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Analytical Biochemistry 288, 89–96 (2001)doi:10.1006/abio.2000.4885, available online at http://www.idealibrary.com on

Measurement of Trace Elements in Proteins Extractedfrom Liver by Size Exclusion Chromatography-InductivelyCoupled Plasma-Mass Spectrometry with a MagneticSector Mass Spectrometer

Jin Wang,*,1 Dawn Dreessen,†,2 Daniel R. Wiederin,†,3 and R. S. Houk*,4

*Department of Chemistry, Ames Laboratory U.S. Department of Energy, Iowa State University, Ames, Iowa 50011; and†Transgenomic CETAC Technologies, Inc., 5600 S. 42nd Street, Omaha, Nebraska 68107

Received October 5, 2000

Proteins are extracted from liver into aqueousbuffer at pH 7 and separated by size exclusion chro-matography (SEC). Inductively coupled plasma massspectrometry (ICP-MS) with a magnetic sector massspectrometer is used to identify those protein frac-tions that contain Cu, Zn, Mn, Fe, Cd, S, P, Mo, Co, Ca,or Mg. The experimental setup provides very high sen-sitivity. Measurements at medium spectral resolutionremove polyatomic interferences for some difficult el-ements like Fe, S, and P. Some elements are found indifferent molecular weight proteins; for example, cad-mium binds to four different protein fractions (>400kDa, 70 kDa, and metallothionein). Other elementslike Mo, Ca, and Mg are present only in low-molecular-weight proteins or other small molecules. © 2001 Academic

ress

Key Words: metalloproteins; ICP-MS; size exclusionhromatography.

Elemental speciation in biological samples providescrucial evidence for determination of the toxicity, bio-availability, and environmental behavior of the ele-ments (1). The various chemical forms of the inorganicelements are not equally active biologically. For exam-ple, methyl mercury is more toxic than inorganic mer-cury salts, arsenobetaine is much less toxic than inor-

1 Present address: Baylor College of Medicine, Department of Bio-chemistry and Cell Biology, Houston, TX 77030.

2 Present address: American Laboratories, Omaha, NE.3 Present address: Elemental Scientific Inc., P.O. Box 310396,

Omaha, NE 68131.4

To whom correspondence should be addressed. Fax: 515-294-

5233. E-mail: rshouk@iastate.edu.

0003-2697/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

ganic arsenic, heme iron is much more valuablebiologically than inorganic iron salts, and cobalt is akey component of cyanocobalamin, an important vita-min (2, 3).

The liver is an important organ that performs manymetabolic functions. It synthesizes serum proteins (al-bumin, antibodies, fibrinogen), urea, prothrombin, andother coagulation factors. The liver is also a detoxifi-cation center and clears endogenous and exogenousloads. It plays a key role in the intermediary metabo-lism of carbohydrates, lipids, and proteins. It is impor-tant for the metabolism of hormones. The liver plays arole in vitamin economy by serving as a storage organfor vitamins A and B12. It also stores glycogen, fat, andprobably proteins. As a storage organ, the liver re-sponds to chronic situations by either repleting or de-pleting its reserves over extended periods. Animalfeeding experiments with orally administered Cd–met-allothionein (Cd–MT) and ionic cadmium show thatthese two forms of Cd are transported differently fol-lowing absorption. Cd–MT is deposited in the kidney(the target organ for cadmium toxicity), while ionic Cdgoes to the liver (4).

Investigation of elemental speciation in food showsthat the metals can be present in the ionic form and/orcomplexed to various binding proteins (5). An analyti-cal methodology that can measure these various ele-mental forms quickly for a wide range of elements withminimal sample preparation has many scientific andmedical applications. SEC–ICP-MS5 is such a method.The general advantage of the ICP as an ion source forsuch measurements is that it generates primarily mon-

5

Abbreviations used: SEC, size exclusion chromatography; ICP-MS, inductively coupled mass spectrometry.

89

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90 WANG ET AL.

atomic positive ions from most elements. Thus, theelements present can be identified from their atomicmass spectra without the many isotope peaks thatcomplicate the mass spectra of parent ions of biopoly-mers. The ICP is also a multielement ion source; sev-eral elements can be identified in the same chromato-graphic peak merely by scanning or hopping the massanalyzer.

SEC has been used previously for ICP-MS with alow-resolution mass analyzer in studies of elementalspeciation in biological samples (6–10). For some im-portant elements, such as Fe, S, and P, ICP-MS with aquadrupole mass analyzer suffers from polyatomic in-terference problems. The use of a magnetic sector massanalyzer can overcome many of these spectral interfer-ence problems (11–13). Compared to quadrupoles, sec-tor instruments also provide extremely low instrumen-tal background, improved sensitivity in low resolutionfor isotopes that are not prone to spectral interference,and very good detection limits (0.1 to 1 ppt) for nearlyall elements.

Two previous papers from our group describe the useof SEC–ICP-MS with a magnetic sector instrument foranalysis of serum and DNA solutions (14, 15). Manyunusual elements can be measured with very goodsignal-to-noise ratio without preconcentration proce-dures, despite the dilution that accompanies the chro-matographic separation. Sanz-Medel and co-workers(16) have described related work with other types ofchromatographic separations. Although the quality ofthe separation is usually inferior to that obtained withother types of chromatography, SEC has the advantagethat there is a regular relationship between the molec-ular weight of the protein and its retention time. Thus,at least the molecular weight of proteins that containthe elements of interest in mixtures can be determinedwithout pure standards of the same proteins. The SECseparation is also very robust and is well suited todirect injection of difficult samples without extensivepretreatment procedures.

In this paper we demonstrate the extension of thisSEC–ICP-MS method to identify the molecular weightfractions that contain particular elements of interest inaqueous extracts from liver. Accurate quantification ofelemental concentrations in individual protein frac-tions should also be possible but is not addressed here.Gentle extraction conditions at physiological pH (7) arechosen to minimize changes to the proteins during theanalysis.

MATERIALS AND METHODS

Sample preparation. Bovine liver 1577a standardeference material (National Institute of Standardsnd Technology, NIST) was used because it is easy to

btain and handle. A 1.3-g batch of the lyophilized solid

was added to 30 ml of the SEC buffer, 50 mM Tris–HClat pH ;7. The mixture was ultracentrifuged at 25,000rpm and 10°C for 2 h. The supernatant was decantedand filtered through a 0.45-mm filter. The filtrate wasinjected onto the column without further treatment.Only about 20% of the total mass of the liver samplewas extracted by this procedure, so the estimated con-centrations in protein fractions pertain only to theextract and cannot be compared with certificate valuesfor total concentrations of the elements in the solid.

SEC conditions. Most separations were carried outwith a TSK-GEL G3000SWXl column (Tosohaas, PA, 5mm particle size, 7.8 mm i.d. 3 30 cm long, injectionvolume 20 ml). The eluent was 50 mM aqueous Tris–HCl buffer (Certified ACS Grade, Fisher Scientific)with 0.05% NaN3 (Fisher Scientific) at 0.5 ml/min, pH7.3. The column is suitable for separating proteins withmolecular weight (MW) from 10,000 to 500,000 Da.This column is larger than those used previously (14,15) and is capable of better separations, but it requireslarger samples and has a longer void time. Chromato-grams for Ca and Mg showed only small moleculescontaining these elements, so the shorter, faster col-umn described previously was adequate.

Sample introduction. The liquid flow from the SECcolumn is converted into aerosol droplets by a mi-croconcentric nebulizer (M2, Transgenomic CETACTechnologies, aerosol gas flow rate 0.80 L min21, makeup gas flow rate 0.10 L min21) with a single pass,conical spray chamber. These droplets are dried andthe bulk of the solvent is removed by a desolvator(heater temperature 140°C, condenser temperature0°C). To minimize band broadening in the liquid phase,it is important to make the tubing between the SECcolumn and the nebulizer as short and narrow as pos-sible.

ICP-MS conditions. A Finnigan MAT ELEMENTICP-MS was used (12, 13, 17). ICP conditions: outergas flow rate 14 L min21, auxiliary gas flow rate 0.8 Lmin21, forward power 1.25 kW, sampling position 10mm from load coil, on center, unshielded load coil. TheICP conditions, ion lens voltages, etc. were adjusted tomaximize the signal for analyte ions from standardsolutions injected postcolumn before the chromato-graphic experiments.

This device has three fixed slit widths and thus threeresolution settings: low (m/Dm ; 300), medium (m/Dm ; 4000), and high resolution (m/Dm ; 8000). Forthe ions studied in the present work, either low ormedium resolution was adequate (14, 15). The acceler-ating voltage was nominally 8 kV. To monitor a set ofelements of similar m/z value in one injection, themagnetic field strength was kept constant and the ac-celerating voltage was either hopped (in low resolution)

or scanned (in medium resolution) to move the mass

twou

91SPECTROMETRIC MEASUREMENT OF TRACE ELEMENTS IN PROTEIN

analyzer to the m/z value of interest. To monitor 59Coand 98Mo from the same sample injection (Fig. 7), themagnetic field setting was changed automatically bythe software. Approximate concentrations for the ele-ments of interest were estimated by comparison ofpeak areas with the usual sensitivity observed for sim-ple aqueous standards of typical elements injectedpostcolumn. Internal standards were not used. The Naand K from the sample elute after the protein peaks(17), which should minimize matrix interference ef-fects.

The retention window of the column was calibratedwith a UV–VIS absorbance detector (Rainin DynamaxUV-C, 280 nm). Data were acquired and stored by aLabview program written in-house.

RESULTS AND DISCUSSION

Molecular weight calibration. Figure 1 shows chro-

FIG. 1. Chromatograms of standard proteins obtained with UV–concentration of ;1000 ppm, injection volume 20 ml. In cases where

sed for the molecular weight calibration.

matograms for several standard proteins recorded by

UV–VIS absorption at 280 nm. The concentration ofeach protein is approximately 1 mg/ml. Thyroglobulin(669 kDa) is outside the retention window and elutes inthe exclusion volume. Other smaller proteins thenelute based on their apparent molecular weight, eachwith a certain retention time. The good separationperformance of this column is shown in Fig. 1. A cali-bration curve can be established for the relationshipbetween the retention time and molecular weight ofprotein. The same calibration curve can also be deter-mined using the ICP-MS device if proteins with knownelements are used.

Figure 1 shows that double peaks are observed forapoferritin and IgG. For these two proteins, the reten-tion time of the most intense peak is used in the mo-lecular weight calibration. We believe the most intensepeaks at longer retention time are the monomers,while the weaker peaks at shorter retention time are

ble absorbance detection at 280 nm. Each protein is injected at apeaks are observed, the retention time of the more intense one was

visi

dimers or other complexes.

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92 WANG ET AL.

Effect of ionic strength and selection of mobile phase.The ionic strength of the mobile phase buffer plays animportant role in maximizing the molecular sievingmechanism and minimizing secondary effects such asionic and hydrophobic interactions between the sampleand the column packing materials. Hydrophobic inter-actions may occur at high ionic strength, while ionicinteractions are possible at low ionic strength, espe-cially with small solutes (18).

Figure 2 shows the effect of ionic strength of themobile phase on the separation of proteins in liverextract. With 50 mM Tris/HCl and 0.05% NaN3 as

obile phase, six fractions can be separated, the lastour of which are fairly close together. With 0.1 M NaClnd 1 mM EDTA added to the mobile phase, the reten-ion times increase slightly and the resolution islightly worse.Figure 2 shows that increasing the ionic strength of

he mobile phase does not improve the chromato-raphic separation for these liver extracts. For ICP-MShe salt load should be kept as low as possible toinimize interferences, matrix effects, and deposition

f material on the sampler and skimmer orifices. Theobile phase of choice for this work is 50 mM Tris/HClith 0.05% NaN3, pH 7, from the standpoint of both the

chromatographic separation and detection by ICP-MS.The lack of organic solvent and use of physiological pHfor the mobile phase should also help keep those metalsthat are bound to proteins intact therein during thechromatographic separation.

Figure 2 also shows that there are several separableprotein fractions in the liver extract and that the most

FIG. 2. Effect of salt strength on separation of proteins in liverextract. Solid line: usual eluent, 50 mM Tris/HCl, 0.05% NaN3.Dashed line: usual eluent 1 0.1 M NaCl 1 1 mM EDTA.

abundant fractions are at relatively long retentionZs

time and therefore low molecular weight. The questionof which fractions contain particular elements of inter-est requires ICP-MS, which replaces the absorbancedetector for the results shown below.

Copper and zinc. Figure 3 shows Cu and Zn chro-matograms from liver extract. For the detection of63Cu1 and 64Zn1, medium spectral resolution (R 5m/Dm 5 4000) is used to remove interferences such as31P16O2

1 and 32S16O21. For Cu there is one sharp chro-

matographic peak at MW . 500 kDa, one larger peakt 13 kDa and two small peaks corresponding to MW ,0 kDa. Most of the copper exists in the smaller proteinractions. The concentration of copper corresponding tohe major chromatographic peak (13 kDa) is estimatedo be ;5 ppb. The peak volume is ;2.5 ml, while the

injected volume was only 20 ml, so the compounds arediluted considerably by the chromatographic separa-tion.

For Zn there is one main chromatographic peak at 13kDa. The concentration of zinc in the major peak isestimated to be ;4 ppb. The sensitivity for Zn is lowerthan that for Cu because (a) Zn is ionized less exten-sively in the plasma (19), and (b) the isotopic abun-dance of 64Zn is less than that of 63Cu. The chromato-rams for Cu and Zn are from the same injection of one0-ml sample, as is the case for each of the pairs of

isotopes in each subsection presented below.The peak widths in these element-selective chro-

matograms are similar to those from the absorbancedetector (Figs. 1 and 2). Thus, the nebulizer and des-olvation system do not broaden the chromatographicpeaks greatly, at least for the relatively low-resolutionseparations obtained by SEC, in agreement with pre-vious work (14, 15).

In common with many other elements, there is a

FIG. 3. Chromatograms for 63Cu (69.2% isotopic abundance) and64Zn (48.6%) in liver extract, spectral resolution 5 4000. Both Cu and

n were monitored from the same injection of sample. The verticalcale is expanded by a factor of 5 for retention times below 1200 s.

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93SPECTROMETRIC MEASUREMENT OF TRACE ELEMENTS IN PROTEIN

substantial baseline for Cu and Zn that is well abovethe instrumental background of the device. This base-line is caused by Cu and Zn in the eluent or washingcontinuously off the column. The Cu and Zn bound tothe protein can still be discerned because they elute atspecific retention times, as long as the levels are abovethe noise in the background.

Iron and manganese. Figure 4 shows the Mn andFe chromatograms from liver extract. Medium spectralresolution is used for both elements; the main interfer-ences are probably 39K16O1, 38Ar16OH1, and 40Ar16O1.

or Mn there is only one chromatographic peak at 13Da. The corresponding concentration of Mn is about 3pb.For iron there are two small peaks corresponding toW . 500 and 150 kDa and a third major Fe fraction

t MW , 10 kDa. For a given element the efficiency oftomization and ionization is not expected to differreatly in the various proteins, so the relative amountsf Fe in the three fractions can be estimated from theelative peak areas. The concentration of iron corre-ponding to the major peak at 13 kDa is ;12 ppb. Inig. 4 the baseline at m/z 56 is elevated, which is

probably 56Fe1 ions from the stainless-steel column orluent.Templeton and co-workers (20) studied excess iron

ccumulation in human and animal tissues by chro-atographic separation of proteins and detection by

CP-MS and reported the iron distribution in healthyat liver. The sensitivity and spectral resolution areuperior in the present work.The NIST certificate for the liver sample lists the

FIG. 4. Chromatograms for 55Mn (100%) and 56Fe (91.7%) in liverextract, spectral resolution 5 4000.

ollowing values for total concentrations (mg/g in the

solid): Cu (158), Fe (194), Mn (9.9), and Zn (123). It istempting to compare these with the estimated concen-trations from the chromatograms, but only ;20% ofthe original liver material is extracted. It appears thata higher fraction of the Mn is in an extractable formthan is the case for Fe, Cu, or Zn.

Cadmium. In ICP-MS, spectral interferences areless severe when the analyte element occurs at m/zvalues well above those of the matrix elements. Figure5 shows chromatograms for 112Cd1 and 114Cd1 in liverextract detected at low spectral resolution. Four chro-matographic peaks are well separated: two small peaksat MW . 500 kDa, one small, broad peak correspond-ing to MW ;30 to 100 kDa, and one sharp peak at 13kDa. Thus, cadmium binds four different protein frac-tions: two at high molecular weight (.500 kDa), one at

70 kDa, with most of the Cd in the fraction at 13 kDa.he concentration of cadmium corresponding to theajor peak at 13 kDa is estimated to be 2 ppb. The

ame chromatographic peaks are produced by either ofhe two Cd isotopes monitored, which happen to be ofearly equal isotopic abundance.Cadmium species in cooked pig kidney were mea-

ured by Crews et al. by SEC–ICP-MS with a quadru-ole instrument (21). The measurement of cadmium inhis work again provides better sensitivity.

Phosphorous and sulfur. Compared to the transi-ion metals studied so far in this paper, many nonmet-ls have high ionization energies and are not as effi-iently ionized in the ICP. There are also largeackground peaks at m/z 31 and 32. Nevertheless, Pnd S are fairly abundant and can be measured readilyn these samples using medium spectral resolution.here is sufficient S for use of the minor isotope 34S1,

112 114

FIG. 5. Chromatograms for Cd (24.1%) and Cd (28.7%) in liverextract, spectral resolution 5 300.

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94 WANG ET AL.

which prevents interference from the long front edge ofthe background peak from 16O2

1.Figure 6 shows the chromatogram for 34S in liver

extract. There are three S1 peaks with MW ; 500, 13,and ,10 kDa. Most of the sulfur is bound to the twolight protein fractions. The concentration of sulfur cor-responding to the last chromatographic peak (MW ,10 kDa) is estimated to be ;30 ppb.

Figure 6 also shows the corresponding chromato-gram for P1. There is one small peak corresponding toMW . 500 kDa, one small, broad peak at 70 kDa, andne larger peak at 11 kDa, followed by a large peakrom smaller molecules. Only a small amount of P isound to larger protein fractions; most of the P is in themall protein fraction (MW 5 11 kDa). The concentra-

tion of P corresponding to the peak at 11 kDa is esti-mated to be 20 ppb. Phosphorus is also found in the 70

FIG. 6. Chromatograms for 34S (4.2%) and 31P (100%) in liverextract, spectral resolution 5 4000.

kDa protein fraction, which does not have much S. The

chromatographic peak for this protein(s) has the samebroad shape as that for Cd in Fig. 5.

Assignment of metallothionein. Of the metals de-cribed above, Cu, Zn, and Cd bind to protein(s) at aetention time ;1270 s, which corresponds to MW ; 13

kDa. Figure 6 shows that the protein(s) at this reten-tion time contain S but not P. Metallothioneins arecommon proteins that contain Cu, Zn, Cd, and S, do notcontain P, and would be expected to be abundant inthese liver extracts (22, 23). A metallothionein stan-dard also produces a weak UV absorption peak at aretention time corresponding to an apparent molecularweight of 13 kDa, instead of the true molecular weightof ;6 kDa (data not shown). As noted by Vallee andco-workers (24), metallothioneins have a relativelyopen “dumbbell” shape and occupy a larger volumethan globular proteins at MW ;6 kDa. Since the elu-tion order in SEC is based on volume of the protein, notstrictly its molecular weight, metallothioneins oftenelute earlier from a SEC column than do globular pro-teins of comparable molecular weight.

We therefore feel that metallothioneins are at leastpartly responsible for the chromatographic peak con-taining Cd, Zn, Cu, and S at retention time of ;1270 s.There is also at least one protein containing Mn thatelutes at this retention time.

Cobalt and molybdenum. Figure 7 shows the Coand Mo chromatograms from liver extract. For Mo,there is only one narrow chromatographic peak atMW 5 11 kDa. Thus, Mo is bound only to a distinctgroup of small proteins. The concentration of Mo cor-responding to the major peak is estimated to be ;2ppb.

There is one very sharp Co peak (MW 5 155 kDa)and several small peaks corresponding to MW , 10kDa. Assuming the sensitivity for Co is about the samefrom any of the various proteins, the first Co chromato-graphic peak at 155 kDa has about half of the totalcobalt. The total concentration of cobalt bound to pro-tein is estimated to be 0.4 ppb. The very sharp peakprobably represents a single, well-defined protein thatcontains Co. Many of the broader peaks shown forother elements could represent families of proteins.The very sharp peak for Co also indicates that thecolumn, nebulizer, and desolvator are capable of goodchromatographic resolution.

In the other chromatograms, the m/z values of theanalyte ions are near one another, so different ele-ments can be monitored simply by switching or scan-ning the accelerating voltage at constant magnetic fieldstrength. However, the magnetic field must be changedto monitor both 59Co1 and 98Mo1 from the same sampleinjection. Figure 7 shows that this can be done repro-ducibly on a time frame fast enough to monitor these

chromatographic peaks. Although the background at

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95SPECTROMETRIC MEASUREMENT OF TRACE ELEMENTS IN PROTEIN

m/z 59 and 98 is substantial, the chromatographiceaks for Co and Mo from the sample can still bebserved readily.Calcium and magnesium. A different column (GPC

00 at 0.1 ml/min) (14, 15) was adequate for the mea-urement of Ca and Mg. Proteins elute much fasterrom this smaller column than for the one used for theesults shown above.

Figure 8 shows chromatograms for Ca and Mg iniver extract. Two isotopes are monitored for each ele-

ent. For both elements, medium resolution is used toeparate analyte signal from interferences such as

40Ar1H21, 12C16O2

1, and 12C21. There is only one chromato-

raphic peak for Ca and Mg, which occurs at longetention time and corresponds to Ca and Mg in smallolecules. The concentrations are estimated to be ;4

pb Ca and 70 ppb Mg. Neither Ca nor Mg yield theong, tailed peaks characteristic of “free” alkali metalons (15).

The relative peak areas for the two Ca and Mg iso-

FIG. 7. Chromatograms for 59Co (100%) and 98Mo (24.1%) in liverxtract, spectral resolution 5 300.

opes are close enough to the expected natural ratios to0

orroborate the assumption that the peaks are actuallyue to these elements. For both Ca and Mg there is toouch of the heavier isotope, probably due to mass bias,hich generally favors the heavier isotope (19, 25). No

pecial effort was made to optimize the instrument forsotope ratio measurements.

CONCLUSION

SEC with a double-focusing ICP-MS device has beensuccessfully applied to the study of elemental distribu-tions in liver extract. Several elements can be moni-tored from a single injection of sample. A magneticsector MS eliminates many polyatomic ion interfer-ences and provides high sensitivity. For the elementsstudied, sample preconcentration is not needed. Sam-ple preparation procedures that are simple, thatshould not dilute or contaminate the samples or change

FIG. 8. Chromatograms for 42Ca (0.65%), 44Ca (2.1%), 24Mg (79.0%),and 25Mg (10.0%) in liver extract, spectral resolution 5 4000. Fromthe chromatograms, the apparent abundance ratio for 42Ca/44Ca is

24 25

.24, while that for Mg/ Mg is 6.6. The actual ratios are 0.31 and7.9, respectively.

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96 WANG ET AL.

the binding of the trace elements, are adequate. Infor-mation about the approximate molecular weight of pro-tein(s) containing particular element(s) can be ob-tained without using a standard sample of that sameprotein. This is one of the advantages of SEC comparedto other separation modes such as reverse-phase HPLCor ion exchange chromatography.

Compared to our previous SEC–ICP-MS work (14,15), better separations are achieved by using a longercolumn with a narrower molecular weight separationrange. However, the separation take a longer time witha larger void volume. Better separations should bepossible by using SEC as an initial fractionation stepfollowed by other chromatographic or electrophoreticprocedures.

ACKNOWLEDGMENTS

The experiments are supported by the Ames Laboratory, U.S.Department of Energy, Office of Basic Energy Sciences, Division ofChemical Sciences, under Contract W-7405-Eng-82. The measure-ments were conducted at Transgenomic CETAC Technologies, Inc.who provided supplies. The authors also thank Finnigan MAT forproviding the mass spectrometer. The authors are grateful toYongjin Hou who measured the retention time of metallothioneinand John Gering who measured the extraction efficiency.

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