Metallothionein isoform analysis by liquid chromatography-electrospray ionisation mass spectrometry.

Abstract

Metallothionein (MT) is a class of small metal-binding proteins [ l ] whose mass can vary from under 6OOO 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 [2], the deduced N-terminally acetylated apoprotein masses are 5993 (MT-la), 6023 (MT-lb), 6027 (MT-lc) 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. Metallothionein was purified from the liver of a gray-faced sheep which had been injected with Zn. For LC-MS studies, an LDC 4100 MS gradient HPLC system with 2 x 125 mm C,, column and UV detector was coupled to a MAT 900 mass spectrometer fitted with a PATRIC detector and using an electrospray ionisation source (Finnigan Mat GmbH). Separations were performed using a 2-step linear gradient of 030% acetonimle with 0.1% TFA over 5 min. followed by 30-40% acetonitrile with 0.1% TFA during the remaining 40 min. Flow rate was 300 pl/min. and sample injection was 20 p1 at a concentration of 1 pg/pL. Separated components were detected by UV monitoring at 214 nm prior to electrospray at 3 kV (1 pA) with a capillary temperature of 270'C. Mass spectra were collected over the range lo00 2300 amu using a resolution of 1400 at 5 sec/decade. For flow injection, the same MS parameters were used and samples were dissolved in either water/acetonitrile (1/1) or water/acetonitrile (4/6) + 0.1% TFA. They were then analysed using a 5 FL loop injection with a flow rate of 30 pUmin. Analysis of sheep MT-I proteins by on-line HPLCESI-MS revealed components with distinct mass profiles whose chromatograms can be seen in Fig. 1. Mass spectra and derived deconvoluted spectra of the components resolved by chromatography confirmed the presence of proteins whose masses were within a fraction of predicted values for MT-la, -1b and -1c (Fig. 1, inset table). Indeed, the relative abundance of these isoforms was similar to that reported for the corresponding MT mRNA in sheep liver [3] and the translation of all 3 MT-1 proteins is consistent with the evidence for isoform heterogeneity obtained using capillary electrophoresis techniques [4]. A further minor isoform of mass 5951 was detected in MT-1 (Fig. 1). which is consistent with the presence of a form of deacetylated MT la. This may have physiological relevance since the rate of protein degradation may be influenced by the degree of deacetylation. The study of MT-1 by flow injection ESI-MS at acid and neutral pH gave rise to complex spectra (Fig. 2). A repetitive pattern with a repeat interval of 63-64 amu can be seen, with components at neutral pH being of higher mass than those at low pH. This pattern was caused by a varying degree of Zn or Cu dissociation from MT isoforms depending on the combined effect of pH and electrospray conditions.