Identification of Arsenic-Binding Proteins in Human Cells by Affinity Chromatography and Mass Spectrometry

Abstract

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.

Synchrotron X-ray absorption spectroscopy analysis of arsenic chemical speciation in human nail clippings

“…[81] The list of more than 70 proteins extracted from lung cell cultures, that were reported to bind to arsenic, included a few cytoskeletal keratins of type I and type II. [82] In a recent shotgun proteomic analysis of the human nail plate, keratins and keratin-associated proteins comprised the largest part of the soluble fraction, which makes up almost 90 % of the organic mass of the human nail plate. However, many cross-linked cytoplasmic, membrane and junctional proteins and histones, together with keratins, were also identified in the insoluble fraction.…”

Mechanisms underlying the toxic effects of antimony species in human embryonic kidney cells (HEK-293) and their comparison with arsenic species

“…The binding of metals/metalloids to a protein and the protein aggregation induced by metals/metalloids have been studied (Uversky et al, 2001;Tamás et al, 2014), and As(III) is also known to complex with hemoglobin and other proteins (Lu et al, 2004;Yan et al, 2009;Zhang et al, 2007). Protein aggregates in yeast, even those of chaperone proteins, were suggested to the interact using the sulfhydryl groups of Cys (Jacobson et al, 2012).…”

Characterization of the role of protein–cysteine residues in the binding with sodium arsenite

“…Recently, a panel of arsenic-binding proteins (50 nuclear proteins and 24 membrane/organelle proteins) was identified from A549 human lung carcinoma cells using an improved affinity selection technique coupled with mass spectrometry (Yan et al 2009). However, the arsenicbinding proteins identified in our current study (RCN3, HSP27 and GAL1) were not among those identified in this previous report.…”