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Detection of Metallothionein Isoforms from Three Different Species Using On-Line Capillary Electrophoresis–Mass Spectrometry
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
265, 167–175 (1998)
AB982874
Detection of Metallothionein Isoforms from Three Different Species Using On-Line Capillary Electrophoresis±Mass Spectrometry Carsten Boye Knudsen, Inga Bjørnsdottir, Ole Jøns, and Steen Honore´ Hansen1 Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark
Received May 4, 1998
An on-line capillary electrophoresis–mass spectrometry method (CE–MS) for the detection of metallothionein (MT) isoforms is described. The detected masses were usually within 1–1.5 mass units of the expected molecular weights. MT-containing samples from rabbit, sheep, and yeast (Saccharomyces cerevisiae) were subjected to CE–MS analysis. The analysis of rabbit liver MT revealed the masses of 10 proteins/peptides. Five of the detected masses corresponded well with the expected masses calculated from the amino acid sequence of previously described MT isoforms, one was suspected to be a deacetylated form of MT-2A, one was presumed to be a yet unknown isoform, and three masses were classified as non-MT compounds. From the analysis of a fetal sheep liver extract six proteins were detected of which three masses corresponded to previously described MT isoforms. Two purified MT subforms from S. cerivisiae (encoded by the CUP1 locus) were analyzed for their copper content and both forms were found to contain eight copper atoms per molecule. © 1998 Academic Press
Metallothioneins (MT)2 are a class of small metalbinding proteins with molecular weights in the range from 5500 to 7000 Da depending on the living organism from which it originates and on the number and kind of associated metal ions. The proteins have a high content of the amino acid cysteine (20 to 30%) and the metal ions are bound as thiolate complexes (1, 2). In MTs, 1 To whom correspondence should be addressed. Fax: 1 45 35 37 53 76. 2 Abbreviations used: MT, metallothionein; CE, capillary electrophoresis; MS, mass spectrometry; ESI, electrospray ionization; OD, optical density; pE, pyroglutamate; DTT, dithiothreitol.
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
bivalent metal ions like zinc and cadmium form tetrahedral thiolate complexes (3), whereas monovalent metal ions like copper(I) and silver are more likely to form trigonal or digonal thiolate complexes (4 – 6). The biosynthesis of MT is controlled at the level of transcription and it is induced by heavy metal ions, glucorticoid hormones, cytokines, and a variety of nucleophilic agents (7). MTs are thought to play a role in the homeostasis of copper and zinc and in protecting cellular structures from the toxic effects of cadmium and mercury. A possible role as free radical scavenger as well as an involvement in the regulation of the zinccontaining DNA-binding transcription factors has also been proposed (8). Mammalian MTs are historically classified in two main groups that can be separated by anion-exchange chromatography at neutral pH and have been named by their order of elution as MT-1 and MT-2, respectively. However, in a number of species this classification is insufficient since the isoforms can be separated into several subisoforms using either reversed-phase high-performance liquid chromatography or capillary electrophoresis (CE) (9, 10) as observed for sheep and rabbit MTs. Also the fast-developing techniques used in molecular biology have revealed a huge number of genes coding for MT isoforms. In human kidney 10 functional MT coding genes have been identified (11). However, it is not known whether all of them are expressed at the protein level. The primary role of different MT isoforms may well be distinct, but until they can be rapidly and routinely analyzed, their expression and hence their function will remain difficult to establish. CE has proved to be a fast highresolution separation technique for the analysis of MTs (12, 13) and it has the advantage of low sample requirements. However, the nanoliter amount of sample injected in the CE capillary also makes it cumbersome to use fraction collection of the detected peaks for further 167
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analysis. By the use of directly coupled CE–mass spectrometry (MS), specific molecular weight information can be obtained and thereby facilitate the identification of peaks where no reference material is available. In this paper, an on-line CE–MS method for the analysis of MT isoforms as well as subisoforms is presented. The method has been optimized with respect to the separation conditions and MS detection of MTs from three different species. The ability of the developed CE–MS method to detect and tentatively identify six MT isoforms from rabbit and three MT isoforms from a fetal sheep liver extract is demonstrated. The technique has also been used to detect and to characterize the copper load of two purified MTs originating from wild-type baker’s yeast (Saccharomyces cerevisiae) treated with copper. EXPERIMENTAL
Chemicals and Reagents The water used in this study (Milli-Q) was demineralized water filtered through a Milli-Q Plus 185 treatment system (Millipore, Bedford, MA). Fluorinert, FC-77 (used as capillary coolant), Bis–Tris [bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane], and standards of rabbit liver MT as well as rabbit liver MT-1 and rabbit liver MT-2 were obtained from Sigma Chemical Company (St. Louis, MO) and dissolved in Milli-Q water giving concentrations of 1 mg protein/ml. Yeast extract “Oxoid” was obtained from Unipath Ltd. (Basingstoke, Hampshire, UK). All other chemicals were analytical grade and obtained from Merck (Darmstadt, Germany). The growth medium consisted of 40 mM KH2PO4, 46 mM K2HPO4, 30 mM (NH4)2SO4, 1 mM MgSO4, 25 mM FeSO4, 50 mM ZnSO4, 50 mM MnSO4, 40 g D-glucose/liter, 1.5 mM L-cystine, and 16.0 g yeast extract (Oxoid)/liter. The growth medium was sterile filtrated using a 0.22-mm Millipore filter. The buffers were adjusted with 1 M sodium hydroxide, 5% ammonium hydroxide solution, or 4 M hydrochloric acid to the appropriate pH value. CE System For CE–UV, a P/ACE 5010 system (Beckman, Fullerton, CA) was used and a capillary cartridge was fitted with a fused-silica capillary (57 cm 3 75 mm i.d.; 50 cm to the detector) (Polymicro Technologies, Phoenix, AZ). Prior to use, the capillaries were rinsed with 1 M sodium hydroxide for 60 min, 0.1 M sodium hydroxide for 20 min, distilled water for 20 min, and separation buffer for 10 min. The samples were kept at ambient temperature and injected by the application of a pressure of 0.5 psi (3448 Pa) for 5 s. Ammonium phosphate (40 mM, pH 2.5):2-propanol (95:5, v/v) was used as separation buffer and a constant current of 80
mA was maintained during separation (approx 122 kV) and the temperature of the capillary was kept at 25°C by the means of circulating coolant. The detection was performed at 200 nm. Between the injections, the capillary was flushed with a 0.1 M sodium hydroxide solution for 2 min and run buffer for 2 min. A PC with System Gold software (Beckman) was used for data acquisition. For CE–MS, a Biofocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA) equipped with an on-column diode-array detector was used. A detection wavelength of 214 nm was used for all samples. The separation was performed in a fusedsilica capillary (90 cm 3 50 mm i.d.; 16 cm to the UV detector) (Polymicro Technologies, Phoenix, AZ). Prior to use, the capillary was treated as described for the CE–UV method. Samples were kept at ambient temperature in the autosampler and injected by the application of a pressure of 20 psi for 0.75– 4.5 s. The separation was performed in 40 mM ammonium phosphate (pH 2.5):2-propanol (95:5, v/v) by the application of a potential of 130 kV until the analysis was completed. The temperature of the capillary was maintained at 25°C by means of circulating coolant. Between the injections, the capillary was flushed with a 2.5% (v/v) ammonium hydroxide solution for 2 min and run buffer for 2 min. Ion Trap MS Mass spectrometry was performed on a Finnigan MAT LCQ ion trap (San Jose, CA) equipped with an electrospray ionization (ESI) source. For the connection between the CE and MS, a BioFocus CE/MS interface (Bio-Rad Laboratories) was used. A spray voltage of 15 kV was applied to the ESI needle. From the nozzle the ions entered a capillary heated to 200°C and applied with a voltage of 130 V. The tube lens offset was set to 10 V. A sheath liquid consisting of methanol: water:formic acid (50:50:1, v/v/v) was used to boost the flow through the ESI needle and to provide a conducting path to the terminating electrode. A syringe pump delivered the sheath liquid at a flow rate of 3 ml/min. A flow of nitrogen was introduced between the ESI nozzle and the ESI needle to aid desolvation of the highly charged electrospray droplets and to minimize any solvent cluster formation. Navigator software version 1 (Finnigan MAT, San Jose, CA) was used for data collection. During CE–MS separation the total ion current from m/z 600 to 1600 was monitored at a scan rate of 45 scans/min. Desalting of CE–MS Samples (Off-Line) Bio-Spin columns containing Bio-Gel P-6 polyacrylamide gel (MW exclusion limit, 6000 Da) (Bio-Rad Lab-
DETECTION OF METALLOTHIONEIN ISOFORMS
169
oratories) were used as a fast and nondiluting desalting step. Prior to use the spin columns were equilibrated with 4 volumes of Milli-Q water. To each of the equilibrated spin columns 100 –150 ml of sample was applied and the columns were centrifuged for 2 min at 1000g using a bucket centrifuge. The collected desalted samples were applied to CE–MS analysis. Crude Extraction of Sheep Liver Metallothionein A crude MT extract from a fetal sheep liver (day 89 of gestation, perturbed) was prepared using a two-step solvent extraction procedure. The extraction procedure has previously been described (14) and was followed except the homogenization, which was carried out in 10 mM sodium phosphate (pH 7.0) containing 100 mM sodium chloride and 1 mM dithiothreitol (DTT). The extract was transferred to a clean microcentrifuge tube and kept frozen (220°C) until desalting and the following analysis by CE–MS. Induction and Purification of Yeast Metallothionein Yeast cell paste (3.2 g, S. cerevisiae; Danisco, Aalborg, Denmark) were suspended in 250 ml Milli-Q water and diluted to give a concentration of approx 4 mg yeast paste/ml (OD6005 3.0). Fifty milliliters of the suspension was centrifuged at 2000g for 15 min and the precipitated yeast cells were resuspended in 50 ml of Milli-Q water. After being washed, 10 ml of the yeast suspension was added to 25 ml of growth medium (see Chemicals and Reagents), 600 mL of 250 mM copper sulfate, and 14.4 ml of Milli-Q water, giving a final concentration of approx 0.85 mg yeast paste/ml (OD6005 0.6) and 3 mM copper sulfate. After incubation for 48 h at 30°C the cells were harvested and washed in 40 ml of Milli-Q water by centrifugation at 2000g for 15 min (4°C). The precipitated yeast cells were then resuspended in buffer (10 mM sodium phosphate (pH 7.0) containing 100 mM sodium chloride and 1 mM DTT), giving a concentration of 0.5 g wet cells/ ml. Two milliliters of this solution was pipetted to a 3.6-ml polyethylene tube and homogenized using a 400 W Vibra Cell sonicator equipped with a 5-mm microtip (Sonics & Materials, Inc., Donbury, CT) for 2 3 20 s (amplitude 30%) and cooled on ice for 60 s in between homogenization intervals. The homogenate was transferred to a microcentrifuge tube and centrifuged at 20,000g for 30 min (4°C). The supernatant (200 ml) was injected on a Superose 12 HR 10/30 gelfiltration column (Pharmacia Biotech, Uppsala, Sweden) and eluted with buffer (10 mM sodium phosphate (pH 6.5) containing 100 mM sodium chloride and 1 mM DTT) at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected and measured for their content of copper by flame atomic absorption spectrometry on a Perkin–
FIG. 1. Anion-exchange chromatogram of the copper-containing fraction obtained after gel filtration. 2 ml equilibrated with buffer A (5 mM Bis-Tris–HCl (pH 6.5) 1 1 mM DTT) was injected on a HR 5/5 Mono Q column and eluted with buffer B (buffer A 1 1 M NaCl) using the following gradient: 0 –5 min, 0% B; 5– 8 min at 2% B; 8 –10 min 2–5% B; 10 –13 min at 5% B; 13–14 min 5– 8% B; 14 –17 min 8% B; 17–24 min 8 –20% B; 24 –27 min 20 –100% B, and 27–29 min 100% B. Fraction 1 was identified as (CUP 1) yeast MT and fraction 2 as a N-terminal pyroglutamate form of fraction 1 (yeast pE-MT).
Elmer Analyst 100 (Perkin–Elmer, Norwalk, CT). The copper-containing fractions eluting at a Kd of 0.7 (corresponding to a MW about 10,000 Da) were collected and lyophilized overnight. The dry substance obtained was solubilized in 200 ml Milli-Q water and desalted using a fast desalting column HR 10/10 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 5 mM Bis-Tris–HCl (pH 6.5) containing 1 mM DTT (buffer A) and eluted using a flow of 4 ml/min. The high-molecular-weight fraction (approx 2 ml) was collected and injected on a Mono Q column HR 5/5 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with buffer A and eluted with buffer B (buffer A 1 1 M sodium chloride) by the following gradient program: 0 –5 min 0% B, 5– 8 min at 2% B, 8 –10 min 2–5% B, 10 –13 min at 5% B, 13–14 min 5– 8% B, 14 –17 min 8% B, 17–24 min 8 –20% B, 24 –27 min 20 –100% B, and 27–29 min 100% B. The flow rate was 0.5 ml/min. Fraction 1 and 2 (Fig.1) eluting at a conductivity of 3 and 5 mS/cm, respectively, were manually collected, lyophilized, and stored under argon in a freezer at 220°C until desalting and analysis by CE–MS. Depletion of Copper from Yeast MT Five microliters of 0.1 M potassium cyanide were added directly to the sample vial containing 100 ml of 200 mg yeast pE-MT/ml. The mixture was allowed to
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stand for 5 min at room temperature before it was directly injected at 20 psi for 4.5 s and analyzed by CE–MS. No effort was made to keep anaerobic conditions during reaction or reducing eventually oxidized cysteines after reaction. RESULTS AND DISCUSSION
Optimization of CE–MS Conditions During initial CE–UV studies the sample containing rabbit liver MT-2 (1 mg/ml) resulted in the least complex electropherogram of the three rabbit liver MT batches commercially available from Sigma chemicals and was therefore used for the optimization of the CE–MS parameters: the composition of the separation buffer, the composition of the sheath liquid, the sheath liquid flow rate, as well as the spray voltage. Several MS-compatible CE separation buffers were tested in order to achieve the best separation. As found in previous studies (12), acidic separation buffers with a pH , 3.0 resulted in an improved separation compared to the neutral or slightly basic buffers. A 40 mM ammonium phosphate (pH 2.5) buffer resulted in better separation than formic acid, and acetic acid-based buffers and the less volatile phosphoric acid did not increase the MS noise level. Addition of 5% 2-propanol to the separation buffer was found to improve separation further. Furthermore, the acidic buffers caused cadmium and zinc ions to fully dissociate from MT, giving sharper MS signals and thereby facilitating the interpretation of the obtained mass spectra. Finally, the composition and flow rate of the sheath liquid as well as the spray voltage were optimized with respect to the S/N ratio. The tested sheath liquids contained 50 – 60% organic modifier (acetonitrile, methanol, or 2-propanol) and 0.5–5% acetic acid or formic acid. Sheath liquids containing 50% methanol as organic modifier resulted in the best S/N ratio, and sheath liquids containing formic acid were superior to those containing acetic acid. The optimal acid concentration was observed between 0.5 and 2%. The optimal sheath liquid was therefore found to consist of methanol:water:formic acid (50:50:1, v/v/v) using rabbit liver MT-2 as standard and a spray voltage of 5 kV.
FIG. 2. CE–UV electropherograms of (a) rabbit liver MT-2, (b) rabbit liver MT-1, and (c) rabbit liver MT mix, all in concentrations of 1 mg total protein/ml. The separations were performed in 40 mM ammonium phosphate (pH 2.5):2-propanol (95:5, v/v) using a constant current of 80 mA (anode at inlet). The samples were injected for 5 s at 0.5 psi and detected by monitoring the UV absorbance at 200 nm. R1 to R10, refers to the masses obtained from the CE–MS analysis listed in Table 1.
MT eluted in the first fraction and the rest 20% in the following fraction. The performance of the mass spectrometer is greatly reduced if any salt remains in the samples that are analyzed. Therefore, only the first fraction was collected for CE–MS analysis in this study. Compared to the speed and simplicity using spin columns for desalting a loss of 20% was acceptable.
Desalting of CE–MS Samples Using Spin Columns (Off-Line)
CE–MS Analysis of Rabbit MT Isoforms
The exclusion limit of the spin columns (6000 Da) used for the off-line desalting of samples prior to CE–MS analysis was close to the actual MW of the MTs. The elution of MT from the spin columns was therefore evaluated using yeast MT (CUP1), which represented the smallest MT analyzed in this study. CE–UV analysis of the three first 150-ml fractions eluting from the spin column revealed that 80% of the total
The analysis of rabbit liver products from Sigma chemicals (rabbit liver MT mix, MT-1, and MT-2) revealed 10 proteins, of which 6 were tentatively identified as previously purified rabbit MTs, 1 was presumed to be a yet unknown MT isoform, and 3 were not related to the MT family. The proteins are marked R1 to R10 and their migration is indicated on the respective UV electropherograms in Fig. 2. The matching masses and the tentative
171
DETECTION OF METALLOTHIONEIN ISOFORMS TABLE 1
The Detected m/z Ions and the Calculated Molecular Weights ((m-1)z) Obtained from the CE–MS Analysis of Rabbit Liver m/z (z)
m/z (z)
m/z (z)
m/z (z)
m/z (z)
MW found
i.d.
MW Accession Charge at expected No. pH 2.5
(a) Rabbit liver MT-2 (injected for 0.75 s at 20 psi) R1 R2 R3 R4 R5
974.6 892.2 888.9 870.2 879.3
(16) (7) (7) (7) (7)
1039.3 1041.4 1037.4 1015.3 1025.4
(15) (6) (6) (6) (6)
1113.5 1249.0 1244.0 1217.6 1230.3
(14) (5) (5) (5) (5)
1199.1 1561.9 1555.2 1521.7 1537.6
(13) (4) (4) (4) (4)
(5) (5) (1) (1) (5)
1532.3 (4) 1540.2 (4) — — 1548.6 (4)
1298.9 (12) 15575.4 6 1.2 unknown — 6241.1 6 2.3 MT-2E — 6216.4 6 1.6 MT-2D — 6084.0 6 1.4 MT 2A 4 Ac — 6146.9 6 0.8 MT-1A/MT-2B
R6 876.1 (7) R7 880.8 (7) R8 — R9 — R10 —
1022.0 (6) 1027.2 (6) — — 1032.4 (6)
1226.1 1232.6 1295.6 1285.7 1238.9
— — — — —
R2 R3 R5
892.4 (7) 888.7 (7) 878.6 (7)
1040.7 (6) 1036.7 (6) 1025.3 (6)
1249.0 (5) 1243.6 (5) 1229.9 (5)
1561.4 (4) 1555.1 (4) 1537.4 (4)
— 6241.4 6215.3 6083.2 6145.3/ 6146.2 6125.2 6155.2 — — —
— P80292 P80291 P18055 P11957/ P80289 P18055 P80290 — — —
— 1 8.85 1 8.85 1 7.87 1 7.90/ 1 7.85 1 6.87 1 6.87 — — —
6240.6 6 1.9 MT-2E 6241.4 6214.5 6 1.3 MT-2D 6215.3 6144.8 6 1.2 MT-1A/MT-2B 6145.3/ 6146.2 6586.9/ 6586.9 6 0.3 Cd4-MT-1A/ 6587.8 Cd4-MT-2B 6124.9 6 0.9 MT-2A 6125.2
P80292 P80291 P11957/ P80289 P11957/ P80289 P18055
1 8.85 1 8.85 1 7.90/ 1 7.85 1 7.90/ 1 7.85 1 6.87
P80292 P80291 P18055 P11957/ P80289 P18055 P80290 — — —
1 8.85 1 8.85 1 7.87 1 7.90/ 1 7.85 1 6.87 1 6.87 — — —
6125.6 6 0.3 6157.7 6 0.8 1295.6 1285.7 6189.4 6 1.0
MT-2A MT-2C unknown unknown MT-?
(b) Rabbit liver MT-1 (injected for 3.0 s at 20 psi) 781.4 (8) 777.9 (8) —
R59
—
942.0 (7)
1098.8 (6)
1318.3 (5)
1647.8 (4)
R6
—
876.1 (7)
1021.9 (6)
1226.0 (5)
1531.9 (4)
(c) Rabbit liver MT mix (injected for 0.5 s at 20 psi) R2 R3 R4 R5 R6 R7 R8 R9 R10
781.3 (8) — 761.0 (8) — — — — — —
892.8 889.4 870.2 878.9
(7) (7) (7) (7)
876.1 (7) — — — —
(6) (6) (6) (6)
1248.8 1243.5 1217.1 1229.7
(5) (5) (5) (5)
— — 1521.9 (4) 1537.8 (4)
6240.9 6 1.9 6214.8 6 3.5 6082.0 6 1.9 6145.8 6 1.7
MT-2E MT-2D MT-2A4Ac MT-1A/MT-2B
1021.7 (6) 1027.0 (6) — — 1032.5 (6)
1225.7 1232.1 1294.6 1284.8 1238.6
(5) (5) (1) (1) (5)
1532.0 (4) 1540.1 (4) — — 1548.0 (4)
6124.4 6 0.9 6156.0 6 0.5 1294.6 1284.8 6188.3 6 0.6
MT-2A MT-2C Unknown Unknown MT-?
1040.9 1036.5 1014.6 1025.5
6241.4 6215.3 6083.2 6145.3/ 6146.2 6125.2 6155.2 — — —
Note. The expected molecular weights were calculated from the respective amino acid sequence of the MT isoform in question assuming an N-terminal acetylation (9) (except R4, which was suspected to be a deacetylated MT isoform). The accession number refers to the SWISS-PROT database, and the theoretical charges of the MT isoforms were calculated using the following pKa-values (17): pKa 5 10.0 (lysines), pKa 5 12.0 (arginines), pKa 5 3.85 (aspartic acid), pKa 5 4.25 (glutamic acid), and pKa 5 7.5 (free N-terminal).
identification, when possible, are listed in Tables 1a–1c. Generally, MT-2E (R2), -2D (R3), -1A/-2B (R5), and -2A (R6) were present in all investigated rabbit liver MT batches. MT-2D and -2E migrated together under the separation conditions used in this study. However, the MT-2D:MT-2E ratios could be elucidated using the MS data and were determined to be 100:60 (MT-2 batch), 38:100 (MT-1 batch), and 76:100 (MT mix batch). R7 was detected in the rabbit liver MT-2 and MT mix batches where it represented masses of 6157.7 6 0.8 and 6156.0 6 0.5, respectively. Due to the close resemblance in migration time, the detected mass was suspected to originate from the same compound and was tentatively identified as MT-2C. R7 migrated close to a peptide impurity with a mass of 1295 Da (R8) and by the use of data obtained by the MS, the ratios between R7 and R8 were
found to be 100:15 and 20:100 in the MT-2 and MT mix batch, respectively. R4, representing a mass of 6084 6 1.4 Da and found in the MT-2 and MT mix batch, was presumed to be the N-deacetylated form of MT-2A. This assumption corresponds with the mass loss of 42 Da and the migration just before MT-1A/MT-2B (R5) due to an extra positive charge obtained at the free N-terminal. N-deacetylated MT subforms have not yet been described in rabbit liver but has previous been reported from sheep liver and Chang liver cells (15, 16). The physiological relevance of the N-deacetylated subforms are yet unknown, although they have been suggested to play a role in the degradation of MT. R5 in Fig. 2, representing detected masses ranging from 6145 to 6147 (Table 1), can be assigned as both MT-1A and as MT-2B. The difference between MT-1A and MT-2B is a substitution of an as-
FIG. 3. (a) CE–MS electropherogram of a fetal sheep liver extract (2.0 mg MT-1/ml and 0.7 mg MT-2/ml), (b) the mass spectrum of the MT-2-containing peak, and (c) the mass spectrum of the MT-1-containing peak. The separation was performed in 40 mM ammonium phosphate (pH 2.5):2-propanol (95:5, v/v) using a voltage of 30 kV (anode at inlet). The sample was injected for 0.75 s at 20 psi and detected by monitoring the total ion current from m/z 5 600 to 1600 using a scan rate of 45 scans/min. S1 to S6 refers to the detected masses listed in Table 2.
172 KNUDSEN ET AL.
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DETECTION OF METALLOTHIONEIN ISOFORMS TABLE 2
The Detected m/z Ions and the Calculated Molecular Weights ((m-1)z) Obtained from the CE–MS Analysis of Fetal Sheep Liver (Injected for 0.75 s at 20 psi)
S1 S2 S3 S4 S5 S6 —
m/z (z)
m/z (z)
m/z (z)
m/z (z)
MW found
i.d.
MW expected
Accession No.
Charge at pH 2.5
765.1 (13) 1041.3 (15) 893.8 (12) 868.0 (7) 857.2 (7) 862.2 (7) —
828.7 (12) 1115.6 (14) 974.8 (11) 1012.4 (6) 999.6 (6) 1006.2 (6) —
903.9 (11) 1201.2 (13) 1072.3 (10) 1214.9 (5) 1199.4 (5) 1206.9 (5) —
994.2 (10) 1301.2 (12) 1191.2 (9) 1518.7 (4) 1498.7 (4) 1507.7 (4) —
9932.4 6 0.6 15603.5 6 1.1 10712.6 6 0.9 6069.4 6 1.0 5992.0 6 1.1 6029.0 6 1.9 N.D.
Unknown Unknown Unknown MT-2 MT-1A MT-1C MT-1B
— — — 6070.1 5993.1 6027.1 6023.1
— — — P09579 P04902 P09578 P09577
— — — 1 7.85 1 6.91 1 6.91 1 6.91
Note. The expected molecular weights, accession numbers, and the theoretical charge were obtained as described in the legend to Table 1.
pargine with an aspargic acid in amino acid position 11 (9). This substitution represents a mass difference of 1 Da, which is within the resolution limits of the mass spectrometer. At pH 2.5 the aspargic acid will only have limited contribution to the overall charge and the two isoforms would be expected to migrate close to each other, which makes the discrimination between MT-1A and -2B infeasible at the separation conditions used in this study. R59 migrates together with R5 and represents a mass of 6587 Da. The a-domain of MT (aa31–aa61) can form a thermodynamic stable Cd4S11 a-cluster (4, 18) with the 11 cysteinyl thiolates represented in this region. The Cd4S11 a-cluster would have a net negative charge of 3 and therefore three associated protons would be expected for neutralization of the cluster. The mass difference between R5 and R59 corresponds to the difference predicted between apoMT-1A/apoMT-2B and Cd4-MT-1A/Cd4MT-2B where the Cd21 ions are bound in the a-domain along with three associated protons. R10 with a migration time of approx 11 min in Figs. 2a and 2c, represents a mass of 6189 Da, which could indicate a yet unidentified rabbit MT isoform. However, the concentration was to low for further investigations. R1, R8, and R9 were not identified by search in the SWISS-PROT database, but the masses of 15575, 1295, and 1285 Da indicate that they are non-MT components. Several other peaks observed with the CE–UV analysis remained undetected by CE–MS (the unmarked peaks in Fig. 2). The differences might result from the limited m/z window used for MS detection (600–1600) or by different detection limits of the compounds in UV and MS detection. CE–MS Analysis of Sheep MT Isoforms A sample of fetal sheep liver subjected to a two-step solvent extraction procedure was analyzed by CE–MS. The resulting MS electropherogram is shown in Fig. 3 along with the mass spectra of the MT-containing peaks. The analysis revealed the masses of six proteins (Table 2), of which three were tentatively identified as MTs (S4–S6) by comparison with the expected masses elucidated from their amino acid composition (19). The 14 to
17 ions of MT-2 are readily identified from the mass spectrum of S4. Additional 14 to 17 ions representing a mass of 6091 6 1 Da were also detected and the mass difference of approx 22 6 2 Da was presumed to originate from a sodium ion associated to MT-2. These sodium ion adducts were also observed in the mass spectrum of the MT-1 isoforms (S5 and S6) and complicated the interpretation. However, masses corresponding to MT-1A and MT-1C were observed as components in the MT-1 peak and an individual ratio of 100:36 could be elucidated from their relative abundance. MT-1B has not been detected in the investigated sample, which might be a consequence of the resolution limits of the mass spectrometer since the m/z peaks would appear in between the m/z peaks from MT-1C and the sodium adduct of MT-1A. Along with the MT isoforms, three non-MT proteins were detected in the liver extract and their masses are listed in Table 2. CE–MS Analysis of Yeast MT The two isolated fractions from copper treated S. cerevisiae (Fig. 1) were analyzed by CE–MS. The mass spectrum of fraction 1 (Fig. 4a) revealed a mass of 6165.4 6 0.8 Da which corresponds to the yeast MT, encoded by the CUP1 locus, binding eight copper atoms. The expected mass of yeast MT binding eight copper atoms would be 6166.5 Da (20). The identification as yeast CUP1 MT was confirmed by sequence analysis by Edman degradation of the first 27 amino acids. In contrast, fraction 2 was nonreactive in the sequence analysis and the mass spectrum revealed a mass of 6149.4 6 0.2 (Fig. 4b). Hence, the mass difference of 16 6 1 Da and the nonreactivity toward Edman degradation suggested a formation of a pyroglutamate by the cyclization of the N-terminal glutamine of yeast MT (CUP1). The detected mass of 6149.4 6 0.2 indicated the prescence of eight copper ions in the metal cluster of the pyroglutamate form (yeast pE-MT), which would result in an expected mass of 6149.5 Da. N-terminal positioned glutamines are known to spontaneously deamidate, forming pyroglutamates (17), but whether this N-terminal cyclization of yeast MT
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FIG. 4. Mass spectrum of (a) yeast Cu8–MT representing a mass of 6165.460.8 Da (0.4 mg/ml) (fraction 1, Fig. 1) and (b) the N-terminal pyroglutamate form of yeast Cu8–MT representing a mass of 6149.460.2(0.3 mg/ml) (fraction 2, Fig. 1). With the exception of a sample injection of 3 s at 20 psi, the CE–MS conditions were the same as those described in the legend to Fig. 3.
DETECTION OF METALLOTHIONEIN ISOFORMS
arises during the purification process or whether it is formed naturally and has biological relevance is unknown. However, pE-MT has been detected 1 h after homogenization directly after the gelfiltration step by CE–UV using a neutral separation buffer (30 mM ammonium phosphate, pH 6.5) and was found to account for 30 –50% of the total MT (MT 1 pE-MT) in the investigated samples. To verify the copper load, pE-MT was reanalyzed after copper depletion using potassium cyanide as reagent. The most abundant ions represented apo-pE-MT (5640.0 6 0.0 Da), Cu4-pE-MT (5894.6 6 2.0 Da) and Cu8-pE-MT (6149.9 6 1.3 Da). The mass differences of approx 12 and 6 Da from the theoretical MW of the apo- and Cu4-complexes, respectively, were thought to be a result of oxidation of the cysteines, which has been reported after copper depletion using potassium cyanide (5). The number of copper atoms in the metal cluster of yeast MT has been a subject for speculation since the first isolation and characterization. The stoichiometry of the copper cluster has been investigated by several methods, i.e., proteolytic assays, spectroscopic assays, metal to protein measurements, and luminescence measurements, all indicating eight ligated copper atoms present in wildtype yeast MT, which also has been the case for a truncated form lacking the last five amino acid residues, including two cysteines (T48) (21, 22). However, recent heteronuclear NMR studies where the resonance silent Cu(I) has been substituted with 109Ag(I), a Ag7Cys10 metal cluster has been identified. Comparing the nuclear Overhauser effect data between Ag-MT and Cu–MT, a similar cluster stoichiometry was also proposed for the Cu–MT complex (23, 24). Nevertheless, our results, based on CE–MS, show the presence of eight copper atoms in yeast MT. The difference between our findings and the stoichiometry proposed by the NMR study could be a result of the size difference between the Cu(I) and Ag(I) ions, which could result in space for an extra Cu(I) ion within the metal cluster. With reference to studies of the T48 and wild-type yeast MT, which were found to have similar metal–MT stoichiometry, and the similarity of the NMR data for Ag–MT and Cu–MT, which indicated that only 10 cysteines were involved in the cluster formation, we therefore propose a Cu8Cys10 metal cluster in yeast MT. CONCLUSION
In this paper we have demonstrated the application of an on-line CE–MS method optimized for the detection of MTs from a variety of species. The obtained masses were usually below 1–1.5 mass unit from the calculated MW based on the published amino acid sequences, and the method could therefore be used as a powerful tool in the search for yet undetected MTs where the sequence is known only from genetic studies.
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The method can also be used to determine the migration order of MT isoforms where pure standard materials are difficult to obtain. In addition, by the use of CE–MS, it was possible to characterize the number of copper ions within the metal cluster of CUP1 encoded yeast MT. Both yeast MT and pE-MT were found to bind eight copper ions within their metal cluster. ACKNOWLEDGMENTS The authors thank John H. Beattie at the Rowett Research Institute, Aberdeen, UK, who kindly provided the samples of fetal sheep livers, and Jytte Pedersen and Irene Simonsen at Novo Nordisk A/S, Gentofte, Denmark, for their assistance in the sequence analysis of yeast MT.
REFERENCES 1. Ka¨gi, J. H. R., and Kojima, Y. (1987) Exp. Suppl. 52, 25– 62. 2. Ka¨gi, J. H. R., and Scha¨ffer, A. (1988) Biochemistry 27, 8509 – 8515. 3. Good, M., Hollenstein, R., and Vasa´k, M. (1991) Eur. J. Biochem. 197, 655– 659. 4. Presta, A., Green, A. R., Zelazowski, A., and Stillman, M. J. (1995) Eur. J. Biochem. 227, 226 –240. 5. Winge, D. R. (1991) Methods Enzymol. 205, 458 – 469. 6. Li, H., and Otvos, J. D. (1996) Biochemistry 35, 13929 –13936. 7. Ka¨gi, J. H. R. (1991) Methods Enzymol. 205, 613– 626. 8. Vallee, B. L. (1995) Neurochem. Int. 27, 23–33. 9. Hunziker, P. E., Kaur, P., Wan, M., and Ka¨nzig, A. (1995) Biochem. J. 306, 265–270. 10. Richards, M. P., and Beattie, J. H. (1995) J. Chromatogr.B 669, 27–37. 11. Mididoddi, S., McGuirt, J. P., Sens, M. A., Todd, T. J., and Sens, D. A. (1996) Toxicol. Lett. 85, 17–27. 12. Richards, M. P., and Beattie, J. H. (1993) J. Chromatogr. 648, 459 – 468. 13. Virtanen, V., Bordin, G., and Rodriguez, A.-R. (1996) J. Chromatogr. A 734, 391– 400. 14. Knudsen, C. B., and Beattie, J. H. (1997) J. Chromatogr. A 792, 463– 473. 15. Beattie, J. H., Lomax, J., Richards, M. P., Self, R., Pesch, R., and Munster, H. (1996) Biochem. Soc. Trans. 24, 220S. 16. Hunziker, P. E., Zehnder, C., Cavigelli, M., Hess, D., and Studer, R. (1997) Proc. 4th International Metallothionein Meeting, in press. 17. Chreigton, T. E. (1993) Proteins—Structures and Molecular Properties, 2nd ed., pp. 1–9, Freeman, New York. 18. Petterson, M. G., Hannan, F., and Mercer, J. F. B. (1988) Eur. J. Biochem. 174, 417– 424. 19. Winge, D. R., Nielson, K. B., Gray, W. R., and Hamer, D. H. (1985) J. Biol. Chem. 260, 14464 –14470. 20. Chen, P., Munoz, A., Nettesheim, D., Shaw, C. F., and Petering, D. H. (1996) Biochem. J. 317, 395– 402. 21. Byrd, J., Bergers, R. M., McMillin, D. R., Wright, C. F., Hamer, D. H., and Winge, D. R. (1988) J. Biol. Chem. 263, 6688 – 6694. 22. Goerge, G. N., Byrd, J., and Winge, D. R. (1988) J. Biol. Chem. 263, 8199 – 8203. 23. Narula, S. S., Winge, D. R., and Armitage, I. M. (1993) Biochemistry 32, 6773– 6787. 24. Peterson, C. W., Narula, S. S., and Armitage, I. M. (1996) FEBS Lett. 379, 85–93.
265, 167–175 (1998)
AB982874
Detection of Metallothionein Isoforms from Three Different Species Using On-Line Capillary Electrophoresis±Mass Spectrometry Carsten Boye Knudsen, Inga Bjørnsdottir, Ole Jøns, and Steen Honore´ Hansen1 Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark
Received May 4, 1998
An on-line capillary electrophoresis–mass spectrometry method (CE–MS) for the detection of metallothionein (MT) isoforms is described. The detected masses were usually within 1–1.5 mass units of the expected molecular weights. MT-containing samples from rabbit, sheep, and yeast (Saccharomyces cerevisiae) were subjected to CE–MS analysis. The analysis of rabbit liver MT revealed the masses of 10 proteins/peptides. Five of the detected masses corresponded well with the expected masses calculated from the amino acid sequence of previously described MT isoforms, one was suspected to be a deacetylated form of MT-2A, one was presumed to be a yet unknown isoform, and three masses were classified as non-MT compounds. From the analysis of a fetal sheep liver extract six proteins were detected of which three masses corresponded to previously described MT isoforms. Two purified MT subforms from S. cerivisiae (encoded by the CUP1 locus) were analyzed for their copper content and both forms were found to contain eight copper atoms per molecule. © 1998 Academic Press
Metallothioneins (MT)2 are a class of small metalbinding proteins with molecular weights in the range from 5500 to 7000 Da depending on the living organism from which it originates and on the number and kind of associated metal ions. The proteins have a high content of the amino acid cysteine (20 to 30%) and the metal ions are bound as thiolate complexes (1, 2). In MTs, 1 To whom correspondence should be addressed. Fax: 1 45 35 37 53 76. 2 Abbreviations used: MT, metallothionein; CE, capillary electrophoresis; MS, mass spectrometry; ESI, electrospray ionization; OD, optical density; pE, pyroglutamate; DTT, dithiothreitol.
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
bivalent metal ions like zinc and cadmium form tetrahedral thiolate complexes (3), whereas monovalent metal ions like copper(I) and silver are more likely to form trigonal or digonal thiolate complexes (4 – 6). The biosynthesis of MT is controlled at the level of transcription and it is induced by heavy metal ions, glucorticoid hormones, cytokines, and a variety of nucleophilic agents (7). MTs are thought to play a role in the homeostasis of copper and zinc and in protecting cellular structures from the toxic effects of cadmium and mercury. A possible role as free radical scavenger as well as an involvement in the regulation of the zinccontaining DNA-binding transcription factors has also been proposed (8). Mammalian MTs are historically classified in two main groups that can be separated by anion-exchange chromatography at neutral pH and have been named by their order of elution as MT-1 and MT-2, respectively. However, in a number of species this classification is insufficient since the isoforms can be separated into several subisoforms using either reversed-phase high-performance liquid chromatography or capillary electrophoresis (CE) (9, 10) as observed for sheep and rabbit MTs. Also the fast-developing techniques used in molecular biology have revealed a huge number of genes coding for MT isoforms. In human kidney 10 functional MT coding genes have been identified (11). However, it is not known whether all of them are expressed at the protein level. The primary role of different MT isoforms may well be distinct, but until they can be rapidly and routinely analyzed, their expression and hence their function will remain difficult to establish. CE has proved to be a fast highresolution separation technique for the analysis of MTs (12, 13) and it has the advantage of low sample requirements. However, the nanoliter amount of sample injected in the CE capillary also makes it cumbersome to use fraction collection of the detected peaks for further 167
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analysis. By the use of directly coupled CE–mass spectrometry (MS), specific molecular weight information can be obtained and thereby facilitate the identification of peaks where no reference material is available. In this paper, an on-line CE–MS method for the analysis of MT isoforms as well as subisoforms is presented. The method has been optimized with respect to the separation conditions and MS detection of MTs from three different species. The ability of the developed CE–MS method to detect and tentatively identify six MT isoforms from rabbit and three MT isoforms from a fetal sheep liver extract is demonstrated. The technique has also been used to detect and to characterize the copper load of two purified MTs originating from wild-type baker’s yeast (Saccharomyces cerevisiae) treated with copper. EXPERIMENTAL
Chemicals and Reagents The water used in this study (Milli-Q) was demineralized water filtered through a Milli-Q Plus 185 treatment system (Millipore, Bedford, MA). Fluorinert, FC-77 (used as capillary coolant), Bis–Tris [bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane], and standards of rabbit liver MT as well as rabbit liver MT-1 and rabbit liver MT-2 were obtained from Sigma Chemical Company (St. Louis, MO) and dissolved in Milli-Q water giving concentrations of 1 mg protein/ml. Yeast extract “Oxoid” was obtained from Unipath Ltd. (Basingstoke, Hampshire, UK). All other chemicals were analytical grade and obtained from Merck (Darmstadt, Germany). The growth medium consisted of 40 mM KH2PO4, 46 mM K2HPO4, 30 mM (NH4)2SO4, 1 mM MgSO4, 25 mM FeSO4, 50 mM ZnSO4, 50 mM MnSO4, 40 g D-glucose/liter, 1.5 mM L-cystine, and 16.0 g yeast extract (Oxoid)/liter. The growth medium was sterile filtrated using a 0.22-mm Millipore filter. The buffers were adjusted with 1 M sodium hydroxide, 5% ammonium hydroxide solution, or 4 M hydrochloric acid to the appropriate pH value. CE System For CE–UV, a P/ACE 5010 system (Beckman, Fullerton, CA) was used and a capillary cartridge was fitted with a fused-silica capillary (57 cm 3 75 mm i.d.; 50 cm to the detector) (Polymicro Technologies, Phoenix, AZ). Prior to use, the capillaries were rinsed with 1 M sodium hydroxide for 60 min, 0.1 M sodium hydroxide for 20 min, distilled water for 20 min, and separation buffer for 10 min. The samples were kept at ambient temperature and injected by the application of a pressure of 0.5 psi (3448 Pa) for 5 s. Ammonium phosphate (40 mM, pH 2.5):2-propanol (95:5, v/v) was used as separation buffer and a constant current of 80
mA was maintained during separation (approx 122 kV) and the temperature of the capillary was kept at 25°C by the means of circulating coolant. The detection was performed at 200 nm. Between the injections, the capillary was flushed with a 0.1 M sodium hydroxide solution for 2 min and run buffer for 2 min. A PC with System Gold software (Beckman) was used for data acquisition. For CE–MS, a Biofocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA) equipped with an on-column diode-array detector was used. A detection wavelength of 214 nm was used for all samples. The separation was performed in a fusedsilica capillary (90 cm 3 50 mm i.d.; 16 cm to the UV detector) (Polymicro Technologies, Phoenix, AZ). Prior to use, the capillary was treated as described for the CE–UV method. Samples were kept at ambient temperature in the autosampler and injected by the application of a pressure of 20 psi for 0.75– 4.5 s. The separation was performed in 40 mM ammonium phosphate (pH 2.5):2-propanol (95:5, v/v) by the application of a potential of 130 kV until the analysis was completed. The temperature of the capillary was maintained at 25°C by means of circulating coolant. Between the injections, the capillary was flushed with a 2.5% (v/v) ammonium hydroxide solution for 2 min and run buffer for 2 min. Ion Trap MS Mass spectrometry was performed on a Finnigan MAT LCQ ion trap (San Jose, CA) equipped with an electrospray ionization (ESI) source. For the connection between the CE and MS, a BioFocus CE/MS interface (Bio-Rad Laboratories) was used. A spray voltage of 15 kV was applied to the ESI needle. From the nozzle the ions entered a capillary heated to 200°C and applied with a voltage of 130 V. The tube lens offset was set to 10 V. A sheath liquid consisting of methanol: water:formic acid (50:50:1, v/v/v) was used to boost the flow through the ESI needle and to provide a conducting path to the terminating electrode. A syringe pump delivered the sheath liquid at a flow rate of 3 ml/min. A flow of nitrogen was introduced between the ESI nozzle and the ESI needle to aid desolvation of the highly charged electrospray droplets and to minimize any solvent cluster formation. Navigator software version 1 (Finnigan MAT, San Jose, CA) was used for data collection. During CE–MS separation the total ion current from m/z 600 to 1600 was monitored at a scan rate of 45 scans/min. Desalting of CE–MS Samples (Off-Line) Bio-Spin columns containing Bio-Gel P-6 polyacrylamide gel (MW exclusion limit, 6000 Da) (Bio-Rad Lab-
DETECTION OF METALLOTHIONEIN ISOFORMS
169
oratories) were used as a fast and nondiluting desalting step. Prior to use the spin columns were equilibrated with 4 volumes of Milli-Q water. To each of the equilibrated spin columns 100 –150 ml of sample was applied and the columns were centrifuged for 2 min at 1000g using a bucket centrifuge. The collected desalted samples were applied to CE–MS analysis. Crude Extraction of Sheep Liver Metallothionein A crude MT extract from a fetal sheep liver (day 89 of gestation, perturbed) was prepared using a two-step solvent extraction procedure. The extraction procedure has previously been described (14) and was followed except the homogenization, which was carried out in 10 mM sodium phosphate (pH 7.0) containing 100 mM sodium chloride and 1 mM dithiothreitol (DTT). The extract was transferred to a clean microcentrifuge tube and kept frozen (220°C) until desalting and the following analysis by CE–MS. Induction and Purification of Yeast Metallothionein Yeast cell paste (3.2 g, S. cerevisiae; Danisco, Aalborg, Denmark) were suspended in 250 ml Milli-Q water and diluted to give a concentration of approx 4 mg yeast paste/ml (OD6005 3.0). Fifty milliliters of the suspension was centrifuged at 2000g for 15 min and the precipitated yeast cells were resuspended in 50 ml of Milli-Q water. After being washed, 10 ml of the yeast suspension was added to 25 ml of growth medium (see Chemicals and Reagents), 600 mL of 250 mM copper sulfate, and 14.4 ml of Milli-Q water, giving a final concentration of approx 0.85 mg yeast paste/ml (OD6005 0.6) and 3 mM copper sulfate. After incubation for 48 h at 30°C the cells were harvested and washed in 40 ml of Milli-Q water by centrifugation at 2000g for 15 min (4°C). The precipitated yeast cells were then resuspended in buffer (10 mM sodium phosphate (pH 7.0) containing 100 mM sodium chloride and 1 mM DTT), giving a concentration of 0.5 g wet cells/ ml. Two milliliters of this solution was pipetted to a 3.6-ml polyethylene tube and homogenized using a 400 W Vibra Cell sonicator equipped with a 5-mm microtip (Sonics & Materials, Inc., Donbury, CT) for 2 3 20 s (amplitude 30%) and cooled on ice for 60 s in between homogenization intervals. The homogenate was transferred to a microcentrifuge tube and centrifuged at 20,000g for 30 min (4°C). The supernatant (200 ml) was injected on a Superose 12 HR 10/30 gelfiltration column (Pharmacia Biotech, Uppsala, Sweden) and eluted with buffer (10 mM sodium phosphate (pH 6.5) containing 100 mM sodium chloride and 1 mM DTT) at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected and measured for their content of copper by flame atomic absorption spectrometry on a Perkin–
FIG. 1. Anion-exchange chromatogram of the copper-containing fraction obtained after gel filtration. 2 ml equilibrated with buffer A (5 mM Bis-Tris–HCl (pH 6.5) 1 1 mM DTT) was injected on a HR 5/5 Mono Q column and eluted with buffer B (buffer A 1 1 M NaCl) using the following gradient: 0 –5 min, 0% B; 5– 8 min at 2% B; 8 –10 min 2–5% B; 10 –13 min at 5% B; 13–14 min 5– 8% B; 14 –17 min 8% B; 17–24 min 8 –20% B; 24 –27 min 20 –100% B, and 27–29 min 100% B. Fraction 1 was identified as (CUP 1) yeast MT and fraction 2 as a N-terminal pyroglutamate form of fraction 1 (yeast pE-MT).
Elmer Analyst 100 (Perkin–Elmer, Norwalk, CT). The copper-containing fractions eluting at a Kd of 0.7 (corresponding to a MW about 10,000 Da) were collected and lyophilized overnight. The dry substance obtained was solubilized in 200 ml Milli-Q water and desalted using a fast desalting column HR 10/10 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with 5 mM Bis-Tris–HCl (pH 6.5) containing 1 mM DTT (buffer A) and eluted using a flow of 4 ml/min. The high-molecular-weight fraction (approx 2 ml) was collected and injected on a Mono Q column HR 5/5 (Pharmacia Biotech, Uppsala, Sweden) equilibrated with buffer A and eluted with buffer B (buffer A 1 1 M sodium chloride) by the following gradient program: 0 –5 min 0% B, 5– 8 min at 2% B, 8 –10 min 2–5% B, 10 –13 min at 5% B, 13–14 min 5– 8% B, 14 –17 min 8% B, 17–24 min 8 –20% B, 24 –27 min 20 –100% B, and 27–29 min 100% B. The flow rate was 0.5 ml/min. Fraction 1 and 2 (Fig.1) eluting at a conductivity of 3 and 5 mS/cm, respectively, were manually collected, lyophilized, and stored under argon in a freezer at 220°C until desalting and analysis by CE–MS. Depletion of Copper from Yeast MT Five microliters of 0.1 M potassium cyanide were added directly to the sample vial containing 100 ml of 200 mg yeast pE-MT/ml. The mixture was allowed to
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stand for 5 min at room temperature before it was directly injected at 20 psi for 4.5 s and analyzed by CE–MS. No effort was made to keep anaerobic conditions during reaction or reducing eventually oxidized cysteines after reaction. RESULTS AND DISCUSSION
Optimization of CE–MS Conditions During initial CE–UV studies the sample containing rabbit liver MT-2 (1 mg/ml) resulted in the least complex electropherogram of the three rabbit liver MT batches commercially available from Sigma chemicals and was therefore used for the optimization of the CE–MS parameters: the composition of the separation buffer, the composition of the sheath liquid, the sheath liquid flow rate, as well as the spray voltage. Several MS-compatible CE separation buffers were tested in order to achieve the best separation. As found in previous studies (12), acidic separation buffers with a pH , 3.0 resulted in an improved separation compared to the neutral or slightly basic buffers. A 40 mM ammonium phosphate (pH 2.5) buffer resulted in better separation than formic acid, and acetic acid-based buffers and the less volatile phosphoric acid did not increase the MS noise level. Addition of 5% 2-propanol to the separation buffer was found to improve separation further. Furthermore, the acidic buffers caused cadmium and zinc ions to fully dissociate from MT, giving sharper MS signals and thereby facilitating the interpretation of the obtained mass spectra. Finally, the composition and flow rate of the sheath liquid as well as the spray voltage were optimized with respect to the S/N ratio. The tested sheath liquids contained 50 – 60% organic modifier (acetonitrile, methanol, or 2-propanol) and 0.5–5% acetic acid or formic acid. Sheath liquids containing 50% methanol as organic modifier resulted in the best S/N ratio, and sheath liquids containing formic acid were superior to those containing acetic acid. The optimal acid concentration was observed between 0.5 and 2%. The optimal sheath liquid was therefore found to consist of methanol:water:formic acid (50:50:1, v/v/v) using rabbit liver MT-2 as standard and a spray voltage of 5 kV.
FIG. 2. CE–UV electropherograms of (a) rabbit liver MT-2, (b) rabbit liver MT-1, and (c) rabbit liver MT mix, all in concentrations of 1 mg total protein/ml. The separations were performed in 40 mM ammonium phosphate (pH 2.5):2-propanol (95:5, v/v) using a constant current of 80 mA (anode at inlet). The samples were injected for 5 s at 0.5 psi and detected by monitoring the UV absorbance at 200 nm. R1 to R10, refers to the masses obtained from the CE–MS analysis listed in Table 1.
MT eluted in the first fraction and the rest 20% in the following fraction. The performance of the mass spectrometer is greatly reduced if any salt remains in the samples that are analyzed. Therefore, only the first fraction was collected for CE–MS analysis in this study. Compared to the speed and simplicity using spin columns for desalting a loss of 20% was acceptable.
Desalting of CE–MS Samples Using Spin Columns (Off-Line)
CE–MS Analysis of Rabbit MT Isoforms
The exclusion limit of the spin columns (6000 Da) used for the off-line desalting of samples prior to CE–MS analysis was close to the actual MW of the MTs. The elution of MT from the spin columns was therefore evaluated using yeast MT (CUP1), which represented the smallest MT analyzed in this study. CE–UV analysis of the three first 150-ml fractions eluting from the spin column revealed that 80% of the total
The analysis of rabbit liver products from Sigma chemicals (rabbit liver MT mix, MT-1, and MT-2) revealed 10 proteins, of which 6 were tentatively identified as previously purified rabbit MTs, 1 was presumed to be a yet unknown MT isoform, and 3 were not related to the MT family. The proteins are marked R1 to R10 and their migration is indicated on the respective UV electropherograms in Fig. 2. The matching masses and the tentative
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DETECTION OF METALLOTHIONEIN ISOFORMS TABLE 1
The Detected m/z Ions and the Calculated Molecular Weights ((m-1)z) Obtained from the CE–MS Analysis of Rabbit Liver m/z (z)
m/z (z)
m/z (z)
m/z (z)
m/z (z)
MW found
i.d.
MW Accession Charge at expected No. pH 2.5
(a) Rabbit liver MT-2 (injected for 0.75 s at 20 psi) R1 R2 R3 R4 R5
974.6 892.2 888.9 870.2 879.3
(16) (7) (7) (7) (7)
1039.3 1041.4 1037.4 1015.3 1025.4
(15) (6) (6) (6) (6)
1113.5 1249.0 1244.0 1217.6 1230.3
(14) (5) (5) (5) (5)
1199.1 1561.9 1555.2 1521.7 1537.6
(13) (4) (4) (4) (4)
(5) (5) (1) (1) (5)
1532.3 (4) 1540.2 (4) — — 1548.6 (4)
1298.9 (12) 15575.4 6 1.2 unknown — 6241.1 6 2.3 MT-2E — 6216.4 6 1.6 MT-2D — 6084.0 6 1.4 MT 2A 4 Ac — 6146.9 6 0.8 MT-1A/MT-2B
R6 876.1 (7) R7 880.8 (7) R8 — R9 — R10 —
1022.0 (6) 1027.2 (6) — — 1032.4 (6)
1226.1 1232.6 1295.6 1285.7 1238.9
— — — — —
R2 R3 R5
892.4 (7) 888.7 (7) 878.6 (7)
1040.7 (6) 1036.7 (6) 1025.3 (6)
1249.0 (5) 1243.6 (5) 1229.9 (5)
1561.4 (4) 1555.1 (4) 1537.4 (4)
— 6241.4 6215.3 6083.2 6145.3/ 6146.2 6125.2 6155.2 — — —
— P80292 P80291 P18055 P11957/ P80289 P18055 P80290 — — —
— 1 8.85 1 8.85 1 7.87 1 7.90/ 1 7.85 1 6.87 1 6.87 — — —
6240.6 6 1.9 MT-2E 6241.4 6214.5 6 1.3 MT-2D 6215.3 6144.8 6 1.2 MT-1A/MT-2B 6145.3/ 6146.2 6586.9/ 6586.9 6 0.3 Cd4-MT-1A/ 6587.8 Cd4-MT-2B 6124.9 6 0.9 MT-2A 6125.2
P80292 P80291 P11957/ P80289 P11957/ P80289 P18055
1 8.85 1 8.85 1 7.90/ 1 7.85 1 7.90/ 1 7.85 1 6.87
P80292 P80291 P18055 P11957/ P80289 P18055 P80290 — — —
1 8.85 1 8.85 1 7.87 1 7.90/ 1 7.85 1 6.87 1 6.87 — — —
6125.6 6 0.3 6157.7 6 0.8 1295.6 1285.7 6189.4 6 1.0
MT-2A MT-2C unknown unknown MT-?
(b) Rabbit liver MT-1 (injected for 3.0 s at 20 psi) 781.4 (8) 777.9 (8) —
R59
—
942.0 (7)
1098.8 (6)
1318.3 (5)
1647.8 (4)
R6
—
876.1 (7)
1021.9 (6)
1226.0 (5)
1531.9 (4)
(c) Rabbit liver MT mix (injected for 0.5 s at 20 psi) R2 R3 R4 R5 R6 R7 R8 R9 R10
781.3 (8) — 761.0 (8) — — — — — —
892.8 889.4 870.2 878.9
(7) (7) (7) (7)
876.1 (7) — — — —
(6) (6) (6) (6)
1248.8 1243.5 1217.1 1229.7
(5) (5) (5) (5)
— — 1521.9 (4) 1537.8 (4)
6240.9 6 1.9 6214.8 6 3.5 6082.0 6 1.9 6145.8 6 1.7
MT-2E MT-2D MT-2A4Ac MT-1A/MT-2B
1021.7 (6) 1027.0 (6) — — 1032.5 (6)
1225.7 1232.1 1294.6 1284.8 1238.6
(5) (5) (1) (1) (5)
1532.0 (4) 1540.1 (4) — — 1548.0 (4)
6124.4 6 0.9 6156.0 6 0.5 1294.6 1284.8 6188.3 6 0.6
MT-2A MT-2C Unknown Unknown MT-?
1040.9 1036.5 1014.6 1025.5
6241.4 6215.3 6083.2 6145.3/ 6146.2 6125.2 6155.2 — — —
Note. The expected molecular weights were calculated from the respective amino acid sequence of the MT isoform in question assuming an N-terminal acetylation (9) (except R4, which was suspected to be a deacetylated MT isoform). The accession number refers to the SWISS-PROT database, and the theoretical charges of the MT isoforms were calculated using the following pKa-values (17): pKa 5 10.0 (lysines), pKa 5 12.0 (arginines), pKa 5 3.85 (aspartic acid), pKa 5 4.25 (glutamic acid), and pKa 5 7.5 (free N-terminal).
identification, when possible, are listed in Tables 1a–1c. Generally, MT-2E (R2), -2D (R3), -1A/-2B (R5), and -2A (R6) were present in all investigated rabbit liver MT batches. MT-2D and -2E migrated together under the separation conditions used in this study. However, the MT-2D:MT-2E ratios could be elucidated using the MS data and were determined to be 100:60 (MT-2 batch), 38:100 (MT-1 batch), and 76:100 (MT mix batch). R7 was detected in the rabbit liver MT-2 and MT mix batches where it represented masses of 6157.7 6 0.8 and 6156.0 6 0.5, respectively. Due to the close resemblance in migration time, the detected mass was suspected to originate from the same compound and was tentatively identified as MT-2C. R7 migrated close to a peptide impurity with a mass of 1295 Da (R8) and by the use of data obtained by the MS, the ratios between R7 and R8 were
found to be 100:15 and 20:100 in the MT-2 and MT mix batch, respectively. R4, representing a mass of 6084 6 1.4 Da and found in the MT-2 and MT mix batch, was presumed to be the N-deacetylated form of MT-2A. This assumption corresponds with the mass loss of 42 Da and the migration just before MT-1A/MT-2B (R5) due to an extra positive charge obtained at the free N-terminal. N-deacetylated MT subforms have not yet been described in rabbit liver but has previous been reported from sheep liver and Chang liver cells (15, 16). The physiological relevance of the N-deacetylated subforms are yet unknown, although they have been suggested to play a role in the degradation of MT. R5 in Fig. 2, representing detected masses ranging from 6145 to 6147 (Table 1), can be assigned as both MT-1A and as MT-2B. The difference between MT-1A and MT-2B is a substitution of an as-
FIG. 3. (a) CE–MS electropherogram of a fetal sheep liver extract (2.0 mg MT-1/ml and 0.7 mg MT-2/ml), (b) the mass spectrum of the MT-2-containing peak, and (c) the mass spectrum of the MT-1-containing peak. The separation was performed in 40 mM ammonium phosphate (pH 2.5):2-propanol (95:5, v/v) using a voltage of 30 kV (anode at inlet). The sample was injected for 0.75 s at 20 psi and detected by monitoring the total ion current from m/z 5 600 to 1600 using a scan rate of 45 scans/min. S1 to S6 refers to the detected masses listed in Table 2.
172 KNUDSEN ET AL.
173
DETECTION OF METALLOTHIONEIN ISOFORMS TABLE 2
The Detected m/z Ions and the Calculated Molecular Weights ((m-1)z) Obtained from the CE–MS Analysis of Fetal Sheep Liver (Injected for 0.75 s at 20 psi)
S1 S2 S3 S4 S5 S6 —
m/z (z)
m/z (z)
m/z (z)
m/z (z)
MW found
i.d.
MW expected
Accession No.
Charge at pH 2.5
765.1 (13) 1041.3 (15) 893.8 (12) 868.0 (7) 857.2 (7) 862.2 (7) —
828.7 (12) 1115.6 (14) 974.8 (11) 1012.4 (6) 999.6 (6) 1006.2 (6) —
903.9 (11) 1201.2 (13) 1072.3 (10) 1214.9 (5) 1199.4 (5) 1206.9 (5) —
994.2 (10) 1301.2 (12) 1191.2 (9) 1518.7 (4) 1498.7 (4) 1507.7 (4) —
9932.4 6 0.6 15603.5 6 1.1 10712.6 6 0.9 6069.4 6 1.0 5992.0 6 1.1 6029.0 6 1.9 N.D.
Unknown Unknown Unknown MT-2 MT-1A MT-1C MT-1B
— — — 6070.1 5993.1 6027.1 6023.1
— — — P09579 P04902 P09578 P09577
— — — 1 7.85 1 6.91 1 6.91 1 6.91
Note. The expected molecular weights, accession numbers, and the theoretical charge were obtained as described in the legend to Table 1.
pargine with an aspargic acid in amino acid position 11 (9). This substitution represents a mass difference of 1 Da, which is within the resolution limits of the mass spectrometer. At pH 2.5 the aspargic acid will only have limited contribution to the overall charge and the two isoforms would be expected to migrate close to each other, which makes the discrimination between MT-1A and -2B infeasible at the separation conditions used in this study. R59 migrates together with R5 and represents a mass of 6587 Da. The a-domain of MT (aa31–aa61) can form a thermodynamic stable Cd4S11 a-cluster (4, 18) with the 11 cysteinyl thiolates represented in this region. The Cd4S11 a-cluster would have a net negative charge of 3 and therefore three associated protons would be expected for neutralization of the cluster. The mass difference between R5 and R59 corresponds to the difference predicted between apoMT-1A/apoMT-2B and Cd4-MT-1A/Cd4MT-2B where the Cd21 ions are bound in the a-domain along with three associated protons. R10 with a migration time of approx 11 min in Figs. 2a and 2c, represents a mass of 6189 Da, which could indicate a yet unidentified rabbit MT isoform. However, the concentration was to low for further investigations. R1, R8, and R9 were not identified by search in the SWISS-PROT database, but the masses of 15575, 1295, and 1285 Da indicate that they are non-MT components. Several other peaks observed with the CE–UV analysis remained undetected by CE–MS (the unmarked peaks in Fig. 2). The differences might result from the limited m/z window used for MS detection (600–1600) or by different detection limits of the compounds in UV and MS detection. CE–MS Analysis of Sheep MT Isoforms A sample of fetal sheep liver subjected to a two-step solvent extraction procedure was analyzed by CE–MS. The resulting MS electropherogram is shown in Fig. 3 along with the mass spectra of the MT-containing peaks. The analysis revealed the masses of six proteins (Table 2), of which three were tentatively identified as MTs (S4–S6) by comparison with the expected masses elucidated from their amino acid composition (19). The 14 to
17 ions of MT-2 are readily identified from the mass spectrum of S4. Additional 14 to 17 ions representing a mass of 6091 6 1 Da were also detected and the mass difference of approx 22 6 2 Da was presumed to originate from a sodium ion associated to MT-2. These sodium ion adducts were also observed in the mass spectrum of the MT-1 isoforms (S5 and S6) and complicated the interpretation. However, masses corresponding to MT-1A and MT-1C were observed as components in the MT-1 peak and an individual ratio of 100:36 could be elucidated from their relative abundance. MT-1B has not been detected in the investigated sample, which might be a consequence of the resolution limits of the mass spectrometer since the m/z peaks would appear in between the m/z peaks from MT-1C and the sodium adduct of MT-1A. Along with the MT isoforms, three non-MT proteins were detected in the liver extract and their masses are listed in Table 2. CE–MS Analysis of Yeast MT The two isolated fractions from copper treated S. cerevisiae (Fig. 1) were analyzed by CE–MS. The mass spectrum of fraction 1 (Fig. 4a) revealed a mass of 6165.4 6 0.8 Da which corresponds to the yeast MT, encoded by the CUP1 locus, binding eight copper atoms. The expected mass of yeast MT binding eight copper atoms would be 6166.5 Da (20). The identification as yeast CUP1 MT was confirmed by sequence analysis by Edman degradation of the first 27 amino acids. In contrast, fraction 2 was nonreactive in the sequence analysis and the mass spectrum revealed a mass of 6149.4 6 0.2 (Fig. 4b). Hence, the mass difference of 16 6 1 Da and the nonreactivity toward Edman degradation suggested a formation of a pyroglutamate by the cyclization of the N-terminal glutamine of yeast MT (CUP1). The detected mass of 6149.4 6 0.2 indicated the prescence of eight copper ions in the metal cluster of the pyroglutamate form (yeast pE-MT), which would result in an expected mass of 6149.5 Da. N-terminal positioned glutamines are known to spontaneously deamidate, forming pyroglutamates (17), but whether this N-terminal cyclization of yeast MT
174
KNUDSEN ET AL.
FIG. 4. Mass spectrum of (a) yeast Cu8–MT representing a mass of 6165.460.8 Da (0.4 mg/ml) (fraction 1, Fig. 1) and (b) the N-terminal pyroglutamate form of yeast Cu8–MT representing a mass of 6149.460.2(0.3 mg/ml) (fraction 2, Fig. 1). With the exception of a sample injection of 3 s at 20 psi, the CE–MS conditions were the same as those described in the legend to Fig. 3.
DETECTION OF METALLOTHIONEIN ISOFORMS
arises during the purification process or whether it is formed naturally and has biological relevance is unknown. However, pE-MT has been detected 1 h after homogenization directly after the gelfiltration step by CE–UV using a neutral separation buffer (30 mM ammonium phosphate, pH 6.5) and was found to account for 30 –50% of the total MT (MT 1 pE-MT) in the investigated samples. To verify the copper load, pE-MT was reanalyzed after copper depletion using potassium cyanide as reagent. The most abundant ions represented apo-pE-MT (5640.0 6 0.0 Da), Cu4-pE-MT (5894.6 6 2.0 Da) and Cu8-pE-MT (6149.9 6 1.3 Da). The mass differences of approx 12 and 6 Da from the theoretical MW of the apo- and Cu4-complexes, respectively, were thought to be a result of oxidation of the cysteines, which has been reported after copper depletion using potassium cyanide (5). The number of copper atoms in the metal cluster of yeast MT has been a subject for speculation since the first isolation and characterization. The stoichiometry of the copper cluster has been investigated by several methods, i.e., proteolytic assays, spectroscopic assays, metal to protein measurements, and luminescence measurements, all indicating eight ligated copper atoms present in wildtype yeast MT, which also has been the case for a truncated form lacking the last five amino acid residues, including two cysteines (T48) (21, 22). However, recent heteronuclear NMR studies where the resonance silent Cu(I) has been substituted with 109Ag(I), a Ag7Cys10 metal cluster has been identified. Comparing the nuclear Overhauser effect data between Ag-MT and Cu–MT, a similar cluster stoichiometry was also proposed for the Cu–MT complex (23, 24). Nevertheless, our results, based on CE–MS, show the presence of eight copper atoms in yeast MT. The difference between our findings and the stoichiometry proposed by the NMR study could be a result of the size difference between the Cu(I) and Ag(I) ions, which could result in space for an extra Cu(I) ion within the metal cluster. With reference to studies of the T48 and wild-type yeast MT, which were found to have similar metal–MT stoichiometry, and the similarity of the NMR data for Ag–MT and Cu–MT, which indicated that only 10 cysteines were involved in the cluster formation, we therefore propose a Cu8Cys10 metal cluster in yeast MT. CONCLUSION
In this paper we have demonstrated the application of an on-line CE–MS method optimized for the detection of MTs from a variety of species. The obtained masses were usually below 1–1.5 mass unit from the calculated MW based on the published amino acid sequences, and the method could therefore be used as a powerful tool in the search for yet undetected MTs where the sequence is known only from genetic studies.
175
The method can also be used to determine the migration order of MT isoforms where pure standard materials are difficult to obtain. In addition, by the use of CE–MS, it was possible to characterize the number of copper ions within the metal cluster of CUP1 encoded yeast MT. Both yeast MT and pE-MT were found to bind eight copper ions within their metal cluster. ACKNOWLEDGMENTS The authors thank John H. Beattie at the Rowett Research Institute, Aberdeen, UK, who kindly provided the samples of fetal sheep livers, and Jytte Pedersen and Irene Simonsen at Novo Nordisk A/S, Gentofte, Denmark, for their assistance in the sequence analysis of yeast MT.
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