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J Biol Inorg Chem (2009) 14:61–74 DOI 10.



Simultaneous Cu-, Fe-, and Zn-specific detection of metalloproteins contained in rabbit plasma by size-exclusion chromatography– inductively coupled plasma atomic emission spectroscopy
Shawn A. Manley Æ Simon Byrns Æ Andrew W. Lyon Æ ¨ Peter Brown Æ Jurgen Gailer

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

Abstract Analytical methods which are capable of determining the plasma or serum metalloproteome have inherent diagnostic value for human diseases associated with increased or decreased concentrations of specific plasma metalloproteins. We have therefore systematically developed 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 saline 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

Parts of the work described in this paper were presented at HPLC 2007 in Ghent, Belgium.

Electronic supplementary material The online version of this article (doi:10.1007/s00775-008-0424-1) contains supplementary material, which is available to authorized users.
S. A. Manley Á S. Byrns Á J. Gailer (&) Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada e-mail: 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

plasma within 30 min of collection results in the detection of one more Cu peak (blood coagulation factor V) than has been 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 (ferritin and transferrin, corresponding to an average of 9 and 91% of total plasma Fe) and approximately five Zn metalloproteins (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 SECICP-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 Á Size-exclusion chromatography Á Inductively coupled plasma atomic emission spectrometry Á Metalloproteins

Introduction All organisms must regularly ingest sufficient quantities of essential trace elements, such as Cu, Fe, and Zn, to maintain 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]. Following the absorption of essential trace elements from the gastrointestinal tract into the systemic blood circulation,


6].47–13. an inductively coupled plasma atomic emission spectrometer (ICP-AES). and reversed-phase chromatography have been employed in conjunction with various elementspecific detectors.1 lg Fe/mL [7.8–3.2–0. Despite this attractive proposition.500 2 1 0. The pioneering work of Dawson et al.1–53. published in 1981. It is well known that numerous genetic human diseases are associated with increased or decreased plasma concentrations of specific metalloproteins [9].6 g/L – – – 1. 0. for instance. an instrumental analytical method which can rapidly determine the major Cu-.62 J Biol Inorg Chem (2009) 14:61–74 Table 1 Molecular properties and relative abundances of the major metalloproteins and metallopeptides in human plasma or serum Metal Fe Cu Metalloprotein or entity which contains bound metal Ferritin Transferrin Blood coagulation factor V Transcuprein Ceruloplasmin Albumin EC-SOD Cu. [16]. 49] [40] – [4. and Zn. All studies which reported on the direct LC analysis of mammalian plasma or serum for metalloproteins are listed in Table 2 (only those which detected at least two of the elements of interest are listed). and Zn metalloproteins [9]. 9] [9] [40] [40] SOD superoxide dismutase.Zn-SOD a Molecular mass (kDa) 450 79. should allow the detection of individual plasma metalloproteins assuming that the metal–protein bond(s) in the latter remain intact during the LC separation process [11–14]. Fe. a graphite furnace atomic absorption spectrometer. Fe-.46–1. 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. the analysis of plasma for the contained major Cu-. and Zn-containing plasma metalloproteins would represent an innovative tool to assist in screening or diagnosing human diseases associated with altered essential trace elements. and 2. Therefore. Wilson’s disease. Therefore. 9] [40. From an analytical point of view. EC-SOD extracellular Cu. 8].6 g/L 36.5 6 1 4 – – 5 1 4 – Plasma or serum protein concentration 10–250 lg/L 1. the direct liquid chromatography (LC) analysis of plasma in conjunction with an element-specific detector. The reconstruction of a Cu. such as endocytosis in the case of Fe [5.1–53.84–1.7 g/L *10 mg/L *180 lg/La 0. comparatively few studies have been carried out to attempt this goal in undiluted mammalian plasma or serum [15]. one Fe. Fe-. and Zn-containing metalloproteins (Table 1) will provide insight into the roles of trace elements in the biochemistry and pathophysiology of both healthy and diseased states. In particular.1–3. such as a flame atomic absorption spectrometer. 32] [9] [4. anion-exchange chromatography (AEX). sizeexclusion chromatography (SEC).45 lg Cu/mL. 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. Among the most abundant transition metals present in human plasma are Cu. and even though numerous investigations have been reported on the speciation of metals and metalloid compounds in other biological fluids and tissues.and Zn-specific chromatogram revealed one Cu and three Zn peaks [16].6 g/L – – References [9] [9] [34] [4. Conversely. Fe. which are present at total concentrations of 0.7 g/L 36. or an inductively coupled plasma mass spectrometer (ICP-MS).Zn-SOD Peptides and amino acids Zn a2 macroglobulin Albumin EC-SOD Cu.Zn superoxide dismutase Rat plasma specific plasma proteins subsequently distribute these elements to internal organs [4]. 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.67 lg Zn/mL. 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].7 330 270 132 66 165 31 \5 725 66 165 31 Number of metal atoms bound per protein B4. regardless of whether the latter have a genetic origin or are the result of exposure to environmental pollutants (chemical or bacterial). where absorption occurs by highly specific uptake mechanisms. and three Zn peaks 123 .

Fe. RP reversed-phase chromatography.02 M NaH2PO4 ? 0.1) – 25 9 0. and Zn-specific detector for human serum analysis using AEX (MonoQ HR) [23. ODS octadecyl silica. SRM standard reference material a b c d Artifact Volume in mL No. Tris tris(hydroxymethyl)aminomethane. 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].02) Human serum (0.5 M NaCl [16] [17] [18] [19] [20] [22] [8] [24] Sephadex G-100 (40–120) 0. AEX.2 60 9 1.4 0.2 mM CHAPS pH 7. 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).25 M NH4Ac in A) SEC size-exclusion chromatography.8. HEPES N-(2-hydroxyethyl)piperazine-N0 -ethanesulfonic acid.4) ? B (0. 4 °C Sephacryl S-300 (25–75) TSK G 3.05 M Tris/HCl pH 7.1 M Tris/HCl pH 7. Fe-.5) Human serum (1.6 – 30 9 0. and Zn metalloprotein peaks that were detected by LC analysis of mammalian plasma or serum (Table 2) and to 123 . and six non-baseline-separated Zn peaks [22].1 M NaCl pH 7.9 Fractogel EMD BioSEC 650 (20–40) Mono Q HR (10) 0.7 25 9 0. the SEC analysis (TSK G 300 SW) of human serum brought to light one Cu.4. 30 °C 15 min linear gradient A (0.1) Cu (1)a Fe (2) AEX Zn (4)a Cu (2) Zn (2) Cu (1) Fe (1) Zn (1) SEC RP Human serum (0.5 mM CaCl2 pH 7. one Fe.1) Human serum SRM (0.1 M HEPES ? 0. Reversed-phase chromatography (octadecyl silica stationary phase) was also employed to analyze human serum using an ICP-MS as the Cu-. and four Zn peaks [19]. With use of a sequential ICP-AES as the online multielement-specific detector.05 M Tris/HCl pH 7. 24] and employing a mobile-phase gradient. In view of the reported variability in the number of Cu.4 0. 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.6 100 9 2.25) Cu (3) Fe (2) Human serum (0.4.1 M Tris/HCl pH 6. and three Zn peaks [18].6 5 9 0.1 M Tris/HCl pH 7. 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]. 22 °C 0.1 M Tris/HCl pH 8. however.4) ? B (0.2 SynChropak GPC 300 (5) 0.0) Human serum (5.25) Rat serum (0.46 5 9 0.3 M NaCl pH 6.5 Sephacryl S-300 (25–75) Mobile-phase composition Reference 0. of peaks Bead diameter in lm [17]. two Fe. AEX anion-exchange chromatography.000 SW (10) TSK G 3.0 ? 0. one Fe. CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate.1 M Tris/HCl ? 2. A double focusing ICP-MS was also employed as an online Cu-.4 Human plasma? (0.J Biol Inorg Chem (2009) 14:61–74 63 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 analyzedb Human plasma (0.2 mM Tris/HCl ? 0. Fe-. More recently. Applying the same stationary phase for the analysis of rat serum in conjunction with an ICP-MS revealed four Cu and three Zn peaks [20]. altered the speciation of Cu and Zn and must therefore be avoided when plasma/serum metalloproteins are to be determined for potential diagnostic applications.5 Superose 12 HR (8–12) CHAPS-coated ODS Mono Q HR (10) [25] [21] Cu (1)a Fe (2) AEX Zn (4)a [23] 15 min linear gradient A (0.05 M Tris/HCl pH 7.000 SW (10) 0.2) Human serum (0. but revealed that all metalloproteins containing these elements were essentially coeluted [21]. and Zn-specific detector.9 100 9 2.002) Elements detectedc Cu (1) Zn (3) Cu (1) Fe (1) Zn (3) Cu (2) Fe (1) Zn (3) Cu (1) Fe (2) Zn (4) Cu (4) Zn (3) Cu (4) Fe (1) Zn (6) Separation Column Stationary phased mechanism dimensions (cm) SEC SEC SEC SEC SEC SEC SEC 57 9 0.0) Human serum (0.25 M NH4Ac in A) 0.

We have systematically developed a novel LC method for the determination of Cu. 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. at room temperature) before ex vivo degradation of certain metalloproteins will occur.35 kDa). With regard to the detection of the separated Cu-. Billerica.45-lm nylon filter membranes (Mandel Scientific. myoglobin (17 kDa). UK). we employed phosphate-buffered saline (PBS. Fe. and vitamin B12 (1. Guelph. CA.0 mL/min (peristaltic pump). Fe-. PBS (10 mM phosphate. and the bicinchoninic acid protein determination kit were purchased from Sigma-Aldrich (St Louis. 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. Fe-. USA).64 J Biol Inorg Chem (2009) 14:61–74 develop this analytical approach into a clinically useful diagnostic tool. Canada). pH 7.5% pure). and Zn. USA) which contained thyroglobulin (670 kDa). it should be examined if plasma and serum generate consistent analytical results (whichever contains more individual metal peaks in the corresponding Cu-. 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 metal—are least likely to occur. Fe. MA. Canada). N. Fe. we systematically screened three commercially available SEC stationary phases (Sephacryl S-500. Canada). and Zn-specific chromatogram inherently contains more information). a standard protein mixture (BSA and lysozyme) was chromatographed before and after the analysis of six consecutive plasma samples. Furthermore.5% salt [27]. 29 and 19) and a UV detector (280 nm). Following these considerations to least disrupt weak metal–protein binding equilibria during the SEC separation process. and Zn-containing metalloproteins in the SEC column effluent.N-dimethyl-p-phenylenediamine monohydrochloride (highly toxic). All solutions were prepared with water from a Simplicity water purification system (Millipore. c-globulin (158 kDa). 2. we chose SEC as the separation mechanism. whereas AEX often requires salt gradients to elute proteins which can—in turn—sever weak transition metal–protein linkages. 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. 26] and to maintain the integrity of the Cu. two key questions must be investigated. dissociation of a metal from its parent plasma protein is minimized. In view of the fact that the binding of transition metals in plasma metalloproteins can be weak [4. In addition.. Ceruloplasmin oxidase activity was measured in collected fractions according to a published procedure [28]. 137 mM NaCl) tablets. ovalbumin (44 kDa). and Zn metalloproteins throughout the entire LC separation process [19]. 2. ON. First. sodium acetate trihydrate (more than 99% pure).7 mM KCl. Second.4) as the isocratic mobile phase. A shift of the retention times 123 . ON. glacial acetic acid (more than 97.7 mM KCl. lysozyme (from chicken egg white). bovine serum albumin (BSA) was from Amersham Pharmacia Biosciences (Little Chalfont. to make 1 M acetic acid) was from Fisher Scientific (Nepean. USA). MO. A mixture of protein standards for SEC column calibration was obtained from Bio-Rad Laboratories (Hercules. To detect irreversible binding of plasma proteins to the stationary phases. heparin (sodium salt). we utilized a state-of-the-art charge injection device based ICP-AES because this multielement-specific detector could be directly hyphenated to the separation column for the simultaneous online detection of Cu. which is required to preclude irreversible adsorption of plasma proteins to the stationary phase. this detector is compatible with LC separations involving mobile phases containing more than 0.5 mL) using various PBS concentrations (39. Each column was equilibrated with at least 50 mL of the mobile phase before plasma was injected. the stability of metalloproteins in plasma/ serum over time must be investigated to establish the maximum time that plasma samples can be kept (e. and Zn metalloproteins in rabbit plasma which can be potentially applied to any mammalian plasma. 25 cm 9 1. PBS of pH 7.4 (10 mM phosphate. and Plasma PURE HCl (34–37%) was from SCP ´ Science (Baie D’Urfe.7% pure. Materials and methods Chemicals and solutions Blue dextran. and Superdex 200 prep grade.g. Superose 6 prep grade. QC. 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. sodium chloride (more than 99. This is because SEC minimizes direct interactions between the analyte molecules and the stationary phase and therefore represents a comparatively gentle separation mechanism for the separation of biomolecules. In addition. Finally. Therefore. SEC can be employed in an isocratic separation mode (which inherently increases sample throughput).0 cm column) for their ability to analyze rabbit plasma (0.

091 nm).5-h-fasted rabbits at approximately 13:30 and was centrifuged at 1.0 mL of water) was used to check the column integrity before and after the injection of six plasma samples. this was obtained from a healthy male volunteer. USA) into BD Vacutainer blood collection tubes (no additive. To establish the interanimal variation. MA.0-cm inner diameter. BD Vacutainer. see the supplementary material). After six consecutive plasma injections. 14% (band 2). 1) consisted of a Waters (Milford.0 cm 9 1. AB. The collection of human blood was approved by the University of Calgary Conjoint Health Research Ethics Board (approval no. Cu (324. 89. one human plasma sample was chromatographed. CA. inner diameter 0.100g (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. and a nebulizer gas 123 . Zn (213. Franklin Lakes.5 mm). with 10. Fe and Zn metalloproteins in collected fractions after the analysis of rabbit plasma. Cu. Canadian Lab Diets. no irreversible protein binding was detected with any stationary phase–mobile phase combination for the seventh injection (protein recovery 99 ± 1%. In particular. Leduc. or at the time points indicated later. 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). a Rheodyne 9010 PEEK injection valve (Rheodyne.940 nm). Superdex 200 prep grade resulted in four protein bands. USA) equipped with a 0.2 and 0.000 for the prepacked Superdex 200 column (30 cm 9 1. Rhonert Park.0-cm inner diameter) which contained 13-lm particles. separates globular proteins between approximately 600 and 10 kDa. and 1% of total protein. 34-lm particles) to approximately 23. we improved the separation of plasma bands by using a prepacked Superdex 200 column (30 cm 9 1. Canada) and fed ad libitum on a ‘‘high-fiber’’ diet (Lab Diet 5321. and a prepacked SuperdexTM 200 10/300 GL TricornTM high-performance column (30. 18 rabbit plasma samples were consecutively analyzed using the SEC-ICP-AES system. Experimental setup of the optimized SEC-ICP-AES system The SEC-ICP-AES system (Fig. NJ. St Louis. Hudson. an RF power of 1. Superdex 200 prep grade and PBS (19) were identified as the ideal stationary phase– mobile phase combination. respectively.3 kW.000 for Superdex 200 prep grade (25.J Biol Inorg Chem (2009) 14:61–74 65 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.856 nm). USA). In contrast. the number of theoretical plates was calculated (using the lysozyme peak) and increased from approximately 1.62 mg in 5. radial-view ICP-AES (Teledyne Leeman Labs. Male New Zealand white rabbits were purchased from Casey Vandermeer (Edmonton. In addition. S (180. high-dispersion. the blood clot was removed by centrifugation (described below). The injection of a heparin blank onto the SEC-ICPAES system (control experiment) revealed no detectable Fe. At least 30 consecutive plasma analyses could be carried out per column without loss of chromatographic resolution of the metal peaks. Only straw-yellow plasma (free of the characteristic red color of hemoglobin from ruptured erythrocytes) was used throughout the study.0 mL) was collected from the marginal ear vein with 20-gauge stainless steel blood collection needles (211 monoject. In view of the fact that stationary phases with a smaller particle size generally result in a better chromatographic resolution. corresponding to 15% (band 1). or Zn (data not shown). NJ. Sherwood Medical.731 nm). USA) at an Ar gas-flow rate of 19 L/min.0-cm inner diameter. Blood was collected from 4.5 mL of the plasma to establish the percentage protein recovery. Piscataway. and P (213. E-21198).618 nm) in the column effluent was achieved with a Prodigy. Canada). 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’’). To qualitatively identify the detected Cu. Again a mixture of BSA and lysozyme (1.4 cm 9 1. with 7 and 93% of total protein. and 2% (band 4) of total protein. NH. the column effluent of each plasma injection was analyzed for total protein (bicinchoninic acid assay) and compared with the total protein contained in 0. For the preparation of serum. Fe (259. Simultaneous multielementspecific detection of C (193. In addition. 69% (band 3). 13-lm particles). Blood (5. AB. Superose 6 prep grade produced only three protein bands. The exit of the SEC column was connected to the Meinhard concentric glass tube nebulizer of the ICP-AES with fluorinated ethylene–propylene Teflon tubing (30 cm.5-mL PEEK injection loop. and Sephacryl S-500 resulted in only two protein bands.5 mg heparin had been added for the preparation of plasma. USA) to which 0. 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. USA) model 510 high-performance LC pump. GE Healthcare.0-cm inner diameter. MO.754 nm).

and Superdex 200 10/300 GL SEC column (1. Furthermore.0 cm 9 30 cm. In general. 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. we did not utilize electrospray ionization mass spectrometry because of the salt content (approximately 1% or approximately 164 mM) of the mobile phase. the salt concentration of aqueous samples that can be analyzed by electrospray ionization mass spectrometry must be below 10 mM. The detector technology utilized in the Prodigy allows the simultaneous measurement of the peak and the background emissions to generate the net emission intensity. The raw data were imported into SigmaPlot 10 and smoothed using the bisquare algorithm. 13-lm particle size) (solid line). Since an antibody-based approach (e. In addition. 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. injection volume 500 lL. and the most abundant plasma protein albumin (which can be easily identified on the basis of the most intense C and S peaks in the chromatogram). and factor V was not readily available.080-s acquisition window). Void volume 600 kDa. 1 The instrumental analytical size-exclusion chromatography (SEC)– inductively coupled plasma atomic emission spectrometer (ICP-AES) setup.. This capability is critical in experiments where the background emission intensity changes (e.0-min delay was implemented between injection and the beginning of data acquisition (1.66 Fig.g.g. These can therefore serve as a rough proxy to estimate the size of a detected metalloprotein. deductions of the molecular mass of an unknown metalloprotein from its retention time alone should be interpreted with caution. a2 macroglobulin. Identification and quantification of metalloproteins in SEC column effluent In terms of qualitatively identifying the separated plasma metalloproteins in the column effluent. Phosphate-buffered saline mobile phase (pH 7. are naturally present in plasma. 2 C-specific chromatograms of rabbit plasma on Superdex 200 prep grade (1. Time scans were performed using the time-resolved-analysis mode (Salsa version 3. Figure 2 depicts the C-specific SECICP-AES chromatograms of rabbit plasma obtained on a Superdex 200 prep grade (34 lm) and a prepacked (13 lm) column.0 cm 9 25 cm. According to the void volume of the Superdex 200 column (blue dextran). together with the ability of the ICP-AES to handle saltcontaining solutions.N-dimethyl-p-phenylenediamine) [28]. such as ferritin and transferrin (Fe metalloproteins). 34-lm particle size) (dashed line).. HPLC highperformance liquid chromatography J Biol Inorg Chem (2009) 14:61–74 pressure of 35 psi. We therefore qualitatively identified the Cu metalloprotein ceruloplasmin in collected fractions using an established ceruloplasmin oxidase activity assay (based on the oxidation of N. makes the Prodigy ideally suited for the LC analysis of solutions containing metalloproteins. the analysis of rabbit blood plasma provided internal molecular weight standards as several proteolytically stable plasma metalloproteins. flow rate 1. inclusion volume 10 kDa 123 . ICPAES detector (C emission at 193.700 proteins). ceruloplasmin (Cu metalloprotein). We size-calibrated the analytical Superdex 200 column with known molecular weight protein standards. it is likely that owing to the sheer complexity of plasma (more than 3. This advantage. Therefore. a 7.0 mL/min.4. transferrin. 22 °C).091 nm). which clearly displays the increased resolution of 80000 Albumin (~66 kDa) the latter stationary phase. enzyme immunoassay) for the identification of rabbit ferritin.0) and a data acquisition rate of one data point per 2 s. 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. an alternative way of identifying these metalloproteins had to be pursued and we therefore analyzed human plasma using Intensity (counts/s) 60000 40000 20000 V0 0 600 800 1000 1200 1400 Time (s) Fig.

which disappears after 0. Fe. correction of the clotting time of factor V-deficient plasma is proportional to the concentration (activity percentage) of factor Va in test plasma. Similarly. To quantify the metal that corresponded to a detected chromatographic metal peak. MA.66 ± 0. and a HemosIL factor V assay protocol supplied by Instrumentation Laboratory USA (Lexington. however. altogether eight fractions were collected of all Zn-containing entities.21 ± 0.99 0. the absence of tailing in the detected Cu. Mississauga. Conceptually. Fe.29 0. QC.0 mL of plasma for comparison with literature data [7. In fact. fractions were collected corresponding to the baseline before and after Cu peak 1 as well as the peak itself. Canada). Factor Va coagulation activity was determined by performing a modified prothrombin time assay. Palo Alto. Finally. these data were used to calculate the number of micrograms of metal that was present in form of a certain metalloprotein per 1. the sheer complexity of the plasma proteome makes it Zn The peak areas of metalloproteins that were not distinct were combined for integration SD standard deviation 123 . the detection of metal peaks within the chromatographic window (between the exclusion volume and the inclusion volume) together with the established stability of the major plasma metalloproteins of Cu. Canada) using the manufacturer’s method and calibrators. reagents. 8]. interpolated from a calibration curve [29]. and Zn-containing metalloproteins (by essentially determining the retention time of the metals corresponding to these metalloproteins). Human ferritin was quantified in the collected fractions by microparticle enzyme immunoassay technology with an Axsym analyzer (Abbott Diagnostics. the average relative standard deviation for all Table 3 Average concentration of Cu. extremely difficult to extract relevant information about the organism’s health status.46 ± 0. which was used to calculate the total amount of metal (in micrograms per 0. This would require the separation of these metalloproteins from each other.73 0. Fractions were collected for each Fe peak at time points corresponding to the maximum. Laval. Fe-. and Zn associated with metalloproteins derived from size-exclusion chromatography–inductively coupled plasma atomic emission spectrometry analysis of rabbit plasma samples (N = 18) Metal Protein(s) Average metal concentration (lg/mL plasma) ± SD 0. Fe. which—since the molecular masses of all major metalloproteins in plasma are known—can be achieved by choosing a SEC stationary phase with the appropriate fractionation range.65 ± 0. and Zn peaks would further substantiate that each metalloprotein remained intact during the entire LC separation process.5 h). USA) using factor V-deficient plasma. With regard to the analysis of rabbit plasma. ON. The very complexity of analyzing plasma for the proteins contained within it. and at the baseline before and after each peak. CA. Mississauga. Following this basic approach. and Zn [1. Factor Va coagulation activity was determined using an ACL TOP analyzer (Beckman Coulter. we injected increasing doses of each metal onto the chromatographic system without a column and measured the area under each ‘‘peak’’ using SigmaPlot. Excluding the standard deviation of the diagnostically inadequate Cu peak 1 (factor V. The two major Fe-containing proteins and all Zn-containing species had essentially the same retention times as those obtained for rabbit plasma.85 ± 0.J Biol Inorg Chem (2009) 14:61–74 67 the SEC-ICP-AES system and collected fractions for protein identification purposes. interanimal variation was expected and experimentally quantified (Table 3).84 ± 0. 31–33] would imply that each detected metal peak corresponds to a plasma metalloprotein.24 Cu Factor V and transcuprein Ceruloplasmin Albumin Small molecular weight Fe Ferritin Transferrin a2-Macroglobulin and unidentified peaks 2–4 Albumin 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. This allowed us to establish a calibration curve.19 1. Human transferrin was measured by immunoturbidometric assay with a Cobas Integra 700 analyzer (Roche Diagnostics Canada. Fe. we have developed a rapid SEC-ICP-AES method to directly analyze plasma for the major Cu-. shoulders on either side of the maximum. Furthermore.10 ± 0. In this assay. USA).14 0.5 mL plasma) that was associated with a detected metal peak (based on its peak area) in the subsequent analysis. which poses an almost insurmountable problem from an analytical separation viewpoint. the human serum proteome comprises at least 3.700 proteins [30].16 2.27 ± 0. OC. such as the ‘‘metalloproteome’’ (in the context of this paper this term refers to all major plasma proteins with bound Cu. and Zn). can be reduced dramatically if one is able to selectively analyze for a subproteome. With regard to the identification of factor V. and human a2-macroglobulin was measured with a Dade Behring BN2 Prospect rate nephelometric immunoassay using the manufacturer’s reagents (Dade Behring Canada. Canada) using the manufacturer’s method and calibrators.12 0.

First. and vitamin B12 1. if neither of these techniques is applicable because the metalloprotein has no inherent enzymatic activity (e. ceruloplasmin. can be indicative of its hydrodynamic radius and thus its approximate molecular mass (assuming minimal protein–protein interactions). In contrast to this. However. The latter involves the utilization of information that is derived from the Cu-. if it functions exclusively as a transport protein) or because no enzyme immunoassay is readily available (for the organism of interest. In this instance. Fe. myoglobin 17 kDa. in our case rabbits).g. and transferrin in collected fractions by various enzyme-based assays (see ‘‘Materials and methods’’) is indicated by horizontal bars metalloprotein relative to a known and abundant protein. we applied the SEC-ICP-AES 123 . In addition. In principle. S at 180. and Zn-specific chromatogram in conjunction with literature data. Fe.731 nm (orange). 3 Simultaneous multielement-specific chromatograms of rabbit plasma on a Superdex 200 10/300 GL (13 lm particle size) SEC column with a phosphatebuffered saline mobile phase (pH 7. c-globulin 158 kDa. Emission lines for a C at 193.754 nm (green). and P at 213. and Zn at 213. Plasma versus serum metalloprotein analysis To establish whether plasma or serum contains a larger number of individual Cu-. it can be definitively identified on the basis of either its enzymatic activity or a specific antibody target site on its surface (e. The task of qualitatively identifying the detected plasma metalloproteins is simplified as only approximately ten major Cu.618 nm (pink) and for b Cu at 324. two strategies can be employed to qualitatively identify an individual metalloprotein. and Zn metalloproteins was 39%. and Zn metalloproteins have so far been reported in mammalian plasma (Table 1) [1]. injection volume 500 lL.4.0 mL/min.68 J Biol Inorg Chem (2009) 14:61–74 detected Cu.. The qualitative identification of the metalloproteins factor V. and Zn-containing entities and therefore more information with regard to the health status of an organism.940 nm (blue). using an enzyme immunoassay). 22 °C). The SEC column was size-calibrated with a mixture of standards (thyroglobulin 670 kDa. flow rate 1.35 kDa). ovalbumin 44 kDa. such as albumin (66 kDa). a2-macroglobulin. the second strategy to tentatively identify a metalloprotein must be used. the experimentally determined relative retention time and abundance of both metalloproteins can be compared with their known molecular mass and abundance from handbooks on human metalloproteins [9] to tentatively identify both metalloproteins.856 nm (red). Fe-. ICP-AES detector. For instance. Fe-. the method reproducibility is excellent (see ‘‘Stability of plasma metalloproteins’’). ferritin.g.. Fe at 259. Both a and b were obtained from the same rabbit plasma sample.091 nm (black). the intensity of a metal peak (corresponding to a metalloprotein) relative to another metal peak (of a different metalloprotein containing the same metal) contains information about the relative abundance of metal atoms that are associated with these two proteins in plasma. the retention time of an unknown Fig.

and P using the ICPAES resulted in the three-element-specific chromatogram shown at the top of Fig. 3 and revealed four major C-containing protein bands.5. Cu-. Fe-. 1. 22 °C). Stability of plasma metalloproteins Cu 200 Counts/s 150 100 Vo 50 0 Fe 300 0.5-h intervals at room temperature and the emission lines of each element (Cu at 324. are only the two most likely explanations and at present the exact cause is unknown. it is known to self-associate to form higher multimers [36.0 h 200 Vo 100 To address a possible degradation of metalloproteins at room temperature (22 °C) over time. 1.5-h time point.856 nm) were plotted on top of each other C-.5. flow rate 1. and 2 h after blood collection. by specific adsorption) involved in the blood clotting process. These results strongly suggest that the corresponding metalloproteins are stable and—more importantly—that the analytical method itself produces results that are sufficiently reproducible for diagnostic applications. This Cu peak could possibly represent blood coagulation factor V. S-.J Biol Inorg Chem (2009) 14:61–74 69 250 Counts/s method developed to analyze plasma (n = 3) and serum (n = 3) of 4. the Cu metalloprotein corresponding to Cu peak 1 must be either directly or indirectly (e. which could explain its elution in essentially the void volume..4. 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. Therefore. and P-specific chromatogram of plasma The analysis of plasma with the method developed and the simultaneous online detection of C. and Zn at 213. the latter must be analyzed within 0. plasma was analyzed using the SEC-ICP-AES system at 0. These. Fe at 259. Fe-. This experiment was carried out twice and the results essentially showed the same overall trend. ICP-AES detector.5 h 1.0 mL/min.5-h-fasted rabbits. and Zn-specific time-course chromatograms are shown in Fig.5 h 2. Typical Cu-. which is a single-chain glycoprotein that contains one Cu per molecule [34] and is known to be sensitive to proteolysis [35]. injection volume 500 lL. The S-specific chromatogram 123 . Fe-.g. 0 140 Zn 120 100 Counts/s 80 60 Vo 40 20 0 600 800 1000 1200 1400 Retention Time (s) Fig. S.5 h after blood collection. see the discussion below) increased to some extent. 4 Simultaneous Cu-. Although factor V has a molecular mass of 330 kDa. the Cu that was eluted prior to 800 s disappeared from plasma after the 0. 4.0 h 1. whereas the most intense Cu peak (ceruloplasmin. 37].754 nm. plasma contains more information than serum for the desired application of the instrumental analytical method developed for diagnostic purposes. if one aims to detect all Cu metalloproteins (including the labile ones) in plasma. As depicted in this chromatogram.200 s (small molecular weight Cu. however. On the basis of these results. Cu peak 1 in the plasma chromatogram at the bottom of Fig. 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 phosphatebuffered saline mobile phase (pH 7. The intensity of the Fe and Zn peaks remained virtually unchanged over the 2-h time period (Fig. 4). 3 was absent in the serum chromatogram (data not shown). see the discussion below) and the one corresponding to the Cu that was eluted at approximately 1.940 nm. Because the number and intensity of all other detected peaks remained unchanged (data not shown). see the discussion below) decreased. the peak corresponding to Cu that was eluted at approximately 900 s (it likely corresponds to albumin-bound Cu. In addition. and Znspecific chromatograms were obtained in 0.

approximately 0. Fe. it was tentatively identified as factor V. the total rabbit plasma Cu concentration was higher than what has been reported for other mammalian species (range 0. On the basis of the observation that Cu peak 1 was absent from serum and disappeared from plasma after 30 min (Fig. and Zn-containing entities that are contained in rabbit plasma. and Zn peaks in the chromatograms at the bottom of Fig. the last Zn peak. Fe-. 32]. which coincides with the elution of the small molecular weight C band 4. extended beyond this peak. the individual Cu. peak 4: approximately 890 s. At first glance. approximately 19% of total Cu. the P-specific chromatogram displayed an elevated P baseline throughout the entire chromatographic window (Fig.g. and Cu peaks 2 and 4 displayed a hump on the long retention end. the fourth S-containing entity was eluted before the fourth C-containing entity (both of these correspond to small molecular weight peptides and amino acids). which is known to be labile. Fe. which is one more than has been reported in other studies (Table 2) (peak 1: approximately 515 s. which may explain the rapid disappearance of Cu peak 1 in the time-course experiments (Fig. We note. However. and is present in human plasma at approximately 10 mg/L [35]. Owing to the utilization of a P-containing mobile phase (PBS). and Zn using the ICP-AES resulted in a three-element-specific chromatogram. however.70 J Biol Inorg Chem (2009) 14:61–74 closely resembled that of the first three C-containing protein bands. Fe.210 s. peak 2: approximately 605 s. approximately 13% of total Cu. 3. 3 correspond to individual metalloproteins and—since Cu peak 5 was in the small molecular mass region— metallopeptides. 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 Cuspecific chromatograms of previous studies [20. the most prominent C band—band 3—must be predominantly composed of albumin (a BSA standard had the same retention time). can be easily explained by the fact that previous studies analyzed either aged plasma or serum. by a surfactant effect).9 lg Cu/mL. which can be rationalized by the elution of a slightly smaller metalloprotein containing the same metal. we calculated a plasma concentration of 4. 4).. approximately 13% of total Cu.2–2. which suggests that the metals did not dissociate from their parent protein during the chromatographic separation process. The detection of the albumin peak did not affect the intensity of the P-emission line. In addition. 3. approximately 28% of total Cu. peak 5: approximately 1. 3. a representative of which is shown at the bottom of Fig. this is not unexpected.0 lg/mL) [7]. and therefore corresponds to the injected plasma ‘‘plug’’ (which contains less P than the mobile phase) reaching the detector. 123 . which can be rationalized by the fact that under the circumstances of blood sampling factor V is expected to be activated into factor Va. which was highest at the maximum intensity of Cu peak 1. the second Fe peak. which is higher than the average total Cu concentration (2. approximately 0. Nevertheless. Importantly. however. two Fe-containing. which is several-fold higher than its concentration in human plasma and is therefore in apparent disagreement. five Cu-containing. the P-specific chromatogram also revealed a characteristic dip at a retention time of about 1. peak 3: approximately 775 s. This discrepancy. 3. 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 majority of the detected metal peaks displayed an ideal peak shape. The Cu-specific chromatogram revealed five peaks. 4) [39]. 22. Thus. The activity. According to our analytical data and assuming an identical stoichiometry in rabbit and human factor V (Table 1). Factor V is known to be very labile and prone to proteolysis. contains Cu [34]. which has a smaller molecular mass (approximately 221 kDa) and could explain the observed tailing of the activity. and approximately five poorly separated Zncontaining entities were detected. Fig.3 lg Cu/mL. and Zn metalloproteins in plasma The analysis of plasma (eight different animals) by SEC and the simultaneous online detection of Cu. which is expected since most mammalian proteins contain the S-containing amino acids L-cysteine and/or L-methionine. Given the inherent limitations of SEC with regard to the chromatographic resolution of proteins of almost similar size from each other. approximately 27% of total Cu. 4). which according to our investigations both lack Cu peak 1 (Fig. The sum of all Cu peaks in this particular plasma sample amounted to 3. approximately 0.0 lg Cu/mL plasma.6 lg Cu/mL.14 lg/mL) in the 18 plasma samples that were analyzed (Table 3). bottom). 3. top).3 lg Cu/mL. The P-emission intensity also provided an effective measure of the mass transfer of droplets from the nebulizer chamber to the plasma. bottom). which is an important prerequisite to accurately measure the total metal that is associated with an eluting plasma metalloprotein. however. Interestingly.7 g/L. which demonstrates that the injected total protein did not adversely affect the mass transfer from the nebulizer to the plasma (e. A similar phenomenon has previously been observed [38].230 s.9 lg Cu/mL. the detection of approximately 12 metalloproteins demonstrates that the optimized mobile phase–stationary phase combination is well suited to separate the major Cu-. Cu. and in contrast to the peaks in the chromatograms at the top Fig. approximately 0. This was corroborated by analyzing collected fractions for factor V coagulation activity (bar in Fig. approximately 0.

approximately 0. 3. 24. approximately 0. peak 2: approximately 870 s. and small molecular weight Cu. On the basis of the simultaneous appearance of Cu peak 5 (Fig. 3. Zn peak 1 likely represents the 725-kDa a2macroglobulin [46] since between 12 and 31% of human plasma Zn has previously been reported to be tightly incorporated in this protein.31 g/L. 3. With regard to the retention time of this metalloprotein (Fig. e. approximately 770 s. On the basis of the experimentally determined total Cu associated with ceruloplasmin in 1. Fe peak 1 is identified as ferritin and Fe peak 2 as transferrin. Cu peak 3 had ceruloplasmin oxidase activity (bar in Fig. 3. which is also known to dimerize (540 kDa) [4]. which also identified the first Zn compound that was eluted from a SEC column as a2macroglobulin [31]. which has inherent diagnostic value that cannot be obtained by conventional antibody-based enzyme assays. sum of all Fe peaks: 2. which indicates that Fe is not bound to peptides and amino acids in rabbit plasma. 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 [4.8 g/L. which would therefore explain its elution after transferrin. 43. approximately five non-baselineseparated peaks were detected (peak 1: approximately 613 s. sum of all Zn peaks: 1.. bottom). 3. 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 experimentally derived total Fe associated with ferritin and transferrin in 1. bottom) with the last C band (Fig. This Cu peak had a small shoulder on the long retention end. it was eluted 123 . 11% of total Fe. Cu peak 2 was tentatively identified as the 270-kDa protein transcuprein. 3. approximately 0. The Fe-specific chromatogram revealed two baselineseparated peaks. bottom). bottom) it is noteworthy that dimerization of this protein has been observed by others [36. ferritin is attributable to (1) 50% apoferritin and 50% fully loaded holoferritin or (2) the case where all ferritin is 50% loaded with Fe. our method allows us to determine the distribution of a metal among various metalloproteins. Even though the putative a2-macroglobulin was eluted before ferritin (450 kDa). peaks 2–4: approximately 655 s. Nevertheless. 3. 37]. 66 kDa (albumin). top was different from that for Cu peak 5 in Fig. bottom). which is in accord with the Cu-specific chromatogram in Fig. On the basis of the previously reported order of elution of Cu plasma metalloproteins from a SEC column [32]. bottom) and is in general agreement with other studies [32]. 32]. It is therefore impossible to distinguish if the Fe that is associated with. 540 kDa (transcuprein dimer). On the basis of previous studies which demonstrated that Fe is bound to human serum albumin in serum [23].0 mL of plasma and assuming that both Fe metalloproteins were fully loaded with Fe (Table 1). which is within the concentration range reported for human serum. such as L-histidine [42] (the retention time for S band 4 in Fig. 89% of total Fe. 34% of total Zn.6 lg Fe/mL. this additional Fecontaining entity could be albumin-bound Fe especially since albumin is approximately 14 kDa smaller than transferrin. 10% of total Zn. bottom) and is in accord with another study.J Biol Inorg Chem (2009) 14:61–74 71 that evidence in favor of significant interanimal species differences of certain plasma metalloprotein concentrations have been reported [40].8 lg Zn/mL plasma) (Fig. 3. 31]. bottom). 45]. approximately 2. 132 kDa (ceruloplasmin). 19. This peak assignment was confirmed by enzyme immunoassay and immunoturbidometric assay (bars in Fig. On the basis of the known molecular size and plasma abundance of the two major Fe-containing metalloproteins ferritin and transferrin (Table 1). which is more than the average number of Zn peaks that has previously been reported (Table 2) [31. 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). 3. In contrast to Cu. 56% of total Zn. With respect to Zn. 44] (peak 1: approximately 670 s. approximately 1.9 lg Fe/mL plasma) (Fig.1 lg Zn/mL. 23.1 lg Zn/mL. approximately 700 s. the plasma ceruloplasmin concentration was calculated at 0. which is identical to the maximum number of Fe peaks that was previously reported (Table 2) [8. 3. a different metalto-protein stoichiometry between human and rabbit factor V could significantly affect the calculated plasma concentration.0 mL of plasma and the known stoichiometry of Cu in this protein (Table 1). which is in accord with our results [25. no detectable Fe was eluted in the small molecular weight range. Cu peak 4 was comparatively broad and appeared 11 s after albumin (dotted line in Fig.g. however. This peak assignment was confirmed by enzyme immunoassay (bars in Fig. we point out that the method developed cannot inherently determine the metal loading of a metalloprotein in which the metal loading may vary. bottom) and was therefore identified as the glycoprotein ceruloplasmin. This finding can be rationalized with the higher-resolution SEC column that was used in the present study (13-lm particles) compared with earlier studies. 4. the rabbit plasma concentration of ferritin was calculated as 535 lg/L and that of transferrin as 1. The distinct shoulder on the long retention end of Fe peak 2 indicates the presence of another Fe-containing entity. top). 44. 3. peak 5: approximately 880 s. this Cu peak represents Cu bound to small non-S-containing peptides and amino acids.3 lg Fe/mL.6 lg Zn/mL. Furthermore. Even though these results are in overall accord with the established concentrations of these metalloproteins in mammalian plasma/serum (Table 1).

and Zn-specific detector. which is composed of approximately 12 metalloproteins and metallopeptides. Similar to the results obtained for Fe. by a spectrophotometric activity assay). this Zn entity likely represents albumin-bound Zn (transferrin does not bind Zn2? [44]). 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 metalloprotein containing the metal of interest is coeluted) from a single analysis in a given amount of time than is possible with other methods that are currently in use. Zn peaks 2–4 and the Zn shoulder on the long retention end of Zn peak 5 could not be qualitatively identified. On the basis of the identical retention times for Zn peak 5 and albumin (dotted lines in Fig.. Similar behavior could also occur between the components of plasma and a2-macroglobulin and subsequently the stationary phase in our experiments. Fe. no Zn was detected bound to small molecular weight peptides and amino acids in rabbit plasma. however. let alone those of more than one element simultaneously. To this end. few methods have been reported that can simultaneously determine all metalloproteins of one element.or advanced-stage human diseases by the direct analysis of human plasma or serum [50–57]. Using the experimentally determined total Zn that is contained in 1. We note. This latter application appears particularly relevant since bioinorganic 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. From a clinical perspective. 3) and since albuminbound Zn represents the major Zn entity in plasma (56% of total plasma Zn in this study. the existence of a 165-kDa extracellular secretory glycoprotein Cu. However. which is in good accord with previous studies on humans [26. 60].Zn superoxide dismutase (EC-SOD) and that of a 31-kDa 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 123 . individually and—more importantly—cumulatively. we calculated the rabbit plasma a2macroglobulin concentration (using the stoichiometry delineated in Table 1) at 222 mg/L. that significant interanimal species differences regarding certain plasma metalloprotein concentrations have been reported [40. Therefore. which served as the simultaneous Cu-. such as toxic metals and metalloid compounds. such as ceruloplasmin (e.72 J Biol Inorg Chem (2009) 14:61–74 after the void volume. Fe.g.Zn-SOD) have been reported in guinea pig and human plasma [40. Thus. which is approximately one tenth of its concentration reported for human plasma (Table 1). Fe-. Conclusions The daunting analytical task of extracting health-relevant information from plasma can be considerably simplified by determining a ‘‘subproteome’’. and Zn metalloproteome. Fe. this simple and rapid technique to establish the Cu. the detection of the majority of the expected Cu-. and Zn metalloproteome within approximately 24 min. in turn. Even though assays exist to quantify individual plasma metalloproteins.Zn superoxide dismutase (Cu. We note. can be helpful to more accurately diagnose the severity of a disease since Wilson’s disease. for instance. 49]. This. and Zn metalloproteome. in the mammalian bloodstream to better understand their chronic toxicity. such as the Cu. but can also result in an increased plasma concentration of the Fe metalloprotein hemoglobin during episodes of acute hemolysis [52]. 31] and pigs [44]).Zn-SOD. is not only associated with decreased plasma concentrations of the Cu metalloprotein ceruloplasmin [58]. within approximately 24 min. The first application is its utilization as a clinical tool to screen for early. we have developed a rapid SEC-based separation of the metalloproteins contained in rabbit plasma followed by the online analysis of the column effluent by an ICP-AES. which is in discord with what would be predicted if its retention were solely based on its molecular mass. Practical applications Owing to the fact that the SEC-ICP-AES method developed allows one to determine the plasma Cu. however.1 lg Zn/mL). Fe-. two major practical applications of this method can be envisioned.0 mL of plasma in form of this metalloprotein (approximately 0. and Zn metalloproteome offers important advantages over individual metalloprotein assays since much more information can be extracted with this method from a single plasma sample. This novel SEC-ICP-AES method has allowed us to directly analyze rabbit plasma in order to generate the Cu. 48]. this method has the obvious advantage of extracting more information (namely. It is therefore likely that EC-SOD represents one of the unidentified Zn peaks 2–4 and that the shoulder on the long retention end of Zn peak 5 could possibly be Cu. The second application is the utilization of the method developed to probe the nonenzymatic bioinorganic chemistry of environmentally abundant pollutants. that nonideal interactions 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 stationary phase.

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