HPLC-ICP-MS and ESI-Q-TOF analysis of biomolecules induced in Brassica juncea during arsenic accumulation

Abstract

Arsenic (As) bioaccumulation by plants can be used as a strategy to detoxify arsenic polluted sites. Genetic engineering may provide a means of optimizing this natural process to increase its efficiency. However, this approach requires a thorough understanding of As metabolism and detoxification in plants. Identifying As-containing metabolites in plants is an important first step in elucidating As metabolism. Brassica juncea (Indian mustard) is studied here as a model for As accumulation in terms of total metalloid accumulation and its elemental speciation. A study on extraction conditions using 25 mM ammonium acetate buffer at increasing pH of 4.4, 5.6 and 7.8 has been performed. Those extracting solutions were also employed as mobile phases for the separation of the As species formed by size exclusion chromatography with inductively coupled plasma mass spectrometry (ICP-MS) as a selective As detector. Two main As containing species have been found in Brassica tissues (one of them at about 2 kDa and the other below 1.2 kDa). The first As species was found to be associated to thiol groups (monitoring 32S with double focusing ICP-MS). This can be ascribed to the presence of As-phytochelatin complexes. Electrospray-quadrupole-time of flight (ESI-Q-TOF) results indicated the presence of phytochelatins (apo-forms), the main metal bioligands in plants, which have also been shown to be induced by As. Oligomers of two, three and four sub-units, respectively (PC2, PC3 and PC4), with internal oxidation of the SH groups, have been extracted from Brassica leaves as well as a potential As–PC4 complex. These species have been further identified by collisional induced dissociation (CID).