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Exploration of unknown nickel-containing proteins from plants by liquid chromatography–inductively coupled plasma mass spectrometry

ABSTRACT Nickel (Ni) is an essential cofactor for various microbial enzymes, but in higher plants, few Ni-containing enzymes (e.g., urease) have been identified. Here, we explored the use of liquid chromatography–inductively coupled plasma mass spectrometry (ICPMS) to identify novel Ni-containing proteins in Arabidopsis thaliana. Water-soluble proteins were extracted from the roots of A. thaliana, and metal-containing proteins in the extracts were separated and detected by means of a two-dimensional procedure involving size-exclusion chromatography–ICPMS and anion-exchange chromatography–ICPMS. Using this procedure, we obtained five separated Ni peaks that were present both in the wild type and a Urease-defective mutant, indicating the presence of Ni-containing proteins other than urease. One of the Ni peaks showed a significant correlation with an iron (Fe) peak, suggesting that a protein contained in this peak incorporated both Ni and Fe. To identify this protein, we used liquid chromatography–tandem mass spectrometry to perform a proteome analysis of the Ni and Fe peak. Among the 269 proteins detected, we identified acireductone dioxygenase 2; the murine and bacterial acireductone dioxygenases have two distinct metalloforms containing either Ni or Fe. Thus, acireductone dioxygenase 2 is most likely a novel Ni-containing enzyme in A. thaliana. Our findings suggest that most of the Ni-containing proteins in higher plants remain uncharacterized.