Separation of metallothionein isoforms extracted from isoform-specific knockdown cells on two-dimensional micro high-performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry

Abstract

A two-dimensional (2D) micro HPLC-inductively coupled plasma mass spectrometry (ICP-MS) system consisting of a gel filtration column and an anion-exchange column was constructed to separate two metallothionein (MT) isoforms. MT-I and MT-II genes were specifically knocked down with the RNA interference (RNAi) technique, and the effects of the knockdown were evaluated by the reverse transcription-polymerase chain reaction (RT-PCR) and the 2D micro HPLC-ICP-MS system. The minimum cell number required by our 2D micro HPLC-ICP-MS system was calculated to be 2.0 × 10³, and the detection limit of MT protein was estimated to be 22.2 μg L⁻¹.