Date of Award

January 2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Scott Garrett

Abstract

Current methods for detecting metallothionein (MT) protein expression lack the specificity to distinguish between all twelve human isoforms. Each, however, can be distinguished by the masses of their acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a mass spectrometry-based method for their simultaneous quantification.

Human kidney HK-2 epithelial cells expressing recombinant MT-3 were grown in the presence or absence of cadmium. Cytosolic proteins were alkylated with 14N- or 15N-iodoacetamide and digested with trypsin. The N-terminal MT peptides were enriched by two-dimensional liquid chromatography and analyzed by MALDI-TOF/TOF mass spectrometry. Relative expression was measured by combining 14N-labeled control and 15N-labeled cadmium-treated cytosols before trypsin digestion and determining monoisotopic peak ratios of the targeted peptides. Absolute quantification was accomplished with 15N-labeled synthetic peptides.

Seven isoforms (MT-1E, MT-1F, MT-1G2, MT-1M, MT-1X, MT-2, and MT-3) were detected and quantified. The dynamic range was sufficient to detect 67-fold differences in abundance and 12-fold induction for some isoforms. Combined, MTs represented 0.3% to 1.5% of total cytosolic protein. The mRNA isoform expression levels differed in both the rank order and fold induction.

The assay was also applied to four malignant (MCF-7, Hs578T, T-47D, and MDA-MB-231) and one non-malignant (MCF-10A) human breast cell lines. The malignant cell lines were either estrogen receptor positive (ER+) or estrogen receptor negative (ER-). Three MT isoforms (MT-2, MT-1E, and MT-1X) were quantified in the breast cells. In regards to the ER status of the cells, the ER+ malignant cells had low MT protein levels while the ER- malignant cells had a significant overexpression of MTs compared to the control. When comparing the ER+ to ER- cells, there was between a 5.5 - 14.5 fold increase in protein expression of the individual MT isoforms or a 38-fold difference in abundance between all MT isoforms. This method expands the usefulness of human MT isoforms as potential biomarkers for specific diseases or environmental exposures to heavy metals.

Download

Share

COinS