Simple Method for Identification of Metallothionein Isoforms in Cultured Human Prostate Cells by MALDI-TOF/TOF Mass Spectrometry

Abstract

The present paper describes a rapid method for identification and characterization of human metallothionein (MT) isoforms in complex cell cultures using high-resolution matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF). In the proposed method, the sample preparation of MTs from cultured cells is both simple and fast. It is accomplished by trypsin cleavage of cell proteins into small peptide species, the majority of which are subsequently removed by gel filtration using beads with an exclusion limit of 4000 Da. In contrast to most cell proteins, MTs remain intact (undigested) upon being treated with trypsin, being excluded by the gel beads and thus recovered by low-speed centrifugation. To identify the protein constitutes of the MT preparation, the MT sample is divided into two parts, one for intact protein accurate mass measurement, the other for tryptic digestion followed by MS and MS/MS analyses. In the latter case, the MT proteins are denatured by the addition of EDTA which strips heavy metals from MTs and renders them susceptible to tryptic digestion. The obtained accurate mass with the unique peptide sequences of each MT isoform allows for unambiguous identification of MT isoforms in the prepared mixture. The method has been applied to RWPE-1 cells derived from normal human prostate epithelium. Four MT isoforms, 1E, 1G, 1X, and 2A, have been confidently identified, being primarily acetylated at N-termini. These results are in agreement with the expression of MT mRNAs in RWPE-1 cells determined by real-time reverse-transcription polymerase chain reaction (RT-PCR).