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Title: IDENTIFICATION AND METAL-BINDING CHARACTERIZATION OF SHEEP METALLOTHIONEIN ISOFORMS BY LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONISATION MASS SPECTROMETRY

Author
item BEATTIE JOHN H - THE ROWETT RESEARCH INST
item LOMAX J A - THE ROWETT RESEARCH INST
item Richards, Mark
item SELF R - UNIVERSITY OF EAST ANGLIA
item PESCH R - FINNIGAN MAT GMBH
item MUNSTER H - FINNIGAN MAT GMBH

Submitted to: Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics
Publication Type: Proceedings
Publication Acceptance Date: 5/29/1994
Publication Date: N/A
Citation: N/A

Interpretive Summary: Trace elements such as zinc and copper are required to support growth and development in all animal species, including humans. There has been a great deal of interest in the scientific community in developing indicators of zinc and copper status. One such status indicator is a zinc- and copper-binding protein called metallothionein (MT). Most animal species, as well as, humans have a more than one functioning MT gene. Although many MT genes have been cloned and sequenced, accurate estimates of the size of the gene products (called MT isoforms) do not exist because of difficulties encountered in the purification of these proteins. The purpose of this study was to evaluate the effectiveness of high accuracy, high resolution electrospray mass spectrometry in determining the size of the various forms of MT (isoforms) form sheep liver. Four MT isoforms were detected and their masses accurately determined. From the data it was also possible to determine relative abundance of each isoform of MT, the nature of the metals bound to the isoforms and to detect a specifically modified type of MT (deacetylated). The results of this study emphasize the feasibility of coupling mass spectrometry with other specialized protein separation techniques such as high-performance liquid chromatography and capillary electrophoresis. Together, these results offer scientists working in the area of trace element metabolism a new analytical tool with which to study the relationship between MT isoforms and changes in trace element status.

Technical Abstract: Metallothionein (MT) is a class of small metal-binding protein whose mass can vary from under 6000 to over 7000 daltons depending on a) variation in non-conserved amino acid regions b) the degree of metal saturation and c) the species of metal bound. Many mammalian species have several functioning genes of MT and accurate mass values of the corresponding protein isoforms can be deduced from published gene sequences. However, the translated product of each gene has yet to be confirmed in purified MT protein samples because the isoforms can be difficult to separate by conventional chromatography or electrophoresis. In the case of sheep which have at least 4 functioning MT genes, the deduced N-terminally acetylated apoprotein masses are 5993 (MT-1a), 6023 (MT-1b), 6027 (MT-1c) and 6070 (MT-2). The objective of this work was to study the proteins by high accuracy, high resolution electrospray mass spectrometry and to confirm their predicted mass values.