A Mass Spectrometry-Based Method For Quantification Of Human Metallothionein Isoforms And Usefulness In Biomarker Detection Of Cancers And Metal Toxicity

<p>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. </p> <p>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. </p> <p>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.</p> <p>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.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biomedical Sciences

Year

2013

Author dc:creator

• Mehus, Aaron Andrew

Contributors dc:contributor

• Scott Garrett

• Breast Cancer, Cadmium, MALDI, Mass Spectrometry, Metallothionein, Protein Isoforms

Repository record dc:identifier

https://commons.und.edu/theses/1454

OAI identifier oai:identifier

oai:commons.und.edu:theses-2455