Simultaneous Cu-, Fe-, And Zn-Specific Detection of Metal Lo Proteins

8/8/2019 Simultaneous Cu-, Fe-, And Zn-Specific Detection of Metal Lo Proteins

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O R I G I N A L P A P E R

Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteinscontained in rabbit plasma by size-exclusion chromatography

inductively coupled plasma atomic emission spectroscopy

Shawn A. Manley Simon Byrns Andrew W. Lyon Peter Brown Ju rgen Gailer

Received: 16 April 2008 / Accepted: 23 August 2008 / Published online: 10 September 2008

SBIC 2008

Abstract Analytical methods which are capable of deter-

mining the plasma or serum metalloproteome have inherentdiagnostic value for human diseases associated with

increased or decreased concentrations of specific plasma

metalloproteins. We have therefore systematically devel-

oped a method to rapidly determine the major Cu-, Fe-, and

Zn-containing metalloproteins in rabbit plasma (0.5 mL)

based on size-exclusion chromatography (SEC; stationary

phase Superdex 200, mobile phase phosphate-buffered sal-

ine pH 7.4) and the simultaneous online detection of Cu, Fe,

and Zn in the column effluent by an inductively coupled

plasma atomic emission spectrometer (ICP-AES). Whereas

most previous studies reported on the analysis of serum, our

investigations clearly demonstrated that the analysis of

plasma within 30 min of collection results in the detection of

one more Cu peak (blood coagulation factor V) than hasbeen previously reported (transcuprein, ceruloplasmin,

albumin-bound Cu, and small molecular weight Cu). The

average amount of Cu associated with these five proteins

corresponded to 21, 18, 21, 30 and 10% of total plasma Cu,

respectively. In contrast, only two Fe metalloproteins (fer-

ritin and transferrin, corresponding to an average of 9 and

91% of total plasma Fe) and approximately five Zn metal-

loproteins (a2-macroglobulin and albumin-bound Zn, which

corresponded to an average of plasma Zn) were detected.

Metalloproteins were assigned on the basis of the coelution

of the corresponding metal and protein identified by

immunoassays or activity-based enzyme assays. The SEC-

ICP-AES approach developed allowed the determination of

approximately 12 Cu, Fe, and Zn metalloproteins in rabbit

plasma within approximately 24 min and can be applied to

analyze human plasma, which is potentially useful for

diagnosing Cu-, Fe-, and Zn-related diseases.

Keywords Blood plasma Size-exclusion

chromatography Inductively coupled plasma atomic

emission spectrometry Metalloproteins

Introduction

All organisms must regularly ingest sufficient quantities of

essential trace elements, such as Cu, Fe, and Zn, to main-

tain the continuous in vivo assembly of biologically active

metalloproteins, which are inherently associated with

health [1, 2]. In humans, for instance, about 1% of the total

body Zn content is replenished daily by the diet [3]. Fol-

lowing the absorption of essential trace elements from the

gastrointestinal tract into the systemic blood circulation,

Parts of the work described in this paper were presented at HPLC

2007 in Ghent, Belgium.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00775-008-0424-1 ) contains supplementarymaterial, which is available to authorized users.

S. A. Manley S. Byrns J. Gailer (&)

Department of Chemistry,

University of Calgary,

2500 University Drive NW,

Calgary, AB T2N 1N4, Canadae-mail: [email protected]

A. W. Lyon

Department of Pathology and Laboratory Medicine,

University of Calgary and Calgary Laboratory Services,

9, 3535 Research Rd NW,

Calgary, AB T2L 2K8, Canada

P. Brown

Teledyne Leeman Labs,

6 Wentworth Drive,

Hudson, NH 03051, USA

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J Biol Inorg Chem (2009) 14:6174

DOI 10.1007/s00775-008-0424-1
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specific plasma proteins subsequently distribute these ele-

ments to internal organs [4], where absorption occurs by

highly specific uptake mechanisms, such as endocytosis in

the case of Fe [5, 6]. Among the most abundant transition

metals present in human plasma are Cu, Fe, and Zn, which

are present at total concentrations of 0.841.45 lg Cu/mL,

0.461.67 lg Zn/mL, and 2.4713.1 lg Fe/mL [7, 8].

Therefore, the analysis of plasma for the contained major

Cu-, Fe-, and Zn-containing metalloproteins (Table 1) will

provide insight into the roles of trace elements in the bio-

chemistry and pathophysiology of both healthy and

diseased states.

It is well known that numerous genetic human diseases

are associated with increased or decreased plasma

concentrations of specific metalloproteins [9]. Wilsons

disease, for instance, is a Cu-overload disease associated

with low plasma Cu [10] and hereditary hemochromatosis

is an Fe-overload disease associated with elevated plasma

Fe [6]. Conversely, it is chemically feasible that the

exposure of humans to certain environmental pollutants or

that the physiological response to infection could also

result in increased or decreased plasma concentrations of

specific Cu, Fe, and Zn metalloproteins [9]. Therefore, an

instrumental analytical method which can rapidly deter-

mine the major Cu-, Fe-, and Zn-containing plasma

metalloproteins would represent an innovative tool to assist

in screening or diagnosing human diseases associated with

altered essential trace elements, regardless of whether the

latter have a genetic origin or are the result of exposure to

environmental pollutants (chemical or bacterial).

From an analytical point of view, the direct liquid

chromatography (LC) analysis of plasma in conjunction

with an element-specific detector, such as a flame atomic

absorption spectrometer, a graphite furnace atomic

absorption spectrometer, an inductively coupled plasma

atomic emission spectrometer (ICP-AES), or an induc-

tively coupled plasma mass spectrometer (ICP-MS), should

allow the detection of individual plasma metalloproteins

assuming that the metalprotein bond(s) in the latter

remain intact during the LC separation process [1114].

Despite this attractive proposition, and even though

numerous investigations have been reported on the speci-

ation of metals and metalloid compounds in other

biological fluids and tissues, comparatively few studies

have been carried out to attempt this goal in undiluted

mammalian plasma or serum [15].

All studies which reported on the direct LC analysis of

mammalian plasma or serum for metalloproteins are lis-

ted in Table 2 (only those which detected at least two

of the elements of interest are listed). In particular, size-

exclusion chromatography (SEC), anion-exchange chro-

matography (AEX), and reversed-phase chromatography

have been employed in conjunction with various element-

specific detectors. The pioneering work of Dawson et al.

[16], published in 1981, represents the first study of its

kind to detect metalloproteins in human plasma and

involved SEC analysis (Sephacryl S-300) followed by the

detection of Cu and Zn in the collected fractions by

flame atomic absorption spectrometry. The reconstruction

of a Cu- and Zn-specific chromatogram revealed one Cu

and three Zn peaks [16]. The same approach (Sephadex

G-100) was applied for the analysis of human serum and

graphite furnace atomic absorption spectrometry of the

fractions showed one Cu, one Fe, and three Zn peaks

Table 1 Molecular properties and relative abundances of the major metalloproteins and metallopeptides in human plasma or serum

Metal Metalloprotein or entity which

contains bound metal

Molecular

mass (kDa)

Number of metal atoms

bound per protein

Plasma or serum

protein concentration

References

Fe Ferritin 450 B4,500 10250 lg/L [9]

Transferrin 79.7 2 1.83.7 g/L [9]

Cu Blood coagulation factor V 330 1 *10 mg/L [34]

Transcuprein 270 0.5 *180 lg/La

[4, 32]Ceruloplasmin 132 6 0.20.6 g/L [9]

Albumin 66 1 36.153.6 g/L [4, 9]

EC-SOD 165 4 [40, 49]

Cu,Zn-SOD 31 [40]

Peptides and amino acids \5

Zn a2 macroglobulin 725 5 1.13.7 g/L [4, 9]

Albumin 66 1 36.153.6 g/L [9]

EC-SOD 165 4 [40]

Cu,Zn-SOD 31 [40]

SOD superoxide dismutase, EC-SOD extracellular Cu,Zn superoxide dismutasea Rat plasma

62 J Biol Inorg Chem (2009) 14:6174

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[17]. The separation of human serum on another SEC

stationary phase (Sephacryl S-300) and the utilization of

direct current plasma atomic emission spectrometry

resulted in the detection of two Cu, one Fe, and three Zn

peaks [18]. With use of a sequential ICP-AES as the

online multielement-specific detector, the SEC analysis

(TSK G 300 SW) of human serum brought to light one

Cu, two Fe, and four Zn peaks [19]. Applying the same

stationary phase for the analysis of rat serum in con-junction with an ICP-MS revealed four Cu and three Zn

peaks [20]. Reversed-phase chromatography (octadecyl

silica stationary phase) was also employed to analyze

human serum using an ICP-MS as the Cu-, Fe-, and

Zn-specific detector, but revealed that all metalloproteins

containing these elements were essentially coeluted [21].

Yet another SEC material (SynChropak GPC 300) was

used for the analysis of a reconstituted human serum

standard reference material by an ICP-MS and uncovered

four Cu (one major and three minor), one Fe, and six

non-baseline-separated Zn peaks [22]. Human plasma

analysis by SEC (Fractogel EMD BioSEC 650) with

offline analysis of the fractions by an ICP-AES identified

three Cu and two Fe peaks (all baseline-separated) [8]. A

double focusing ICP-MS was also employed as an online

Cu-, Fe-, and Zn-specific detector for human serum

analysis using AEX (MonoQ HR) [23, 24] and employ-

ing a mobile-phase gradient. AEX, however, altered thespeciation of Cu and Zn and must therefore be avoided

when plasma/serum metalloproteins are to be determined

for potential diagnostic applications. More recently,

human serum analysis by SEC (Superose 12HR) followed

by the online detection of Cu and Zn by an ICP-MS

revealed two Zn and two poorly separated Cu peaks [25].

In view of the reported variability in the number of Cu,

Fe, and Zn metalloprotein peaks that were detected by LC

analysis of mammalian plasma or serum (Table 2) and to

Table 2 Applications of liquid chromatography coupled with element-specific detectors for the identification of metalloproteins in mammalian

plasma/serum reported in the literature (in chronological order)

Biological fluid

analyzedbElements

detectedcSeparation

mechanism

Column

dimensions

(cm)

Stationary phased Mobile-phase composition Reference

Human plasma (0.5) Cu (1) Zn (3) SEC 57 9 0.9 Sephacryl S-300 (2575) 0.1 M Tris/HCl pH 8.0 ? 0.5 M

NaCl

[16]

Human serum (1.0) Cu (1) Fe (1)

Zn (3)

SEC 100 9 2.6 Sephadex G-100 (40120) 0.05 M Tris/HCl pH 7.4, 4 C [17]


Zn (3)

SEC 100 9 2.6 Sephacryl S-300 (2575) 0.1 M Tris/HCl pH 7.4, 22 C [18]


Zn (4)

SEC TSK G 3,000 SW (10) 0.1 M HEPES ? 0.1 M NaCl pH

7.4

[19]

Rat serum (0.1) Cu (4) Zn (3) SEC 30 9 0.7 TSK G 3,000 SW (10) 0.1 M Tris/HCl pH 7.2 [20]

Human serum

SRM (0.002)

Cu (4) Fe (1)

Zn (6)

SEC 25 9 0.2 SynChropak GPC 300 (5) 0.1 M Tris/HCl pH 6.9 [22]

Human plasma? (0.25) Cu (3) Fe (2) SEC 60 9 1.6 Fractogel EMD BioSEC

650 (2040)

0.02 M NaH2PO4 ? 0.3 M NaCl

pH 6.8, 30 C

[8]

Human serum (0.1) Cu (1)a Fe (2)

Zn (4)aAEX 5 9 0.5 Mono Q HR (10) 15 min linear gradient A (0.05 M

Tris/HCl pH 7.4) ? B (0.25 M

NH4Ac in A)

[24]

Human serum (0.2) Cu (2) Zn (2) SEC Superose 12 HR (812) 0.1 M Tris/HCl ? 2.5 mM CaCl2

pH 7.4

[25]


Zn (1)

RP 25 9 0.46 CHAPS-coated ODS 0.2 mM Tris/HCl ? 0.2 mM

CHAPS pH 7.4

[21]

Human serum (0.1) Cu (1)a Fe (2)

Zn (4)aAEX 5 9 0.5 Mono Q HR (10) 15 min linear gradient

A (0.05 M Tris/HCl pH

7.4) ? B (0.25 M NH4Ac in A)

[23]

SEC size-exclusion chromatography, AEX anion-exchange chromatography, RP reversed-phase chromatography, Tris tris(hydroxy-

methyl)aminomethane, HEPES N-(2-hydroxyethyl)piperazine-N0-ethanesulfonic acid, CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-

propanesulfonate, ODS octadecyl silica, SRM standard reference materiala Artifactb Volume in mLc No. of peaksd Bead diameter in lm

J Biol Inorg Chem (2009) 14:6174 63

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develop this analytical approach into a clinically useful

diagnostic tool, two key questions must be investigated.

First, it should be examined if plasma and serum generate

consistent analytical results (whichever contains more

individual metal peaks in the corresponding Cu-, Fe-, and

Zn-specific chromatogram inherently contains more infor-

mation). Second, the stability of metalloproteins in plasma/

serum over time must be investigated to establish themaximum time that plasma samples can be kept (e.g., at

room temperature) before ex vivo degradation of certain

metalloproteins will occur.

We have systematically developed a novel LC method

for the determination of Cu, Fe, and Zn metalloproteins

in rabbit plasma which can be potentially applied to any

mammalian plasma. In view of the fact that the binding

of transition metals in plasma metalloproteins can be

weak [4, 26] and to maintain the integrity of the Cu, Fe,

and Zn metalloproteins throughout the entire LC separa-

tion process [19], we chose SEC as the separation

mechanism. This is because SEC minimizes directinteractions between the analyte molecules and the sta-

tionary phase and therefore represents a comparatively

gentle separation mechanism for the separation of bio-

molecules. In addition, the utilization of physiologically

relevant buffers will provide an environment where

conformational changes of the plasma metalloproteins

which could potentially lead to the loss of the metalare

least likely to occur. Therefore, dissociation of a metal

from its parent plasma protein is minimized. Furthermore,

SEC can be employed in an isocratic separation mode

(which inherently increases sample throughput), whereas

AEX often requires salt gradients to elute proteins which

canin turnsever weak transition metalprotein link-

ages. Finally, the availability of SEC stationary phases

with much smaller particle sizes (approximately 13 lm)

and particle size distributions today compared with those

that were used in some previous studies (Table 2) will

inherently allow better separations owing to the increased

chromatographic resolution. Following these consider-

ations to least disrupt weak metalprotein binding

equilibria during the SEC separation process, we

employed phosphate-buffered saline (PBS, pH 7.4) as the

isocratic mobile phase. With regard to the detection of

the separated Cu-, Fe-, and Zn-containing metalloproteins

in the SEC column effluent, we utilized a state-of-the-art

charge injection device based ICP-AES because this

multielement-specific detector could be directly hyphen-

ated to the separation column for the simultaneous online

detection of Cu, Fe, and Zn. In addition, this detector is

compatible with LC separations involving mobile phases

containing more than 0.5% salt [27], which is required to

preclude irreversible adsorption of plasma proteins to the

stationary phase.

Materials and methods

Chemicals and solutions

Blue dextran, PBS (10 mM phosphate, 2.7 mM KCl, 137

mM NaCl) tablets, sodium chloride (more than 99.5%

pure), sodium acetate trihydrate (more than 99% pure),

N,N-dimethyl-p-phenylenediamine monohydrochloride(highly toxic), lysozyme (from chicken egg white), heparin

(sodium salt), and the bicinchoninic acid protein determi-

nation kit were purchased from Sigma-Aldrich (St Louis,

MO, USA), bovine serum albumin (BSA) was from

Amersham Pharmacia Biosciences (Little Chalfont, UK),

glacial acetic acid (more than 97.7% pure, to make 1 M

acetic acid) was from Fisher Scientific (Nepean, ON,

Canada), and Plasma PURE HCl (3437%) was from SCP

Science (Baie DUrfe, QC, Canada). A mixture of protein

standards for SEC column calibration was obtained from

Bio-Rad Laboratories (Hercules, CA, USA) which con-

tained thyroglobulin (670 kDa), c-globulin (158 kDa),ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12(1.35 kDa). All solutions were prepared with water from a

Simplicity water purification system (Millipore, Billerica,

MA, USA). PBS of pH 7.4 (10 mM phosphate, 2.7 mM

KCl, and 137 mM NaCl) was prepared by dissolving PBS

tablets in the appropriate volume of water (followed by pH

adjustment if necessary) and filtration through 0.45-lm

nylon filter membranes (Mandel Scientific, Guelph, ON,

Canada). Ceruloplasmin oxidase activity was measured in

collected fractions according to a published procedure [28].

Development of an isocratic SEC separation method

of the major plasma proteins using UV detection

Screening of prospective stationary phases

All separations were carried out at room temperature

(22 C) and a mobile phase flow rate of 1.0 mL/min

(peristaltic pump). Since irreversible binding of plasma

proteins to the stationary phase represents a major obstacle

that must be overcome when plasma is to be directly

analyzed by SEC, we systematically screened three com-

mercially available SEC stationary phases (Sephacryl

S-500, Superose 6 prep grade, and Superdex 200 prep

grade, 25 cm 9 1.0 cm column) for their ability to analyze

rabbit plasma (0.5 mL) using various PBS concentrations

(39, 29 and 19) and a UV detector (280 nm). Each

column was equilibrated with at least 50 mL of the

mobile phase before plasma was injected. To detect irre-

versible binding of plasma proteins to the stationary

phases, a standard protein mixture (BSA and lysozyme)

was chromatographed before and after the analysis of six

consecutive plasma samples. A shift of the retention times


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of the standard proteins after the six plasma injections

(compared with those obtained before the six plasma

injections) would indicate irreversible binding of plasma

proteins to the stationary phase. In addition, the column

effluent of each plasma injection was analyzed for total

protein (bicinchoninic acid assay) and compared with the

total protein contained in 0.5 mL of the plasma to establish

the percentage protein recovery. After six consecutiveplasma injections, no irreversible protein binding was

detected with any stationary phasemobile phase combi-

nation for the seventh injection (protein recovery 99 1%,

see the supplementary material). On the basis of the

chromatograms obtained and our objective to separate

plasma proteins into as many chromatographic protein

bands as possible (the term band is used rather than the

term peak since hundreds of proteins constitute a single

chromatographic band), Superdex 200 prep grade and

PBS (19) were identified as the ideal stationary phase

mobile phase combination. In particular, Superdex 200

prep grade resulted in four protein bands, correspondingto 15% (band 1), 14% (band 2), 69% (band 3), and 2%

(band 4) of total protein. In contrast, Superose 6 prep

grade produced only three protein bands, with 10, 89,

and 1% of total protein, and Sephacryl S-500 resulted in

only two protein bands, with 7 and 93% of total protein,

respectively.

Increase of column efficiency by using a higher-resolution

column

The commercially available stationary phase Superdex 200

prep grade is composed of 34-lm particles. In view of the

fact that stationary phases with a smaller particle size

generally result in a better chromatographic resolution, we

improved the separation of plasma bands by using a pre-

packed Superdex 200 column (30 cm 9 1.0-cm inner

diameter) which contained 13-lm particles. Again a mix-

ture of BSA and lysozyme (1.2 and 0.62 mg in 5.0 mL of

water) was used to check the column integrity before and

after the injection of six plasma samples. In addition, the

number of theoretical plates was calculated (using the

lysozyme peak) and increased from approximately 1,000

for Superdex 200 prep grade (25.4 cm 9 1.0-cm inner

diameter, 34-lm particles) to approximately 23,000 for the

prepacked Superdex 200 column (30 cm 9 1.0-cm inner

diameter, 13-lm particles). At least 30 consecutive plasma

analyses could be carried out per column without loss of

chromatographic resolution of the metal peaks.

Animal experiments

The Animal Care Committee of the University of Calgary

approved the procedure to collect blood from New Zealand

white rabbits (Protocol Approval #BI 2005-27). Male New

Zealand white rabbits were purchased from Casey Van-

dermeer (Edmonton, AB, Canada) and fed ad libitum on a

high-fiber diet (Lab Diet 5321, Canadian Lab Diets,

Leduc, AB, Canada). Blood (5.0 mL) was collected from

the marginal ear vein with 20-gauge stainless steel blood

collection needles (211 monoject, Sherwood Medical, St

Louis, MO, USA) into BD Vacutainer blood collectiontubes (no additive, BD Vacutainer, Franklin Lakes, NJ,

USA) to which 0.5 mg heparin had been added for the

preparation of plasma. For the preparation of serum, the

blood clot was removed by centrifugation (described

below). The injection of a heparin blank onto the SEC-ICP-

AES system (control experiment) revealed no detectable

Fe, Cu, or Zn (data not shown). Blood was collected from

4.5-h-fasted rabbits at approximately 13:30 and was cen-

trifuged at 1,100g (22 C) for 10 min and the plasma (or

serum) obtained was analyzed using the SEC-ICP-AES

system within 30 min after blood collection, or at the time

points indicated later. Only straw-yellow plasma (free ofthe characteristic red color of hemoglobin from ruptured

erythrocytes) was used throughout the study. To establish

the interanimal variation, 18 rabbit plasma samples were

consecutively analyzed using the SEC-ICP-AES system.

To qualitatively identify the detected Cu, Fe and Zn

metalloproteins in collected fractions after the analysis of

rabbit plasma, one human plasma sample was chromato-

graphed; this was obtained from a healthy male volunteer.

The collection of human blood was approved by the Uni-

versity of Calgary Conjoint Health Research Ethics Board

(approval no. E-21198).

Experimental setup of the optimized SEC-ICP-AES

system

The SEC-ICP-AES system (Fig. 1) consisted of a Waters

(Milford, MA, USA) model 510 high-performance LC

pump, a Rheodyne 9010 PEEK injection valve (Rheodyne,

Rhonert Park, CA, USA) equipped with a 0.5-mL PEEK

injection loop, and a prepacked SuperdexTM 200 10/300 GL

TricornTM high-performance column (30.0 cm 9 1.0-cm

inner diameter, separates globular proteins between

approximately 600 and 10 kDa; GE Healthcare, Piscataway,

NJ, USA). The exit of the SEC column was connected to the

Meinhard concentric glass tube nebulizer of the ICP-AES

with fluorinated ethylenepropylene Teflon tubing (30 cm,

inner diameter 0.5 mm). Simultaneous multielement-

specific detection of C (193.091 nm), S (180.731 nm), Zn

(213.856 nm), Fe (259.940 nm), Cu (324.754 nm), and P

(213.618 nm) in the column effluent was achieved with a

Prodigy, high-dispersion, radial-view ICP-AES (Teledyne

Leeman Labs, Hudson, NH, USA) at an Ar gas-flow rate of

19 L/min, an RF power of 1.3 kW, and a nebulizer gas


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pressure of 35 psi. The detector technology utilized in the

Prodigy allows the simultaneous measurement of the peak

and the background emissions to generate the net emission

intensity. This capability is critical in experiments where

the background emission intensity changes (e.g., when a

major protein peak reaches the ICP-AES) so the operator is

not mislead into believing that an analytically significant

event has occurred when in fact it has not. This advantage,

together with the ability of the ICP-AES to handle salt-

containing solutions, makes the Prodigy ideally suited forthe LC analysis of solutions containing metalloproteins.

Time scans were performed using the time-resolved-anal-

ysis mode (Salsa version 3.0) and a data acquisition rate of

one data point per 2 s. The raw data were imported into

SigmaPlot 10 and smoothed using the bisquare algorithm.

According to the void volume of the Superdex 200 column

(blue dextran), a 7.0-min delay was implemented between

injection and the beginning of data acquisition (1,080-s

acquisition window). Figure 2 depicts the C-specific SEC-

ICP-AES chromatograms of rabbit plasma obtained on a

Superdex 200 prep grade (34 lm) and a prepacked (13 lm)

column, which clearly displays the increased resolution of

the latter stationary phase. The marginal increase in the

retention time for the small molecular weight C peak in the

13-lm column compared with the 34-lm column is caused

by the difference in column length of approximately 5 cm.

We size-calibrated the analytical Superdex 200 column

with known molecular weight protein standards. In addi-

tion, the analysis of rabbit blood plasma provided internal

molecular weight standards as several proteolytically stable

plasma metalloproteins, such as ferritin and transferrin (Fe

metalloproteins), ceruloplasmin (Cu metalloprotein), andthe most abundant plasma protein albumin (which can be

easily identified on the basis of the most intense C and S

peaks in the chromatogram), are naturally present in

plasma. These can therefore serve as a rough proxy to

estimate the size of a detected metalloprotein. Furthermore,

it is likely that owing to the sheer complexity of plasma

(more than 3,700 proteins), a detected unknown plasma

metalloprotein is unlikely to have the same retention time

as is suggested from a calibration curve because of its

unavoidable interactions with other plasma proteins.

Therefore, deductions of the molecular mass of an unknown

metalloprotein from its retention time alone should beinterpreted with caution.

Identification and quantification of metalloproteins

in SEC column effluent

In terms of qualitatively identifying the separated plasma

metalloproteins in the column effluent, we did not utilize

electrospray ionization mass spectrometry because of the

salt content (approximately 1% or approximately 164 mM)

of the mobile phase. In general, the salt concentration of

aqueous samples that can be analyzed by electrospray

ionization mass spectrometry must be below 10 mM. Wetherefore qualitatively identified the Cu metalloprotein

ceruloplasmin in collected fractions using an established

ceruloplasmin oxidase activity assay (based on the oxida-

tion of N,N-dimethyl-p-phenylenediamine) [28]. Since an

antibody-based approach (e.g., enzyme immunoassay) for

the identification of rabbit ferritin, transferrin, a2 macro-

globulin, and factor V was not readily available, an

alternative way of identifying these metalloproteins had to

be pursued and we therefore analyzed human plasma using

Fig. 1 The instrumental

analytical size-exclusion

chromatography (SEC)

inductively coupled plasma

atomic emission spectrometer

(ICP-AES) setup. HPLC high-

performance liquid

chromatography

Time (s)

600 800 1000 1200 1400

Intensity(counts/s)

0

20000

40000

60000

80000

V0

Albumin (~66 kDa)

Fig. 2 C-specific chromatograms of rabbit plasma on Superdex 200

prep grade (1.0 cm 9 25 cm; 34-lm particle size) (dashed line), and

Superdex 200 10/300 GL SEC column (1.0 cm9 30 cm; 13-lm

particle size) (solid line). Phosphate-buffered saline mobile phase (pH

7.4, 22 C), flow rate 1.0 mL/min, injection volume 500 lL, ICP-

AES detector (C emission at 193.091 nm). Void volume 600 kDa,

inclusion volume 10 kDa


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the SEC-ICP-AES system and collected fractions for pro-

tein identification purposes.

The two major Fe-containing proteins and all Zn-con-

taining species had essentially the same retention times as

those obtained for rabbit plasma. Fractions were collected

for each Fe peak at time points corresponding to the max-

imum, shoulders on either side of the maximum, and at the

baseline before and after each peak. Similarly, altogethereight fractions were collected of all Zn-containing entities.

Human ferritin was quantified in the collected fractions by

microparticle enzyme immunoassay technology with an

Axsym analyzer (Abbott Diagnostics, Mississauga, ON,

Canada) using the manufacturers method and calibrators.

Human transferrin was measured by immunoturbidometric

assay with a Cobas Integra 700 analyzer (Roche Diagnos-

tics Canada, Laval, QC, Canada) using the manufacturers

method and calibrators, and human a2-macroglobulin was

measured with a Dade Behring BN2 Prospect rate nephe-

lometric immunoassay using the manufacturers reagents

(Dade Behring Canada, Mississauga, OC, Canada).With regard to the identification of factor V, fractions

were collected corresponding to the baseline before and

after Cu peak 1 as well as the peak itself. Factor Va

coagulation activity was determined by performing a

modified prothrombin time assay. In this assay, correction

of the clotting time of factor V-deficient plasma is pro-

portional to the concentration (activity percentage) of

factor Va in test plasma, interpolated from a calibration

curve [29]. Factor Va coagulation activity was determined

using an ACL TOP analyzer (Beckman Coulter, Palo Alto,

CA, USA) using factor V-deficient plasma, reagents, and a

HemosIL factor V assay protocol supplied by Instrumen-

tation Laboratory USA (Lexington, MA, USA).

To quantify the metal that corresponded to a detected

chromatographic metal peak, we injected increasing doses

of each metal onto the chromatographic system without a

column and measured the area under each peak using

SigmaPlot. This allowed us to establish a calibration curve,

which was used to calculate the total amount of metal (in

micrograms per 0.5 mL plasma) that was associated with a

detected metal peak (based on its peak area) in the sub-

sequent analysis. Finally, these data were used to calculate

the number of micrograms of metal that was present in

form of a certain metalloprotein per 1.0 mL of plasma for

comparison with literature data [7, 8].

Results and discussion

Although mammalian blood plasma can be easily obtained

and contains critical information about the essential trace

element status of the organism from which it was obtained,

the sheer complexity of the plasma proteome makes it

extremely difficult to extract relevant information about the

organisms health status. In fact, the human serum prote-

ome comprises at least 3,700 proteins [30], which poses an

almost insurmountable problem from an analytical sepa-

ration viewpoint. The very complexity of analyzing plasma

for the proteins contained within it, however, can be

reduced dramatically if one is able to selectively analyze

for a subproteome, such as the metalloproteome (in thecontext of this paper this term refers to all major plasma

proteins with bound Cu, Fe, and Zn). This would require

the separation of these metalloproteins from each other,

whichsince the molecular masses of all major metallo-

proteins in plasma are knowncan be achieved by

choosing a SEC stationary phase with the appropriate

fractionation range. Following this basic approach, we

have developed a rapid SEC-ICP-AES method to directly

analyze plasma for the major Cu-, Fe-, and Zn-containing

metalloproteins (by essentially determining the retention

time of the metals corresponding to these metalloproteins).

Conceptually, the detection of metal peaks within thechromatographic window (between the exclusion volume

and the inclusion volume) together with the established

stability of the major plasma metalloproteins of Cu, Fe, and

Zn [1, 3133] would imply that each detected metal peak

corresponds to a plasma metalloprotein. Furthermore, the

absence of tailing in the detected Cu, Fe, and Zn peaks

would further substantiate that each metalloprotein

remained intact during the entire LC separation process.

With regard to the analysis of rabbit plasma, interanimal

variation was expected and experimentally quantified

(Table 3). Excluding the standard deviation of the diag-

nostically inadequate Cu peak 1 (factor V, which disappears

after 0.5 h), the average relative standard deviation for all

Table 3 Average concentration of Cu, Fe, and Zn associated with

metalloproteins derived from size-exclusion chromatographyinduc-

tively coupled plasma atomic emission spectrometry analysis of

rabbit plasma samples (N= 18)

Metal Protein(s) Average metal

concentration

(lg/mL plasma) SD

Cu Factor V and transcuprein 0.85 0.73

Ceruloplasmin 0.46 0.14

Albumin 0.65 0.29

Small molecular weight 0.21 0.12

Fe Ferritin 0.27 0.16

Transferrin 2.66 0.99

Zn a2-Macroglobulin and

unidentified peaks 24

0.84 0.19

Albumin 1.10 0.24

The peak areas of metalloproteins that were not distinct were com-

bined for integration

SD standard deviation


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detected Cu, Fe, and Zn metalloproteins was 39%. In con-

trast to this, the method reproducibility is excellent (see

Stability of plasma metalloproteins).

The task of qualitatively identifying the detected plasma

metalloproteins is simplified as only approximately ten

major Cu, Fe, and Zn metalloproteins have so far been

reported in mammalian plasma (Table 1) [1]. In principle,

two strategies can be employed to qualitatively identify anindividual metalloprotein. First, it can be definitively

identified on the basis of either its enzymatic activity or a

specific antibody target site on its surface (e.g., using an

enzyme immunoassay). However, if neither of these tech-

niques is applicable because the metalloprotein has no

inherent enzymatic activity (e.g., if it functions exclusively

as a transport protein) or because no enzyme immunoassay

is readily available (for the organism of interest; in our case

rabbits), the second strategy to tentatively identify a

metalloprotein must be used. The latter involves the utili-

zation of information that is derived from the Cu-, Fe-, and

Zn-specific chromatogram in conjunction with literaturedata. For instance, the retention time of an unknown

metalloprotein relative to a known and abundant protein,

such as albumin (66 kDa), can be indicative of its hydro-

dynamic radius and thus its approximate molecular mass

(assuming minimal proteinprotein interactions). In addi-

tion, the intensity of a metal peak (corresponding to a

metalloprotein) relative to another metal peak (of a dif-

ferent metalloprotein containing the same metal) contains

information about the relative abundance of metal atomsthat are associated with these two proteins in plasma. In

this instance, the experimentally determined relative

retention time and abundance of both metalloproteins can

be compared with their known molecular mass and abun-

dance from handbooks on human metalloproteins [9] to

tentatively identify both metalloproteins.

Plasma versus serum metalloprotein analysis

To establish whether plasma or serum contains a larger

number of individual Cu-, Fe-, and Zn-containing entities

and therefore more information with regard to the healthstatus of an organism, we applied the SEC-ICP-AES

Fig. 3 Simultaneous

multielement-specific

chromatograms of rabbit plasma

on a Superdex 200 10/300 GL

(13 lm particle size) SEC

column with a phosphate-

buffered saline mobile phase

(pH 7.4, 22 C); flow rate

1.0 mL/min, injection volume

500 lL, ICP-AES detector.

Emission lines for a C at

193.091 nm (black), S at

180.731 nm (orange), and P at

213.618 nm (pink) and for b Cu

at 324.754 nm (green), Fe at

259.940 nm (blue), and Zn at

213.856 nm (red). Both a and b

were obtained from the same

rabbit plasma sample. The SEC

column was size-calibrated with

a mixture of standards

(thyroglobulin 670 kDa,

c-globulin 158 kDa, ovalbumin

44 kDa, myoglobin 17 kDa, and

vitamin B12 1.35 kDa). The

qualitative identification of the

metalloproteins factor V,

a2-macroglobulin,

ceruloplasmin, ferritin, and

transferrin in collected fractions

by various enzyme-based assays

(see Materials and methods)

is indicated by horizontal bars


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method developed to analyze plasma (n = 3) and serum

(n = 3) of 4.5-h-fasted rabbits. Cu peak 1 in the plasma

chromatogram at the bottom of Fig. 3 was absent in the

serum chromatogram (data not shown). Because the num-

ber and intensity of all other detected peaks remained

unchanged (data not shown), the Cu metalloprotein corre-

sponding to Cu peak 1 must be either directly or indirectly

(e.g., by specific adsorption) involved in the blood clottingprocess. This Cu peak could possibly represent blood

coagulation factor V, which is a single-chain glycoprotein

that contains one Cu per molecule [34] and is known to be

sensitive to proteolysis [35]. Although factor V has a

molecular mass of 330 kDa, it is known to self-associate to

form higher multimers [36, 37], which could explain its

elution in essentially the void volume. On the basis of these

results, plasma contains more information than serum for

the desired application of the instrumental analytical

method developed for diagnostic purposes.

Stability of plasma metalloproteins

To address a possible degradation of metalloproteins at

room temperature (22 C) over time, plasma was analyzed

using the SEC-ICP-AES system at 0.5, 1, 1.5, and 2 h after

blood collection. This experiment was carried out twice

and the results essentially showed the same overall trend.

Typical Cu-, Fe-, and Zn-specific time-course chromato-

grams are shown in Fig. 4.

As depicted in this chromatogram, the Cu that was eluted

prior to 800 s disappeared from plasma after the 0.5-h time

point. In addition, the peak corresponding to Cu that was

eluted at approximately 900 s (it likely corresponds to

albumin-bound Cu; see the discussion below) and the one

corresponding to the Cu that was eluted at approximately

1,200 s (small molecular weight Cu; see the discussion

below) decreased, whereas the most intense Cu peak

(ceruloplasmin; see the discussion below) increased to some

extent. The discrepancy between the reduction in intensity

of some Cu peaks over time versus the increase of the most

intense Cu peak must be attributed to the loss of Cu (net Cu

loss of approximately 30%) either to the container wall (that

the plasma was kept in prior to analysis) or to the stationary

phase of the SEC column. These, however, are only the two

most likely explanations and at present the exact cause is

unknown. Therefore, if one aims to detect all Cu metallo-

proteins (including the labile ones) in plasma, the latter

must be analyzed within 0.5 h after blood collection.

The intensity of the Fe and Zn peaks remained virtually

unchanged over the 2-h time period (Fig. 4). These results

strongly suggest that the corresponding metalloproteins are

stable andmore importantlythat the analytical method

itself produces results that are sufficiently reproducible for

diagnostic applications.

C-, S-, and P-specific chromatogram of plasma

The analysis of plasma with the method developed and the

simultaneous online detection of C, S, and P using the ICP-

AES resulted in the three-element-specific chromatogram

shown at the top of Fig. 3 and revealed four major

C-containing protein bands. The S-specific chromatogram

C

ounts/s

0

50

100

150

200

250

Cu

Vo

Counts/s

0

100

200

300

0.5 h

1.0 h

1.5 h

2.0 h

Fe

Vo

Retention Time (s)

600 800 1000 1200 1400

Counts/s

0

20

40

60

80

100

120

140

Zn

Vo

Fig. 4 Simultaneous Cu-, Fe-, and Zn-specific chromatograms of

rabbit plasma over a 2-h time period (after collection) on a Superdex

200 10/300 GL (13-lm particle size) SEC column with a phosphate-

buffered saline mobile phase (pH 7.4, 22 C); flow rate 1.0 mL/min,

injection volume 500 lL, ICP-AES detector. Cu-, Fe-, and Zn-

specific chromatograms were obtained in 0.5-h intervals at room

temperature and the emission lines of each element (Cu at

324.754 nm, Fe at 259.940 nm, and Zn at 213.856 nm) were plotted

on top of each other


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closely resembled that of the first three C-containing pro-

tein bands, which is expected since most mammalian

proteins contain the S-containing amino acids L-cysteine

and/or L-methionine. Interestingly, the fourth S-containing

entity was eluted before the fourth C-containing entity

(both of these correspond to small molecular weight pep-

tides and amino acids). Since albumin is by far the most

abundant mammalian plasma protein (approximately 50 g/L)and comprises more than half of the total protein in plasma

[9], the most prominent C bandband 3must be pre-

dominantly composed of albumin (a BSA standard had the

same retention time). Owing to the utilization of a P-con-

taining mobile phase (PBS), the P-specific chromatogram

displayed an elevated P baseline throughout the entire

chromatographic window (Fig. 3, top). The P-emission

intensity also provided an effective measure of the mass

transfer of droplets from the nebulizer chamber to the

plasma. The detection of the albumin peak did not affect

the intensity of the P-emission line, which demonstrates

that the injected total protein did not adversely affect themass transfer from the nebulizer to the plasma (e.g., by a

surfactant effect), which is an important prerequisite to

accurately measure the total metal that is associated with

an eluting plasma metalloprotein. In addition, the P-spe-

cific chromatogram also revealed a characteristic dip at a

retention time of about 1,210 s, which coincides with the

elution of the small molecular weight C band 4, and

therefore corresponds to the injected plasma plug (which

contains less P than the mobile phase) reaching the

detector. A similar phenomenon has previously been

observed [38].

Cu, Fe, and Zn metalloproteins in plasma

The analysis of plasma (eight different animals) by SEC

and the simultaneous online detection of Cu, Fe, and Zn

using the ICP-AES resulted in a three-element-specific

chromatogram, a representative of which is shown at the

bottom of Fig. 3. At first glance, five Cu-containing, two

Fe-containing, and approximately five poorly separated Zn-

containing entities were detected. The majority of the

detected metal peaks displayed an ideal peak shape, which

suggests that the metals did not dissociate from their parent

protein during the chromatographic separation process.

However, the second Fe peak, the last Zn peak, and Cu

peaks 2 and 4 displayed a hump on the long retention end,

which can be rationalized by the elution of a slightly

smaller metalloprotein containing the same metal. Given

the inherent limitations of SEC with regard to the chro-

matographic resolution of proteins of almost similar size

from each other, this is not unexpected. Nevertheless, the

detection of approximately 12 metalloproteins demon-

strates that the optimized mobile phasestationary phase

combination is well suited to separate the major Cu-, Fe-,

and Zn-containing entities that are contained in rabbit

plasma. Importantly, and in contrast to the peaks in the

chromatograms at the top Fig. 3, the individual Cu, Fe, and

Zn peaks in the chromatograms at the bottom of Fig. 3

correspond to individual metalloproteins andsince Cu

peak 5 was in the small molecular mass region

metallopeptides.The Cu-specific chromatogram revealed five peaks,

which is one more than has been reported in other studies

(Table 2) (peak 1: approximately 515 s, approximately

28% of total Cu, approximately 0.9 lg Cu/mL; peak 2:

approximately 605 s, approximately 13% of total Cu,

approximately 0.3 lg Cu/mL; peak 3: approximately 775 s,

approximately 27% of total Cu, approximately 0.9 lg

Cu/mL; peak 4: approximately 890 s, approximately

19% of total Cu, approximately 0.6 lg Cu/mL; peak 5:

approximately 1,230 s, approximately 13% of total Cu,

approximately 0.3 lg Cu/mL; Fig. 3, bottom). The sum of

all Cu peaks in this particular plasma sample amounted to3.0 lg Cu/mL plasma, which is higher than the average total

Cu concentration (2.14 lg/mL) in the 18 plasma samples

that were analyzed (Table 3). Thus, the total rabbit plasma

Cu concentration was higher than what has been reported for

other mammalian species (range 0.22.0 lg/mL) [7]. Cu

peak 1 appeared close to the void volume and was not

observed in previous studies as evidenced by comparing the

relative intensities of the observed Cu peaks with the Cu-

specific chromatograms of previous studies [20, 22, 32].

This discrepancy, however, can be easily explained by the

fact that previous studies analyzed either aged plasma or

serum, which according to our investigations both lack Cu

peak 1 (Fig. 4). On the basis of the observation that Cu peak

1 was absent from serum and disappeared from plasma after

30 min (Fig. 4), it was tentatively identified as factor V,

which is known to be labile, contains Cu [ 34], and is present

in human plasma at approximately 10 mg/L [35]. This was

corroborated by analyzing collected fractions for factor V

coagulation activity (bar in Fig. 3, bottom), which was

highest at the maximum intensity of Cu peak 1. The activity,

however, extended beyond this peak, which can be ratio-

nalized by the fact that under the circumstances of blood

sampling factor V is expected to be activated into factor Va,

which has a smaller molecular mass (approximately

221 kDa) and could explain the observed tailing of the

activity. Factor V is known to be very labile and prone to

proteolysis, which may explain the rapid disappearance of

Cu peak 1 in the time-course experiments (Fig. 4) [39].

According to our analytical data and assuming an identical

stoichiometry in rabbit and human factor V (Table 1), we

calculated a plasma concentration of 4.7 g/L, which is sev-

eral-fold higher than its concentration in human plasma and

is therefore in apparent disagreement. We note, however,


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that evidence in favor of significant interanimal species

differences of certain plasma metalloprotein concentrations

have been reported [40]. Furthermore, a different metal-

to-protein stoichiometry between human and rabbit factor V

could significantly affect the calculated plasma con-

centration. With regard to the retention time of this

metalloprotein (Fig. 3, bottom) it is noteworthy that

dimerization of this protein has been observed by others [36,37]. This would result in a 660-kDa entity and could explain

the elution of factor V close to the void volume. On the basis

of the previously reported order of elution of Cu plasma

metalloproteins from a SEC column [32], Cu peak 2 was

tentatively identified as the 270-kDa protein transcuprein,

which is also known to dimerize (540 kDa) [4]. This Cu

peak had a small shoulder on the long retention end, which is

in accord with the Cu-specific chromatogram in Fig. 4. Cu

peak 3 had ceruloplasmin oxidase activity (bar in Fig. 3,

bottom) and was therefore identified as the glycoprotein

ceruloplasmin. On the basis of the experimentally deter-

mined total Cu associated with ceruloplasmin in 1.0 mL ofplasma and the known stoichiometry of Cu in this protein

(Table 1), the plasma ceruloplasmin concentration was

calculated at 0.31 g/L, which is within the concentration

range reported for human serum. Cu peak 4 was compara-

tively broad and appeared 11 s after albumin (dotted line in

Fig. 3, bottom). The misalignment of this Cu peak with the

albumin peak can be rationalized either by a rather weak

binding of Cu to albumin (which has been reported by others

[41]) or by the presence of a smaller Cu-containing entity in

addition to the expected albumin-bound Cu [4, 32]. On the

basis of the simultaneous appearance of Cu peak 5 (Fig. 3,

bottom) with the last C band (Fig. 3, top), this Cu peak

represents Cu bound to small non-S-containing peptides and

amino acids, such as L-histidine [42] (the retention time for S

band 4 in Fig. 3, top was different from that for Cu peak 5 in

Fig. 3, bottom) and is in general agreement with other

studies [32]. We note that the observed order of elution for

all major Cu entities is as expected on a SEC column

and follows decreasing molecular masses from 660 kDa

(putative factor V dimer), 540 kDa (transcuprein dimer),

132 kDa (ceruloplasmin), 66 kDa (albumin), and small

molecular weight Cu.

The Fe-specific chromatogram revealed two baseline-

separated peaks, which is identical to the maximum num-

ber of Fe peaks that was previously reported (Table 2)

[8, 19, 23, 24, 43, 44] (peak 1: approximately 670 s, 11%

of total Fe, approximately 0.3 lg Fe/mL; peak 2: approx-

imately 870 s, 89% of total Fe, approximately 2.6 lg

Fe/mL; sum of all Fe peaks: 2.9 lg Fe/mL plasma) (Fig. 3,

bottom). On the basis of the known molecular size and

plasma abundance of the two major Fe-containing metal-

loproteins ferritin and transferrin (Table 1), Fe peak 1 is

identified as ferritin and Fe peak 2 as transferrin. This peak

assignment was confirmed by enzyme immunoassay and

immunoturbidometric assay (bars in Fig. 3, bottom). On

the basis of the experimentally derived total Fe associated

with ferritin and transferrin in 1.0 mL of plasma and

assuming that both Fe metalloproteins were fully loaded

with Fe (Table 1), the rabbit plasma concentration of fer-

ritin was calculated as 535 lg/L and that of transferrin as

1.8 g/L. Even though these results are in overall accordwith the established concentrations of these metallopro-

teins in mammalian plasma/serum (Table 1), we point out

that the method developed cannot inherently determine the

metal loading of a metalloprotein in which the metal

loading may vary. It is therefore impossible to distinguish

if the Fe that is associated with, e.g., ferritin is attributable

to (1) 50% apoferritin and 50% fully loaded holoferritin or

(2) the case where all ferritin is 50% loaded with Fe.

Nevertheless, our method allows us to determine the dis-

tribution of a metal among various metalloproteins, which

has inherent diagnostic value that cannot be obtained by

conventional antibody-based enzyme assays. The distinctshoulder on the long retention end of Fe peak 2 indicates

the presence of another Fe-containing entity. On the basis

of previous studies which demonstrated that Fe is bound to

human serum albumin in serum [23], this additional Fe-

containing entity could be albumin-bound Fe especially

since albumin is approximately 14 kDa smaller than

transferrin, which would therefore explain its elution after

transferrin. In contrast to Cu, however, no detectable Fe

was eluted in the small molecular weight range, which

indicates that Fe is not bound to peptides and amino acids

in rabbit plasma.

With respect to Zn, approximately five non-baseline-

separated peaks were detected (peak 1: approximately

613 s, 10% of total Zn, approximately 0.1 lg Zn/mL;

peaks 24: approximately 655 s, approximately 700 s,

approximately 770 s, 34% of total Zn, approximately

0.6 lg Zn/mL; peak 5: approximately 880 s, 56% of total

Zn, approximately 1.1 lg Zn/mL; sum of all Zn peaks:

1.8 lg Zn/mL plasma) (Fig. 3, bottom), which is more than

the average number of Zn peaks that has previously been

reported (Table 2) [31, 44, 45]. This finding can be ratio-

nalized with the higher-resolution SEC column that was

used in the present study (13-lm particles) compared with

earlier studies. Zn peak 1 likely represents the 725-kDa a2-

macroglobulin [46] since between 12 and 31% of human

plasma Zn has previously been reported to be tightly

incorporated in this protein, which is in accord with our

results [25, 31]. This peak assignment was confirmed by

enzyme immunoassay (bars in Fig. 3, bottom) and is in

accord with another study, which also identified the first Zn

compound that was eluted from a SEC column as a2-

macroglobulin [31]. Even though the putative a2-macro-

globulin was eluted before ferritin (450 kDa), it was eluted


123
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12/15

after the void volume, which is in discord with what would

be predicted if its retention were solely based on its

molecular mass. We note, however, that nonideal interac-

tions between a2-macroglobulin and a SEC stationary

phase (TSK-G4000SW) have been observed using PBS

when the native protein was treated with chemicals which

exposed hydrophobic amino acids to the surface [47] and

resulted in the adsorption of this protein to the stationaryphase. Similar behavior could also occur between the

components of plasma and a2-macroglobulin and subse-

quently the stationary phase in our experiments. Using the

experimentally determined total Zn that is contained in

1.0 mL of plasma in form of this metalloprotein (approx-

imately 0.1 lg Zn/mL), we calculated the rabbit plasma a2-

macroglobulin concentration (using the stoichiometry

delineated in Table 1) at 222 mg/L, which is approxi-

mately one tenth of its concentration reported for human

plasma (Table 1). We note, however, that significant in-

teranimal species differences regarding certain plasma

metalloprotein concentrations have been reported [40, 48].On the basis of the identical retention times for Zn peak 5

and albumin (dotted lines in Fig. 3) and since albumin-

bound Zn represents the major Zn entity in plasma (56% of

total plasma Zn in this study, which is in good accord with

previous studies on humans [26, 31] and pigs [44]), this Zn

entity likely represents albumin-bound Zn (transferrin does

not bind Zn2? [44]). Zn peaks 24 and the Zn shoulder on

the long retention end of Zn peak 5 could not be qualita-

tively identified. However, the existence of a 165-kDa

extracellular secretory glycoprotein Cu,Zn superoxide

dismutase (EC-SOD) and that of a 31-kDa Cu,Zn super-

oxide dismutase (Cu,Zn-SOD) have been reported in

guinea pig and human plasma [40, 49]. It is therefore likely

that EC-SOD represents one of the unidentified Zn peaks

24 and that the shoulder on the long retention end of Zn

peak 5 could possibly be Cu,Zn-SOD. Similar to the results

obtained for Fe, no Zn was detected bound to small

molecular weight peptides and amino acids in rabbit

plasma.

Practical applications

Owing to the fact that the SEC-ICP-AES method devel-

oped allows one to determine the plasma Cu, Fe, and Zn

metalloproteome within approximately 24 min, two major

practical applications of this method can be envisioned.

The first application is its utilization as a clinical tool to

screen for early- or advanced-stage human diseases by

the direct analysis of human plasma or serum [5057].

Even though assays exist to quantify individual

plasma metalloproteins, such as ceruloplasmin (e.g., by a

spectrophotometric activity assay), few methods have

been reported that can simultaneously determine all

metalloproteins of one element, let alone those of more

than one element simultaneously. Therefore, this method

has the obvious advantage of extracting more information

(namely, the relative abundance of the metalloproteins of

the three major essential trace metals in plasma as well as

the concentration of those metalloproteins in which the

metal-to-protein ratio is fixed given that no other metal-

loprotein containing the metal of interest is coeluted) froma single analysis in a given amount of time than is possible

with other methods that are currently in use. This, in turn,

can be helpful to more accurately diagnose the severity of a

disease since Wilsons disease, for instance, is not only

associated with decreased plasma concentrations of the Cu

metalloprotein ceruloplasmin [58], but can also result in an

increased plasma concentration of the Fe metalloprotein

hemoglobin during episodes of acute hemolysis [52].

The second application is the utilization of the method

developed to probe the nonenzymatic bioinorganic chem-

istry of environmentally abundant pollutants, such as toxic

metals and metalloid compounds, in the mammalianbloodstream to better understand their chronic toxicity,

individually andmore importantlycumulatively. This

latter application appears particularly relevant since bio-

inorganic processes in the mammalian bloodstream are

likely to be fundamentally involved in the origin of

numerous human diseases that are associated with chronic

exposure to toxic metals and metalloid compounds [59,

60].

Conclusions

The daunting analytical task of extracting health-relevant

information from plasma can be considerably simplified

by determining a subproteome, such as the Cu, Fe, and

Zn metalloproteome. To this end, we have developed a

rapid SEC-based separation of the metalloproteins con-

tained in rabbit plasma followed by the online analysis of

the column effluent by an ICP-AES, which served as the

simultaneous Cu-, Fe-, and Zn-specific detector. This

novel SEC-ICP-AES method has allowed us to directly

analyze rabbit plasma in order to generate the Cu, Fe, and

Zn metalloproteome, which is composed of approximately

12 metalloproteins and metallopeptides, within approxi-

mately 24 min. From a clinical perspective, this simple

and rapid technique to establish the Cu, Fe, and Zn me-

talloproteome offers important advantages over individual

metalloprotein assays since much more information can

be extracted with this method from a single plasma

sample. Thus, the detection of the majority of the

expected Cu-, Fe-, and Zn-containing entities in rabbit

plasma by the SEC-ICP-AES system constitutes an

important first step in the development of an instrumental


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analytical technique for the efficient detection of the

plasma metalloproteome for potential diagnostic applica-

tions in humans. The method developed can also be used

to directly probe the bioinorganic chemistry of toxic

metals in whole blood and thus has considerable potential

to provide exciting new insights into the origin of toxic-

metal-related human diseases.

Acknowledgments This research was funded by the Natural Sci-

ences and Engineering Research Council (NSERC) of Canada.

Teledyne Leeman Labs is gratefully acknowledged for funding the

attendance of S.A.M. and J.G. at HPLC 2007 in Ghent, Belgium.

Katie L. Pei is gratefully acknowledged for help regarding the col-

lection of fractions. We would also like to extend thanks to Raymond

J. Turner and especially Arvi Rauk for constructive feedback on the

final draft of the manuscript. The staff of the Animal Health Unit

(LESARC) at the University of Calgary is gratefully acknowledged

for the maintenance of and the drawing of blood from the rabbits. We

would also like to extend sincere thanks to one anonymous reviewer

who provided valuable comments to significantly improve the final

manuscript.

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Simultaneous Cu-, Fe-, And Zn-Specific Detection of Metal Lo Proteins

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