SEC-ICP-MS and ESI-MS/MS for analyzing in vitro and in vivo Cd-phytochelatin complexes in a Cd-hyperaccumulator Brassica chinensis
In this study, in vitro synthesized Cd-phytochelatin (PC) complexes and in vivo Cd–PC complexes in Cd-stressed Brassica chinensis, which has been identified as a Cd hyperaccumulator, were characterized using SEC-ICP-MS and ESI-MS/MS. The PCs (n = 1–5) obtained from Cd-stressed B. chinensis together with CdCl2 were used to synthesize the in vitro Cd–PC complexes, and the formation of CdGS1–2, (CdGS)2, Cd1–2PC2, Cd1–3PC3, Cd1–3PC4 and Cd1–3PC5 was observed. In addition, for the first time, in vivo CdPC3 and CdPC4 complexes, as well as Cd-free PCs (n = 2–5) and desGlu-PC3 were detected in the extracts of Cd-stressed B. chinensis and confirmed by means of their corresponding isotopic peak distribution and MS/MS spectra. Nitrogen saturated ammonium bicarbonate buffer (pH 7.8), instead of Tris-HCl and phosphate buffer, was used as a suitable mobile phase in order to stabilize the Cd–PC complexes and effectively avoid possible oxidation of PC analogues during SEC fractionation. Results obtained in this study give definite evidence elucidating the important roles which PCs play in plant defensive mechanisms to Cd stress. SEC-ICP-MS and ESI-MS/MS were demonstrated as powerful and promising techniques for screening and identifying the in vivo metallopeptides, with accurate isotopic distribution assignment, in metal toxicological studies.