic / Q000737:chemistry*; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data
D002352:Carrier Proteins; D001151:Arsenic