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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Mehus, Aaron Andrew"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Biomedical Sciences","degree_department":null,"school":null,"contributors":["Scott Garrett"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2013,"date_issued":"2013-01-01T08:00:00Z","date_published":"2013-01-01T08:00:00Z","updated_at":"2026-07-24T03:26:24Z","subjects":["Breast Cancer, Cadmium, MALDI, Mass Spectrometry, Metallothionein, Protein Isoforms"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://commons.und.edu/theses/1454","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Scott Garrett"]},{"key":"dc:creator","label":"Author","values":["Mehus, Aaron Andrew"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_discipline","label":"Discipline","values":["Biomedical Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Breast Cancer, Cadmium, MALDI, Mass Spectrometry, Metallothionein, Protein Isoforms"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://commons.und.edu/theses/1454"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["A Mass Spectrometry-Based Method For Quantification Of Human Metallothionein Isoforms And Usefulness In Biomarker Detection Of Cancers And Metal Toxicity"]}]}],"canonical_facts":{"dc:contributor":["Scott Garrett"],"dc:creator":["Mehus, Aaron Andrew"],"dc:description.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.
"],"dc:identifier":["https://commons.und.edu/theses/1454"],"dc:subject":["Breast Cancer, Cadmium, MALDI, Mass Spectrometry, Metallothionein, Protein Isoforms"],"dc:title":["A Mass Spectrometry-Based Method For Quantification Of Human Metallothionein Isoforms And Usefulness In Biomarker Detection Of Cancers And Metal Toxicity"],"thesis:degree_discipline":["Biomedical Sciences"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T03:26:24Z"}