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Originally published In Press as doi:10.1074/mcp.M300080-MCP200 on October 7, 2003.
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Molecular & Cellular Proteomics 2:1306-1318, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Identification of Metal-binding Proteins in Human Hepatoma Lines by Immobilized Metal Affinity Chromatography and Mass Spectrometry*

Yi-Min She{ddagger}, Suree Narindrasorasak{ddagger}, Suyun Yang§, Naomi Spitale{ddagger}, Eve A. Roberts§,¶ and Bibudhendra Sarkar{ddagger},||,**

From the Programs in {ddagger} Structural Biology and Biochemistry and § Metabolism Research, The Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, and the Departments of || Biochemistry and Pediatrics, University of Toronto, Toronto, Ontario M5S 1A8, Canada


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The metalloproteome is defined as the set of proteins that have metal-binding capacity by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu and Zn metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SK-Hep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep G2 cells depleted in vitro of cellular copper. We used matrix-assisted laser desorption ionization and electrospray ionization quadrupole time-of-flight mass spectrometry to analyze peptide sequences of tryptic digests obtained by either in-gel digestion of metal-binding proteins or peptides on an immobilized metal affinity chromatography column loaded with either Cu or Zn. Mainly high abundance proteins were identified. Cu-binding proteins identified included enolase, albumin, transferrin, and alcohol dehydrogenase as well as certain intracellular chaperone proteins. The Cu metalloproteome was not identical to the Zn metalloproteome. Peptide binding experiments demonstrated that Cu coordination prefers the order of residues histidine > methionine > cysteine. Although the Cu metalloproteome was similar from line to line, subtle differences were apparent. Gel profiling showed more extensive variation in expression of annexin II in SK-Hep-1 and Mz-Hep-1 than in Hep G2 and normal liver tissue. Glycerylphosphorylethanolamine was identified as a post-translational modification at residue Glu-301 of elongation factor 1-{alpha} in Hep G2. Intracellular copper depletion was associated with loss of the glycerylphosphoryl side group. These findings suggest that post-translational modification could be affected by intracellular actions of copper. Comparison of the Cu and Zn metalloproteomes in Hep G2 with a published general proteome of Hep G2 disclosed little overlap (Seow, T. K., et al. (2001) Proteomics 1, 1249–1263). Proteins in the metalloproteomes of human hepatocytes can be identified by these methods. Variations in these metalloproteomes may have important physiological relevance.

Metals play a pivotal role in cellular metabolism. They function as the catalytic centers in many biochemical reactions and serve as structural elements for a large number of regulatory proteins (1, 2). Characterization of metal-binding proteins is important for understanding the structure and biological functions of such proteins in metal-associated diseases. In the past few years, a variety of mass spectrometric techniques has been used to probe the metal-protein interactions in metal-containing proteins. These studies have been successfully applied to determining metal binding stoichiometry (3–9), metal-binding sites (10, 11), and metal-dependent structure/conformation changes (12, 13). A number of metal-binding proteins, such as cytochrome c oxidase (14), albumin (3, 7), metallothionein (12, 15, 16), prion protein (PrP) (8, 9, 11, 13, 17), matrilysin (4, 5), and non-heme iron-containing metalloproteins (6), have been individually well characterized by either overall mass measurements on the intact metal-protein complexes or peptide sequencing on the protein digest with tandem mass spectrometry.

Rapid developments in proteomic technology and bioinformatics have permitted identification of proteomes of cell lines and tissue by using mass spectrometry instrumentation (for review, see Refs. 18–22). The specific advances include the use of two-dimensional gel electrophoresis (2DE),1 on-line two-dimensional liquid chromatography-electrospray ionization mass spectrometry (2D liquid chromatography-ESI MS), and off-line liquid chromatography-matrix-assisted laser desorption ionization mass spectrometry (liquid chromatography-MALDI MS). The availability of automatic sample analysis and database searching enables high throughput protein sequence identification and detailed investigation of protein post-translational modifications. MALDI and ESI combined with hybrid quadrupole time-of-flight (QTOF) mass spectrometry have become a powerful tool for current proteomic research due to high sensitivity, resolution, and mass accuracy for analyzing proteins and peptides (23–26). We have used this analytical method to examine the metal-binding proteins in human hepatoma lines and normal human liver tissue.

Hep G2 (27), SK-Hep-1 (28), and Mz-Hep-1 (29) are human hepatoma lines derived from hepatic neoplasia. Hep G2 and Mz-Hep-1 retain numerous cellular functions typical of differentiated, normal hepatocytes (such as synthesis of albumin, transferrin, {alpha}1-antitrypsin, lipoproteins, fibrinogen, and certain other coagulation factors), but SK-Hep-1 has fewer differentiated characteristics (30). In Hep G2 receptor-mediated functions such as lipoprotein and asialoglycoprotein uptake are closely similar to those processes in normal hepatocytes (31). Hep G2 is widely accepted as a valuable and informative model system for studying human hepatocyte function.

Although a general proteomic study in Hep G2 has recently been reported (32), we have attempted to describe the Cu and Zn metalloproteomes in Hep G2, Mz-Hep-1, and SK-Hep-1. In defining such a metalloproteome, we seek to determine the set of proteins that have unique metal binding capacity either by virtue of being metalloproteins or by having metal-binding sites. We used selective enrichment of metal-binding proteins with immobilized metal affinity chromatography (IMAC) and separation and identification by gel electrophoresis and mass spectrometry. We also employed a complementary strategy of examining metal-binding peptides selectively captured on a metal-charged IMAC column in anticipation of investigating low abundance metal-binding proteins and possible metal-binding motifs. Sequence identification studies were performed on metal-binding proteins separated by gel electrophoresis and metal-binding peptide fragments from different human hepatoma lines to further detect any metal-dependent alteration on post-translational modifications.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Enzymes—
All chemicals were from Sigma unless indicated otherwise. Sequencing grade trypsin was purchased from Roche Diagnostics.

Cell Lines, Culture Conditions, and Liver Tissue—
The human hepatoma lines Hep G2 and SK-Hep-1 were originally purchased from American Type Culture Collection (ATCC, Manassas, VA), and Mz-Hep-1 was obtained as a gift from Dr. Wolfgang Dippold (University of Mainz, Mainz, Germany). Normal human liver tissue from a 39-year-old female was obtained from a liver graft excess to that required for use in a segmental liver transplant; it was perfused with University of Wisconsin solution (33) to remove all contaminating blood and maintained in sterile slush at 0–4 °C until it was snap-frozen in liquid nitrogen in 1–2-g aliquots for long term storage at -84 °C. All cell lines were maintained in {alpha}-minimum essential medium with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 95% room air, 5% CO2. Cell lysates were prepared from confluent cultures. These were washed with 0.9% saline and incubated with 0.25% trypsin citrate saline (Difco, BD Biosciences) for 7–10 min at room temperature to lift cells from the plastic. Trypsinization was quenched with medium containing 10% fetal bovine serum. Cells were gently separated and then centrifuged at 180 x g for 5 min. The resulting cell pellet was resuspended in 0.9% saline and washed twice. For lysis the pellet was suspended in 10 mM HEPES-NaOH buffer at pH 7.8 (5 ml/g of cells), placed on ice for 10 min, and centrifuged at 800 x g at 4 °C for 2 min. The resulting pellet was then suspended in 0.6 M sucrose in 10 mM HEPES-NaOH pH 7.8 buffer and homogenized with 30 strokes of a tight fitting Dounce homogenizer. The homogenate was treated with protease inhibitor mixture without EDTA and centrifuged at 8000 x g for 20 min at 4 °C. The resulting supernatant was free of large organelles including nuclei and was used for metalloproteomic analysis.

For preparation of human liver tissue the above protocol was modified by homogenizing the 1.6-g tissue aliquot in 0.25 M sucrose, 10 mM HEPES-NaOH pH 7.8 buffer with 30 strokes of a loose fitting Dounce homogenizer prior to the addition of protease inhibitor mixture and centrifugation. Thereafter, the same preparative methods were used as for the hepatoma lines.

To examine the effect of depleting intracellular copper on the metalloproteome, Hep G2 cells were treated for 48 h with 50 µM tetraethylenepentamine, a copper-chelating agent. Intracellular copper was analyzed by atomic absorption spectrometry (Varian, Mississauga, Ontario, Canada). Cellular copper concentration was determined using the average value of three measurements with comparison to a standard calibration curve of 20–100 µg of Cu/liter.

Sample Pretreatment and Metal-binding Protein Preparation by IMAC—
Cell lysates from Hep G2, SK-Hep-1, Mz-Hep-1, and the normal human liver specimen were sequentially dialyzed with 10 mM Tris-HCl (pH 8.0) and a binding buffer (4 M urea, 0.5 M NaCl, 0.25 M sucrose, 0.5% Triton X-100, 10 mM Tris-HCl, pH 8.0). The metal affinity column was prepared using chelating Sepharose Fast Flow beads (Amersham Biosciences), and copper and zinc were coupled to the columns by applying 50 mM CuCl2 (or 50 mM ZnCl2) solution in 50 mM NaAc and 50 mM NaCl (pH 4). A control experiment on the uncharged column with metal was used for comparison. Excess metal was then removed from each column using either 50 mM NaCl (pH 4) or deionized water (three times) followed by washing with the binding buffer (five times). The sample (1 ml) was then loaded onto each metal-bound column, followed by gentle mixing overnight. Once the column was washed thoroughly with the binding buffer (10 times), the metal-binding proteins were eluted using 50 mM EDTA containing 50 mM NaCl. Further dialysis (3-kDa cut-off) was used to remove small molecules (metal ions, EDTA, NaCl, and detergent), and the purified metal-binding proteins were dried by SpeedVac and stored at -20 °C.

Gel Electrophoresis—
One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) of metal-binding proteins was conducted on 12% polyacrylamide gel, adapted from the protocol of Laemmli (34). The protein bands were visualized with staining using 0.2% Coomassie Blue R-250.

Two-dimensional gel electrophoresis was performed in accordance with guidelines described by Amersham Biosciences. A protein sample (~500 µg) was dissolved in 250 µl of rehydration buffer (7 M urea, 2 M thiourea, 0.5% Triton X-100, 20 mM dithiothreitol, 4% CHAPS, 0.5% IPG buffer) with a small amount of bromphenol blue, and this solution was centrifuged for 5 min to ensure that all proteins were soluble in solution. The first-dimensional isoelectric focusing of the proteins was performed on a 13-cm precast immobilized pH gradient strip (pH 3–10) and an IPGphor isoelectric focusing system (Amersham Biosciences). After separating proteins on the basis of isoelectric points, proteins immobilized on the immobilized pH gradient strip were reduced with 125 mM dithiothreitol solution in 6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl (pH 8.0) for 15 min and were alkylated in the same solution with 125 mM iodoacetamide in place of dithiothreitol for a further 15 min. The second-dimensional SDS-PAGE based on molecular mass separation was performed on 16 or 12% acrylamide gels at 40 V for 23 h.

Silver staining on the resolved 2D gel spots was adopted from the protocol by Jensen et al. (35). Briefly, the gel was treated with two changes of 40% methanol and 10% acetic acid for 15 min followed by sensitization for 30 min in 0.02% sodium thiosulfate solution. The solution was discarded, and the gel was rinsed with two changes of deionized water. After incubation in chilled 0.1% AgNO3 solution for 20–40 min at 4 °C, the gel was developed with a solution of 0.04% formaldehyde, 2% Na2CO3. As soon as the gel turned yellow, the developing solution was immediately removed. Protein spots were visible on the gel following rinses with 1% acetic acid, and finally the silver-stained gel was stored in 1% acetic acid at 4 °C.

In-gel Tryptic Digestion—
The procedure for in-gel tryptic digestion of proteins was used as described previously (36). The Coomassie-stained 1D gel band was excised and destained with 100 mM ammonium bicarbonate/acetonitrile solution, whereas the silver-stained 2D gel spot was destained by chemical reduction with potassium ferricyanide and sodium thiosulfate (37). Gel pieces were dried in a SpeedVac centrifuge (Savant, Fisher, Nepean, Ontario, Canada). Reduction and alkylation of the proteins were performed using 10 mM dithiothreitol and 55 mM iodoacetamide, respectively. In-gel digestion of each protein band or spot was performed using 0.02 µg of trypsin in 25 mM ammonium bicarbonate solution. After overnight digestion at 37 °C, the proteolytic peptides were extracted by sonicating with 0.1% trifluoroacetic acid and acetonitrile and were dried by SpeedVac.

On-column Digestion of Metal-binding Proteins—
Tryptic digestion of the metal-bound proteins was carried out on proteins bound on an IMAC column with 0.1 mg/ml trypsin in 10 mM Tris-HCl buffer (pH 7.6) at 37 °C for 15 min. The proteolytic peptide fragments possessing a metal-binding motif were retained on the column, while the non-binding fragments were washed off. Metal-binding peptides were eluted by 0.5 M acetic acid, subsequently dried by SpeedVac, and redissolved in 100 µl of deionized water.

Peptide Binding onto an IMAC Column—
The metal-binding proteins (~200 µg) separated from the Cu IMAC column were digested with 1 µg of trypsin in 25 mM Tris-HCl buffer (pH 7.6) for 12 h. The resulting digest was mixed with 1 M NaCl (1:1, v/v) to achieve a final buffer concentration at 10 mM Tris-HCl plus 500 mM NaCl, and then was loaded onto the copper IMAC column and incubated overnight. Following washes with the same buffer (10 mM Tris-HCl containing 500 mM NaCl), the binding peptides were eluted with 1 ml of 0.5 M acetic acid. The eluted fraction was treated as above to remove acid for subsequent mass spectrometric analysis.

Mass Spectrometry and Database Searching—
Peptide mapping and MS/MS sequencing were performed either on a Micromass ESI QTOF or a prototype Manitoba/Sciex QStar MALDI QTOF tandem mass spectrometer (38, 39) at the University of Manitoba. All of the in-gel digests were desalted with a C18 ZipTip (Millipore) before ESI analysis, and each peptide was sequenced by MS/MS measurements. Samples were dissolved in a solution of 50% methanol containing 0.1% formic acid and were introduced by nanospray at the capillary voltages of 850–1200 eV. Nitrogen and argon were used as nebulization and collision gases, respectively. Multiple point calibration at m/z scale was performed by MS/MS measurements on the doubly charged ion at m/z 785.84 of Glu-fabrinopeptide using collision energy of 29 eV.

In MALDI mode, the sample (0.5 µl) was mixed with 2,5-dihydroxybenzoic acid matrix in 50% acetone at a ratio of 1:1 (v/v) on the target. The instrument was equipped with a UV nitrogen laser (337 nm), and the acceleration voltage was set to 10 kV. The mass scale was calibrated externally using two standard peptides (dalargin and melittin). Argon was also used as the collision gas in collision-induced dissociation experiments.

Peptide fingerprinting of the in-gel digests was routinely analyzed by database searching with ProFound (129.85.19.192/profound_bin/WebProFound.exe). The identified proteins were verified further by National Center for Biotechnology Information (NCBI) database searching of MS/MS fragments on each peptide with MS-Tag (prospector.ucsf.edu/ucsfhtml4.0/mstagfd.htm). In some cases where no matching protein was found in the databases, manual interpretation of the peptide sequences was carried out on the MS/MS spectra using computer software ProMaC (MDS Sciex, Mississauga, Ontario, Canada). The available partial sequence was then searched against the protein homology database with BLAST (www.ncbi.nlm.nih.gov:80/BLAST/).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Metal-binding Proteins in Hep G2 Cells by 1- or 2D Gel Electrophoresis-MS—
High resolution two-dimensional gel electrophoresis capable of separating complex mixtures of proteins is a well established technique for current proteomic research. Procedures normally involve excision of gel spots of interest, in-gel tryptic digestion, and mass spectrometric analysis of the resulting peptides. Because only a limited amount of peptides can be extracted from gels, especially for small proteins and low abundance proteins, MS/MS measurements are required to achieve reliable protein sequence identification (40). As a starting point, we have used the techniques of IMAC, 1- or 2D gel electrophoresis, MS mapping, and MS/MS sequencing to investigate several abundant Cu- or Zn-binding proteins from human Hep G2 cell lines. Fig. 1 shows the 2D gels of metal-binding proteins eluted from a copper or zinc IMAC column. A total of 38 gel spots of high abundance proteins that bound to copper (Table I) and zinc (Table II) were identified. Most of them appeared as the common metal-binding proteins (albumin, enolase, S100 calcium-binding protein, endoplasmic reticulum luminal calcium-binding protein grp78, and transferrin), nucleic acid-binding proteins (histone, ribosomal, and elongation factor 1-{alpha} (EF-1{alpha})), chaperones (heat shock proteins 60, 70, grp78, 90, and gp96), or redox enzymes (protein-disulfide isomerase (PDI) and peroxiredoxin) (41–43).



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  		FIG. 1. Silver-stained 2D gels of the eluted proteins from either a Cu or Zn IMAC column on 16% SDS-PAGE. The identified proteins, as shown in Table I by 2DE-MS, were labeled on the figure.

 

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  		TABLE I Identification of copper-binding proteins in Hep G2 cells by 1- or 2DE-MS

 

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  		TABLE II Identification of zinc-binding proteins in Hep G2 cells by 1- or 2DE-MS

 
Metal-binding sites in proteins differ with respect to the number of ligands involved and their spatial geometry (2, 44). Site-specific ligands composed of amino acid residues cysteine, histidine, and methionine in the protein sequence preferably bind copper (11, 14). Sequence prediction for the Cu-binding proteins identified in Hep G2 revealed the following putative metal-binding motifs as expected: C(X)mC (m = 2–4) and H(X)nH (n = 0–5). For example, protein-disulfide isomerase contains two structural CXXC motifs, which are similar to the Cu-binding motifs of Wilson disease copper-transporting ATPase (45) and copper chaperone protein ATOX1 (46). The existence of the Cu binding capability of PDI, identified in this study, has not been recognized previously. Peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase, elongation factor 1{alpha}, and heat shock protein gp96 presumably possess one or more histidine-binding motifs with HH, HXXH, HXXXH, or HXXXXXH for binding to copper ions.

Determination of Metal-binding Motifs by On-column Digestion—
The chromatographic buffer conditions chosen in our IMAC experiments for metal-binding protein separation were as closely related to physiological conditions as possible so that only proteins that bind metals in vivo would bind to the IMAC column. However, an important limitation is the fact that some non-metal-binding protein partners, which do not normally bind metals, might be detected along with the metal-binding proteins to which they had formed complexes through protein-protein interactions. To minimize this possibility, MALDI MS analyses were performed following on-column tryptic digestion of metal-bound proteins and removal of unbound peptides. Because histidine is a well known ligand for Cu- and Zn-binding proteins, the formation of strong metal-histidine interactions allows direct identification of metal-binding peptides.

Peptide mapping of the tryptic digests of the metal-binding protein from Hep G2 cells are shown in Fig. 2, and the observed high abundance peptides were analyzed by MS/MS measurements. Table III summarizes the matched peptides, which were sequenced by MS-Tag database searching on the MS/MS fragments of each peptide, except the peptide at m/z 2677.259 (described below). Of the 25 peptides identified, the structural motif in 16 cases contains a multiple histidine sequence consisting of either H(X)mH (m = 0–6) or H(X)nH(X)nH (n = 1, 2). The distance between two histidine residues in a Cu-binding peptide can vary by up to six amino acids, but in general the structure for a Cu-binding peptide bearing H(X)mH motif at m > 3 requires a proline residue to provide the binding geometry. Of the rest of the Cu-binding peptides, three peptides contain one histidine, and five peptides consist of one histidine plus one or two methionines. There is one peptide at m/z 1907.978 containing the MXXXM motif (also proline-containing), as seen in enolase.



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  		FIG. 2. MALDI mass spectra of metal-binding peptides following on-column tryptic digestion of the metal-binding proteins. A, Cu IMAC column. B, Zn IMAC column.

 

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  		TABLE III Identification of metal-binding peptides to either copper or zinc by MALDI MS/MS measurements following on-column tryptic digestion

E*, the glutamic acid (E) was modified by glycerylphosphorylethanolamine.

 
PDI, identified by 2DE-MS, has a structural motif CXXC, but we could not identify this motif by using on-column protein digestion. This could be due to weak interaction of the metal with the motif and release of the peptide in the wash after on-column digestion. Many metallochaperones possessing the CXXC structural motif are known to have weak binding activity for metals (45–47). We therefore conclude that the residue binding affinity for a peptide to copper is histidine > methionine > cysteine.

Analysis of metal-binding peptides has shown the partial metal-binding property of metal-binding proteins in vitro. Accordingly, most of the proteins are consistent with the identification of metal-binding proteins identified by 2DE-MS as shown in Table I, such as actin, enolase, elongation factor 1-{alpha}, heat shock proteins, peroxiredoxin, and glyceraldehyde-3-phosphate dehydrogenase. Some proteins were not observed by 2DE gel but were detected by on-column digestion. They are probably at low abundance in Hep G2. These proteins include alcohol dehydrogenase, M1 isozyme, fatty acid synthase, GTP-binding nuclear protein, and histidine triad nucleotide-binding protein. Of interest, alcohol dehydrogenase, known to be a zinc metalloenzyme, was found to interact with the Cu IMAC column.

An unexpected finding was the abnormal Cu-binding peptide presented at the single charged ion of m/z = 2677.259 in Fig. 2, where MS-Tag search did not match any protein in the database. Manual interpretation of the MS/MS data (Fig. 3) on the singly charged ion at m/z 2677.26 (MALDI) and doubly charged ion at m/z 1339.10 (ESI) defined partial peptide sequences HEA(L/I)S and PGDNVGFNVK based on the N-terminal bn (n = 5–10, 15–23) fragment ions. The National Center for Biotechnology Information (NCBI) database searching with the deduced peptide residues against BLAST matched the protein EF-1{alpha}. The observed tryptic peptide at m/z 2677.259 is supposed to encompass residues 291–313 (SVEMHHEALSEALPGDNVGFNVK). A mass comparison of the theoretically predicted fragments with the measured values identified the peptide sequence except for an undefined residue Glu-301, which contained two inconsistent mass differences of 172.08 and 154.09 Da, as shown in Fig. 3. This overall modification on the residue is 197.05 Da higher than the calculated mass of glutamic acid (129.116 Da). It thus confirms an unusual post-translational modification associated with glycerylphosphorylethanolamine (C5H12O5NP, calculated mass: 197. 045 Da), which localizes at residue Glu-301 in the human EF-1{alpha}. The two mass differences in MS/MS spectra of the parent ion at m/z 2677.26 (Fig. 3) can be rationalized with the loss of neutral glycerylphosphatide molecule (C3H9O6P, calculated mass: 172.014 Da) and the immonium fragment ion of ethanolamine attached to glutamic acid (C7H10O2N2, calculated mass: 154.074 Da). Similar observations had been made by fast atom bombardment tandem mass spectrometry on the rabbit EF-1{alpha} (48), by amino acid analysis of tryptic peptides from a human erythroleukemia cell line (49), and by radiolabeling experiments on proteins from murine lymphocyte lines (50).



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  		FIG. 3. Peptide sequencing by MS/MS measurements. A, the MS/MS spectrum of the singly charged ion at m/z 2677.26 was acquired by Manitoba/Sciex prototype QStar MALDI QTOF tandem mass spectrometer. B, the nanospray ionization MS/MS spectrum of the doubly charged ion at m/z 1339.10 was acquired by Micromass ESI QTOF mass spectrometer followed by computer software MaxEnt 3 translation. The insets show the identified peptide sequence 291–313 and the chemical structure of glycerylphosphorylethanolamine attached on the residue Glu-301 of EF-1{alpha}.

 
Cu-binding Proteins in Other Hepatoma Lines and in Normal Human Liver Tissue—
To examine how typical Hep G2 is of human hepatocytes, we analyzed the Cu metalloproteomes in two other human hepatoma lines (SK-Hep-1 and Mz-Hep-1) and in a sample of normal adult human liver tissue. To examine whether intracellular copper was interfering with detection of Cu-binding proteins, we also studied Cu-depleted Hep G2 cells (designated as HepG2*). When the Hep G2 cells were treated with tetraethylenepentamine for 48 h, intracellular copper dropped by ~50% (from 0.0014 µg/mg of protein at the baseline to 0.00065 µg/mg of protein) as determined by atomic absorption spectrometry. Fig. 4 illustrates the 1D gel profile of the Cu-binding components from the different cell lines, normal tissue, and Cu-depleted Hep G2 cells. Because most of the protein bands in Hep G2 had been analyzed, we excised 35 gel bands from SK-Hep-1, Mz-Hep-1, and the liver tissue specimen for protein identification by time-of-flight mass spectrometry, as summarized in Table IV. The metal-binding proteins enolase, albumin, and transferrin were found in all cell/tissue lysates. In this analysis no significant change in Cu-binding proteins was found between the untreated Hep G2 cells and Cu-depleted Hep G2 cells (termed HepG2* in Fig. 4). In particular, 10 high abundance protein bands excised from both untreated and Cu-depleted Hep G2 cells were found to be identical. Subtle qualitative or quantitative differences between lines could not be excluded.



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  		FIG. 4. SDS-PAGE gel of the Cu-binding proteins in human hepatocyte cells SK-Hep-1, Mz-Hep-1, Hep G2, and Cu-depleted Hep G2 cells (treated with tetraethylenepentamine) with a comparison to a specimen of normal adult liver tissue. Lane 1, molecular weight standards (ST); lane 2, normal human liver tissue (NHL); lane 3, SK-Hep-1 (SK); lane 4, Mz-Hep-1 (MZ); lane 5, Cu-depleted Hep G2 cells (HepG2*); lane 6, untreated Hep G2 cells (HepG2).

 

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  		TABLE IV Identification of copper-binding proteins in different hepatoma lines by in-gel tryptic digestion and mass spectrometric measurements

 
The discernible protein bands (bands 15, 16, and 22 in Fig. 4) clearly visible in the Cu-binding fractions of SK-Hep-1 and Mz-Hep-1 cells were identified as annexin II and its degradation products. Interestingly, this protein was not detected in Hep G2 or in the normal liver tissue. Gel bands 16 and 22 both have a molecular mass of ~39 kDa, which corresponds to the predicted molecular mass for intact annexin II by protein sequence in the database. The high abundance band 15 has a lower molecular mass (~35 kDa), presumably caused by degradation in SK-Hep-1 cells. Close analysis of annexin II tryptic fragments generated from in-gel digestion by either MALDI or ESI MS/MS measurements revealed most of the peptides (residues 13–28, 29–37, 29–47, 29–49, 50–63, and 64–77) in agreement with the predicted protein sequences near the N terminus.

Cu-binding Peptides Determined by MS Analysis after In-solution Tryptic Digestion of Copper-binding Proteins—
We also examined the Cu-binding capacity of proteolytic peptides derived from the various human hepatoma lines. The intact Cu-binding proteins from IMAC separation were digested by trypsin in solution; the resulting peptides were then captured on a copper-charged agarose column, and then unbound peptides were removed. Mapping of the eluted Cu-binding peptides in SK-Hep-1, Mz-Hep-1, Cu-depleted Hep G2 cells, and the normal liver tissue is shown in Fig. 5. Table V summarizes the representative Cu-binding peptides from normal liver tissue and SK-Hep-1 cells, in which the individual peptides were sequenced by MS/MS. The majority of Cu-binding peptides that contained the histidine tag motifs with His, HH, HXH, and HXXH appeared in abundant peaks in the mass spectra. Six peptides containing the MXXH motif were also detected in selenium-binding protein, 70-kDa heat shock protein, enolase, stathmin, and cytokeratin 8.



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  		FIG. 5. MALDI mapping of the copper IMAC-binding peptides in normal human liver tissue (NHL) and in SK-Hep-1, Mz-Hep-1, and Cu-depleted Hep G2 (HepG2*). The copper-binding proteins were isolated by IMAC and subsequently digested by trypsin in solution. The resulting peptides were bound to copper IMAC again, followed by removal of unbound partners with washes.

 

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  		TABLE V Identification of copper-binding peptides resulting from in-solution digests of copper-binding proteins from normal liver tissue and SK-Hep-1 cells by MALDI MS/MS measurements

E*, the residue Glu (E) was modifed by ethanolamine. @, acetylation at the N terminus.

 
The high intensity peptide ion at m/z 2523.239 ± 0.004 was present in all of the samples examined, but MS-Tag searching showed no match to any protein in the database. Again, the partial peptide sequence (residues HEALS) could be inferred from the predominant N-terminal bn (n = 5–10) ions in the MS/MS spectrum (Fig. 6). Because this fragmentation pattern is similar to that in the MS/MS spectrum of the ion at m/z 2677.259 in Fig. 3, a BLAST database search retrieved the same peptide sequence corresponding to the tryptic fragment 291–313 of EF-1{alpha}. The 172.094-Da mass difference between b10 and b11 ions is located at the residue Glu-301. This post-translational modification is caused by ethanolamine alone, which is covalently bound to the residue Glu-301 (calculated mass: 129.043 + 43.042 = 172.085) in the protein sequence. The finding is slightly different from the identification of glycerylphosphorylethanolamine by on-column tryptic digestion of Cu-binding proteins in Hep G2 as described above but agrees with a previous report that a hydrophilic cytosolic protein has been found to incorporate ethanolamine in a variety of different cell lines (51).



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  		FIG. 6. MALDI MS/MS spectrum of the parent ion at m/z 2523.24. The inset shows the chemical structure of post-translationally modified ethanolamine at the residue Glu-301 in the EF-1{alpha} peptide fragment 291–313 (residues SVEMHHEALSEALPGDNVGFNVK).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Protein sequence identification and metal-binding motif analysis are important components in the structural characterization of metal-binding proteins. IMAC is a traditional technology used for separation and purification of histidine-tagged recombinant proteins. This is the first attempt to use proteomics as a strategy for identifying proteins that have metal-binding capacity (metalloproteins or proteins with metal-binding sites). To identify such a metalloproteome, we have attempted to separate metal-binding proteins by an IMAC column prior to analysis by classic proteomic methods. We recognize that this is only one approach and may not identify all proteins in a given metalloproteome. Although in this study we have identified a number of metal-binding proteins in Hep G2 and other human hepatoma lines, many other proteins in the metalloproteome, including low abundance, high mass, and hydrophobic proteins, likely remain unidentified because of technological limitations. In comparison with the currently published proteome of Hep G2 (32), the Cu metalloproteome that we have described here is different. Similarly, differences exist between the general proteome described for SK-Hep-1 and its Cu metalloproteome shown here. Proteomic studies of Mz-Hep-1 have never been reported.

The characterization of Cu-binding peptides by mass spectrometry has revealed the metal-binding ligands that involve histidine, methionine, and cysteine in the peptide sequence. The affinity for a peptide-binding residue to Cu appears to be histidine > methionine > cysteine, which is consistent with thermodynamic results (52). We have found that common motifs for peptides binding to Cu are localized at the sequences HXH, HXXH, and MXXM. The distance between two histidines for Cu binding can vary by as much as 24 residues (enolase in Table V), but at least one proline is maintained within the sequence region for providing appropriate structural geometry. Histidine and methionine can be found in the same structural motif, as HXM or HXXM, for Cu binding. The property of binding of Cu to methionine in a protein compared with cysteine, as we observed, may be considered somewhat unexpected because the mean interatomic metal-ligand bond distances found in metalloproteins are as follows: His, 2.02 Å; Cys, 2.15 Å; and Met, 2.55 Å (53). A plausible explanation could be that the methyl group is acting as an electron donor and that its electron density transfer to neighboring sulfur atom promotes the coordination between Cu and methionine. Also, although the cysteine residue is coordinated to Cu, binding of the thiol side chain is weakened due to rapid reduction of Cu(II) to Cu(I). Analysis of metal-binding peptides offers an alternate methodology for identifying metal-binding proteins, especially those in low abundance. However, the identification of Cu-binding peptides has shown the different selectivity for on-bead digested proteins (i.e. protein binding) and in-solution digests (i.e. peptide binding) as shown in Figs. 2 and 5. Different analytical approaches must be combined to maximize characterization of metal-binding proteins.

The Cu metalloproteome in Hep G2 is comprised both of proteins that are known to bind metals such as Cu and Zn and also of proteins not previously identified to have metal binding capability. PDI is a prominent example of the latter; we have now determined that PDI can actually bind Cu and other metals (54). For complete definition of a metalloproteome, functional studies of the identified proteins are required as an essential complement to proteomics analysis. These functional studies will eliminate any proteins identified nonspecifically. Refinements of the IMAC technique may also reduce the extent of nonspecific inclusion of proteins in a metalloproteome. Improvement in techniques for isolation of low abundance and membrane-bound proteins may enhance the inclusiveness of the metalloproteome.

Gel profiling of Cu-binding proteins showed differences between human hepatoma lines, most prominently involving annexin II. The metal binding properties of proteins in the annexin family, including annexin II, have been described previously (55–57). A pair of amino acid sequence repeats may form a composite binding site for a metal ion and a phospholipid in the structure of annexin II (58). Since expression of annexin II may be reduced or lost in prostate cancer cells in vivo (59, 60), changes as we found in our studies may relate to neoplastic transformation in human hepatoma cells. Another important finding is the identification of a variable post-translational modification in the Cu-binding protein EF-1{alpha} by MS/MS measurements and homology protein database searching. We have detected glycerylphosphorylethanolamine at residue Glu-301 by in-gel tryptic digestion of the protein in Hep G2. Differences in this post-translational modification were found after depletion of copper in Hep G2 cells; namely, the glycerylphosphoryl side group was lost. Although the biological role of modifications between glycerylphosphorylethanolamine and ethanolamine is unknown, the loss of this glycerylphosphoryl group in human EF-1{alpha} suggests that a lack of glycerylphosphorylation at the Cu-binding sequence region may result from copper depletion.

Our studies on the Cu and Zn metalloproteomes in human hepatoma lines by mass spectrometric analyses have identified unique sets of proteins involved in copper and zinc disposition in hepatocytes. We expect that further studies of these metalloproteomes will extend our knowledge of the handling of these metals in cells.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Kenneth G. Standing and Dr. Werner Ens of the University of Manitoba for kind support and for the use of the University of Manitoba/Sciex MALDI QTOF tandem mass spectrometer.


   FOOTNOTES
 
Received, August 20, 2003, and in revised form, October 3, 2003.

Published, MCP Papers in Press, October 7, 2003, DOI 10.1074/mcp.M300080-MCP200

1 The abbreviations used are: 2DE, two-dimensional gel electrophoresis; 2D, two-dimensional; 1D, one-dimensional; ESI, electrospray ionization; IMAC, immobilized metal affinity chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem MS; PDI, protein-disulfide isomerase; QTOF, quadrupole time-of-flight; EF-1{alpha}, elongation factor 1-{alpha}; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Back

* This work was supported in part by Grant MOP1800 from the Canadian Institutes of Health Research and by grants from the Ontario Research and Development Challenge Fund and the Coady Family Fund for Hepatic Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Structural Biology and Biochemistry, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5921; Fax: 416-813-5379; E-mail: bsarkar{at}sickkids.ca


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