Using Immobilized Metal Affinity Chromatography, Two-Dimensional

Jul 16, 2004 - Department of Structural Biology and Biochemistry, Advanced Protein Technology Centre, Metabolism Research Program, The Hospital for Si...
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Using Immobilized Metal Affinity Chromatography, Two-Dimensional Electrophoresis and Mass Spectrometry to Identify Hepatocellular Proteins with Copper-Binding Ability Scott D. Smith,†,‡ Yi-Min She,§ Eve A. Roberts,|,# and Bibudhendra Sarkar*,†,‡ Department of Structural Biology and Biochemistry, Advanced Protein Technology Centre, Metabolism Research Program, The Hospital for Sick Children Research Institute, Toronto, Canada Received March 2, 2004

To further our knowledge of intracellular copper transport, we used a proteomics strategy to search for hepatic proteins with copper-binding ability. Hep G2 cytosolic and microsomal fractions were applied to a copper(II)-loaded immobilized metal-affinity chromatography (IMAC) column. Protein identification was performed with 2-D gel electrophoresis and mass spectrometry. We identified 48 cytosolic proteins and 19 microsomal proteins displaying copper-binding ability. These proteins are diverse in function. Fifty-two of the 67 proteins contain putative metal-binding domains. We have identified many components of the Hep G2 copper metalloproteome including a large number of proteins not previously known to bind copper. Keywords: Copper-binding proteins • Hep G2 • proteomics • peroxiredoxin

Introduction Copper is an essential trace element that plays important structural and functional roles in all aerobic organisms. Excess levels of copper can catalyze the formation of free radicals that are damaging to cellular components. The cell’s handling of copper is thus highly regulated and there is effectively no free copper present in the cytoplasm.1 A large number of known copper-containing enzymes and copper-binding proteins participate in diverse biological functions. Intracellular copper disposition is complex.2-4 The trafficking and delivery of copper to cellular proteins remains poorly understood. This is demonstrated by the continuing emergence of proteins possessing novel copper-binding ability, including the endogenous prion protein (PrP) and protein disulfide isomerase (PDI), a multifunctional oxidative folding catalyst.5,6 Metalloproteomics refers to the identification and detailed characterization of metal-binding proteins and their metalbinding motifs. Since most metals pass through the liver for detoxification, hepatocytes are ideal for the study of proteins involved in intracellular heavy metal metabolism. To date, a * To whom correspondence should be addressed. Department of Structural Biology and Biochemistry, The Hospital for Sick Children, 555 University Ave, M5G 1X8, Toronto, Canada. E-mail: [email protected]. Tel: +1-(416) 813-5921. Fax: +1-(416) 813-5022. † Department of Structural Biology and Biochemistry, The Hospital for Sick Children Research Institute. ‡ Department of Biochemistry, The Hospital for Sick Children Research Institute. § Advanced Protein Technology Centre, The Hospital for Sick Children Research Institute. | Metabolism Research Program, The Hospital for Sick Children Research Institute. # Departments of Paediatrics, Medicine and Pharmacology, The University of Toronto, Toronto, Canada.

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comprehensive study of the hepatocellular proteins that constitute the copper metalloproteome does not exist. Determining this distinct proteome will help uncover novel components of copper transport and extend our understanding of the disease mechanisms of copper-associated diseases. The Hep G2 human hepatoma line is widely used for the study of hepatic function. Hep G2 cells closely resemble differentiated hepatocytes and produce numerous proteins characteristic of normal hepatocytes (albumin, R1-antitrypsin, transferrin, ceruloplasmin, and other proteins).7 The composition of a general Hep G2 proteome was recently described8 and a corresponding 2D map is presented in the SWISS-2D PAGE database9. Here, we report a comprehensive assessment of the copper metalloproteome in Hep G2. We first separate Hep G2 proteins into cytosolic and microsomal fractions, greatly improving the resolution of our 2-dimensional gel electrophoresis (2DE) separation compared to initial attempts with Hep G2 lysate.10 Our metalloproteomic approach employs a copper(II)-IMAC column, 2DE and MALDI QqTOF mass spectrometry (MS).

Experimental Section Tissue Culture and Sample Preparation. The Hep G2 human hepatoma cell line was obtained from the American Type Culture Collection (ATCC HB 8065). Cells were cultured in R-minimal essential medium containing 10% fetal bovine serum in a humidified 5% CO2 incubator at 37 °C. Confluent cells were detached with 0.25% trypsin citrate saline (DIFCO) and washed with phosphate buffered saline without Ca2+ or Mg2+. The harvested cells were resuspended in hypo-osmotic buffer containing 10 mM HEPES-NaOH, pH 7.8 and swelled on ice. Cells were then isolated by centrifugation at 800 × g and resuspended in hyper-osmotic buffer containing 0.6 M 10.1021/pr049941r CCC: $27.50

 2004 American Chemical Society

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sucrose, 10 mM HEPES-NaOH, pH 7.8. Homogenization was performed using a Douane homogenizer (Wheaton Type A) and 30 strokes with the tight fitting pestle. Protease inhibitor cocktail without EDTA (Roche) was added, and cellular debris and larger organelles were removed by centrifugation at 8000 × g. The resulting whole cell extract was separated into cytosolic and microsomal fractions by ultracentrifugation at 150 000 × g for 45 min using a Beckman Optima L-90K ultracentrifuge. The microsomal pellet was resuspended in iso-osmotic buffer containing 0.25 M sucrose and 10 mM HEPES-NaOH, pH 7.8. Immobilized Metal-Affinity Chromatography (IMAC). Copper-IMAC columns were prepared using 2 mL Bio-Spin columns (BioRad) and separation was performed by gravity flow. Copper (II) was coupled to Chelating Sepharose Fast Flow resin (Amersham Pharmacia) by applying a 50 mM CuSO4, 50 mM sodium acetate, pH 4.0, 0.5 M NaCl solution. Excess metal was removed with 50 mM sodium acetate, pH 4.0, 0.5 M NaCl (×3) and the column was equilibrated with equilibration/wash buffer (10 mM Tris-HCl, pH 8.0, 4 M urea, 0.5 M NaCl, 0.25 M sucrose, 0.5% (w/v) Triton X-100) (×3). Hep G2 cytosolic and microsomal preparations (5-10 mg) in equilibration/wash buffer were bound to the column for 2 h at 4 °C with gentle shaking. The column was washed thoroughly with 10 column volumes of equilibration/wash buffer and bound proteins were eluted with 50 mM EDTA. Protein concentrations were determined using the BioRad protein assay and fractions were monitored by 1D SDS-PAGE. 1-Dimensional SDS-PAGE. One-dimensional SDS-PAGE separation of IMAC column fractions was performed as described by Laemmli.11 Proteins were visualized with Coomassie Brilliant Blue R-250 stain. 2DE Separation. Samples were concentrated with Microcon centrifugal filter devices (MWCO 10 kDa) and dialyzed with PlusOne Mini Dialysis tubes (Amersham Pharmacia) (MWCO 8 kDa) into iso-electric focusing (IEF) rehydration buffer (7 M urea, 2 M thiourea, 0.5% (w/v) Triton X-100, 4% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 0.5% (v/v) IPG buffer). IEF was preceded by rehydrating 13 cm immobilized pH gradient (IPG) drystrips, pH 3-10 (Amersham Pharmacia), with rehydration buffer containing appropriate amounts of protein samples. Rehydrated strips were focused overnight with an IPGphor instrument (Amersham Pharmacia). The following morning focused IPG strips were reduced with equilibration buffer A (6 M urea, 30% (v/v) glycerol, 125 mM dithiothreitol (DTT), 2% (w/v) SDS, 50 mM Tris-HCl, pH 8.0) for 15 min and then alkylated with equilibration buffer B (6 M urea, 30% (v/v) glycerol, 125 mM iodoacetamide, 2% (w/v) SDS, 50 mM Tris-HCl, pH 8.0) for an additional 15 min. Reduced IPG strips was then resolved for 24 h at 40 V on a vertical 12% SDS-PAGE gel. Gels were stained with Coomassie Brilliant Blue R-250 and protein spots were excised and stored at -80 °C for MS analysis. In-Gel Tryptic Digestion and MALDI QqTOF MS Analysis. In-gel tryptic digestion and recovery of tryptic peptides were modified from previously described protocols.12 Briefly, gel spots were crushed and destained with 100 mM NH4HCO3 and acetonitrile until colorless. The gel pieces were reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, dehydrated with acetonitrile and dried in a SpeedVac evaporator (Fisher Scientific). Gel pieces were rehydrated with 20 µL of 50 mM NH4HCO3 containing 20 ng trypsin (Promega) and incubated on ice for 30 min. An additional 20-40 µL of 50 mM NH4HCO3 was added to cover the swelled gel pieces and samples were

Figure 1. One-dimensional SDS-PAGE separation of Hep G2 proteins bound to copper-IMAC columns. (A) Hep G2 lysate applied to a control column not charged with metal. (B) Hep G2 microsomal fraction applied to a copper-IMAC column. (C) Hep G2 cytosolic fraction applied to a copper-IMAC column. Columns were washed with 10 column volumes of wash buffer and bound proteins were eluted with 50 mM EDTA. Fractions were separated on 12% SDS-PAGE gels and stained with Coomassie Blue.

incubated at 37 °C overnight. The tryptic peptides were recovered sequentially with (i) 50 mM NH4HCO3, (ii) 50% acetonitrile/1% trifluoric acid (2X) and (iii) 80% acetonitrile. Samples were desalted using µC18 ZipTips (Millipore) prior to MS analysis. The desalted peptides were eluted in 2 µL of matrix, a saturated solution of 2,5-dihydroxybenzoic acid in 50% acetonitrile and 0.1% trifluoroacetic acid and spotted directly onto the MS target plate. Mass spectrometric analysis was performed in the Advanced Protein Technology Centre at the Hospital for Sick Children with an Applied Biosystems/MDS Sciex MALDI QStar XL QqTOF mass spectrometer equipped with a UV nitrogen laser (337 nm) for sample ionization, and an accelerating voltage of 4 kV. Accurate mass determination was achieved by external calibration with two standard peptides (dalargin and melittin). Data Processing. Proteins were identified by searching the experimental peptide masses at the ProFound (http://prowl. rockefeller.edu/profound_bin/WebProFound.exe) and ProteinProspector (www.prospector.ucsf.edu) databases. Experimentally determined tryptic peptide masses, as well as MW and pI values from 2DE gels, were compared to theoretical masses of trypsin digested proteins contained in the SWISS-PROT and NCBI protein databases to identify proteins. Search criteria were selected as follows: monoisotopic masses, one missed cleavage, carbamidomethylation of cysteines and oxidation of methionine residues were accepted as optional modifications. The tryptic peptide masses were compared to the theoretical masses of all documented proteins from all species. Protein candidates identified by MALDI QqTOF mass spectrometry were verified by two highly reproducible gel spots and subsequent MS/MS measurements of peptides to conclusively identify the proteins.

Results and Discussion Copper(II)-IMAC Column Separation of Proteins. Binding of proteins to the copper-IMAC column was monitored by 1D SDS-PAGE and the BioRad protein assay (Figure 1). No protein was bound to a control column not charged with metal. Following washes with 10 column volumes we expected all nonspecifically bound proteins to be removed from the columns. There was a significant amount of protein bound to copper-IMAC columns for both cytosolic and microsomal fractions. Following elution, copper-IMAC columns yielded Journal of Proteome Research • Vol. 3, No. 4, 2004 835

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Table 1. Hep G2 Microsomal Proteins Eluted from a Copper-IMAC Column and Identified by MALDI QqTOF Mass Spectrometry protein name (NCBI or SWISS-PROT accession no.)

albumin (gi:4389275) cell growth regulating nucleolar protein (Q08288) GRP78 (glucose-regulated protein 78 kD); BiP (P11021) PDI (protein disulfide isomerase) A3 precursor (P30101) transferrin (NP_001054) hsc70 (heat shock cognate 71 kD protein) (P11142) 40S ribosomal protein sa; 35/67 kDa laminin receptor (P08865) 40S ribosomal protein s4 (P15880) ASF2; alternative splicing factor 2 (B40040) heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) (P22626) hnRNP H (P31943) hnRNP H3 (P31942) hnRNP K (NP_112553) hnRNP L (P14866) Pro/Glu rich splicing factor; polypyramidine tract-binding protein (P23246) SAP 62; spliceosome-associated protein; splicing factor 3a subunit 2 (NP_009096) γ-actin (P02571) GAPDH; glyceraldehydes-3-phosphate dehydrogenase (P04406) RACK 1; guanine nucleotide binding protein (P25388)

MW (kDa)/pI

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function

known cation-binding proteins 66.0/5.7 22 -transport of Cu(II) in blood 44.1/9.8 26 -cell growth regulation -contains Zn-finger binding motifs 72.3/5.1 22 -ER Ca2+-binding chaperone 57.2/6.0

21

77.1/6.8

24 chaperones 70.9/5.4 22

-an oxidative folding catalyst -binds 4-10 Cu(I)/monomer6 -Fe-binding transport protein

-assists protein folding with binding and release of substrates nucleic acid binding proteins 32.9/4.8 48 -component of 40S ribosomal subunit 31.3/10.4 44 -component of 40S ribosomal subunit 32.0/5.6 30 -splicing factor and a component of the hnRNP complex 37.4/9.0 28 -mRNA splicing and polyadenylation 49.2/5.9 40 -mRNA splicing and polyadenylation 36.9/6.4 32 -mRNA splicing and polyadenylation 51.0/5.2 30 -transcription and translational regulation 60.2/6.7 16 -mRNA export and stability

putative metalbinding motif(s)

17 disulfides 4 CXXC, HH, HX5H none 2 CXXC 4 CXXC, HH MXXH none HX3H HH HH, HX4H HXXH 2 HXXH, HX5H none

76.1/9.1

35

-mRNA splicing factor

66.0/9.7

22

-mRNA splicing factor

CXXC, 2 HXH, 5 HX5H 2 HH, HXH, HXXH, 2 HX3H, 2 HX4H CXXC

-cell motility -glycolytic enzyme

HH HX3H, CX3C

-intracellular receptor of activated PKC

HXH, HXXH

miscellaneous 42.0/5.3 25 35.9/8.4 23 35.1/7.6

400-600 µg of copper-binding proteins for 2DE analysis per 5 mg of Hep G2 protein preparation. 2DE Separation and MS Identification of Hep G2 Microsomal Proteins Eluted from a Copper-IMAC Column. Excellent reproducibility among three microsomal 2DE gels was achieved. We have identified 19 Hep G2 microsomal proteins exhibiting copper-binding ability. Microsomal proteins identified from the copper-IMAC column are listed in Table 1 and Figure 2 illustrates a representative gel of the 3 experiments performed for this analysis. The membrane preparations were devoid of nuclei and mitochondria, and the identified proteins are mostly associated with organelles in the perinuclear region such as the Golgi apparatus and the endoplasmic reticulum (ER). Ten of the identified proteins are nucleic acid binding proteins including 6 heterogeneous ribonucleoproteins (hnRNPs), 2 splicing factors and 2 ribosomal proteins. Two protein chaperones (glucose-regulated protein 78 kD and heat shock cognate 71 kD protein) and 3 known copper-binding proteins (transferrin, albumin, and protein disulfide isomerase) were also identified. Of the 19 microsomal proteins identified, 16 contain putative metal-binding domains (H(X)nH (n ) 0-5) or C(X)mC (m ) 2-4)) or known metal cation-binding motifs. Five of the identified proteins have known metal cation-binding capabilities (transferrin, albumin, protein disulfide isomerase, glucose-regulated protein 78 kD, and cell growth regulating nucleolar protein). Cell growth regulating nucleolar protein was identified due to its high degree of homology to the mouse LYAR gene product (Q08288). 2DE Separation and MS Identification of Hep G2 Cytosolic Proteins Eluted from a Copper-IMAC Column. The three cytosolic 2DE gels used displayed excellent reproducibility. In 836

seq. cov. (%)

55

Figure 2. 2DE map of Hep G2 microsomal proteins displaying copper-binding ability. After elution from a copper-IMAC column the proteins were separated on a pH 3-10 IPG strip and resolved on a vertical 12% SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue R-250 and protein spots were analyzed by MALDI QqTOF mass spectrometry. The identified proteins are listed in Table 1.

total, we have identified 48 cytosolic proteins exhibiting binding to a copper-IMAC column. Hep G2 cytosolic proteins displaying copper-binding ability are listed in Table 2 and Figure 3 shows a representative gel of the 3 experiments used for this analysis. The identified proteins vary widely in function and include 6 oxidoreductases, 6 chaperones, 5 nucleic acid binding proteins, 4 glycolytic enzymes and 4 cytoskeletal proteins. Twelve of the cytosolic proteins are known to bind cations (these consist of

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Table 2. Hep G2 Cytosolic Proteins Eluted from a Copper-IMAC Column and Identified by MALDI QqTOF Mass Spectrometry protein name (NCBI or SWISS-PROT accession no.)

R-actinin (P128167) aldehyde dehydrogenase (P00352) aminoacylase 1 (Q03154) annexin IV (P09525) annexin V (Q8WV69) calreticulin precursor; calregulin (P27797) R-enolase; 2-phosphopyruvate hydratase (P06733) GRP78 (glucose-regulated protein 78 kD); BiP (P11021) PDI (protein disulfide isomerase) (P07237) PDI (A3) precursor (P30101) pyruvate kinase, L-isozyme (P14786) transketolase (P29401) aldehyde reductase; alcohol dehydrogenase (P14550) cytochrome b5 reductase (P00387) GR (glutathione reductase) (P00390) peroxiredoxin (Prx) 1 (Q06830) peroxiredoxin 2 (P32119) peroxiredoxin 6 (P30041) cyclophilin A; peptidylprolyl isomerase A (NP_066953) hsc70 (heat shock cognate 71 kD protein) (P11142) hsp60 (heat shock protein 60 kD) (P10809) hsp86 (heat shock protein 90 kD, R) (P07900) hsp90 co-chaperone; telomerase-binding protein p23 (Q15185) STI1 (stress-induced phosphoprotein 1); hsp70/hsp90 organizing protein (P31948) 40S ribosomal protein sa (34/67 kd laminin receptor) (P08865) eEF-1A (euk. translation elongation factor 1A) (Q96RE1) eIF-3A; p27 BBP protein (NP_852134) eIF-4A (eukaryotic translation initiation factor 4A) (P04765) NDK A (nucleoside diphosphate kinase) (P15531) GAPDH; glyceraldehyde 3-phosphatedehydrogenase (P04406) GPI (glucose-6-phosphate isomerase) (P06744) l-lactate dehydrogenase M chain (P00338) phosphoglycerate kinase I (P00558) R-tubulin (Q13748) β-tubulin (P07437) cofilin (P23528) γ-actin (P02571) 14-3-3 protein; protein kinase C inhibitor protein 1 (P31946) aspartate aminotransferase (P17174) glutathione synthetase (P48637) HDGF (hepatoma-derived growth factor) (P51858) isocitrate dehydrogenase (P50213) NCC27 (nuclear chloride channel protein) (O00299) PA28R (proteasome activator 28R subunit) (Q06323) PEBP (phosphatidylethanolamine binding protein) (P30086) phosphogylycerate mutase I (P18669) RanBP1 (Ran binding protein 1) (P43487) UDP-GlcDH (UDP-glucose dehydrogenase) (O60701)

MW (kDa)/pI

seq. cov. (%)

putative metalbinding motif(s)

function

known cation-binding proteins 103/5.2 MS/MS -contains 2 EF-hand Ca2+ binding domains 55.5/6.3 45 -binds 2 Zn ions per subunit 46.1/5.8 61 -hydrolysis of amino acids -incorporates Zn as a cofactor 36.3/5.6 34 -promotes membrane fusion -Ca2+/phospholipid-binding 35.8/5.0 42 -anticoagulant -Ca2+/phospholipid-binding 48.3/4.3 32 -ER lumenal Ca2+-binding protein 47.2/7.0 36 -Mg2+ as a cofactor -Zn-binding protein 72.3/5.1 28 -ER Ca2+-binding chaperone 57.5/4.8 47 -an oxidative folding catalyst -binds 4-10 Cu(I)/monomer6 57.2/6.0 37 -a PDI precursor 57.9/8.0 21 -Mg2+ and K+ as cofactors 67.9/7.6 55 -binds Ca2+ and Zn2+ redox proteins 36.7/6.3 56 -reduction of xenobiotic aldehydes 30.9/8.9 39 -protects cell against reactive oxygen species 52.2/7.1 36 -maintains high levels of reduced glutathione in cytosol 22.3/8.7 62 -peroxide detoxification -disulfide linked homodimer 21.7/5.7 42 -peroxide detoxification -disulfide linked homodimer 25.1/6.0 73 -a 1-Cys Prx chaperones 18.1/8.1 68 -accelerates protein folding

HXH, HX4H, CX3C HH, HX4H HXH, HXXH, HX4H HXXH none none none none 2 CXXC 2 CXXC 2 HXXH CX3C HH HH HXH, HX4H and interchain disulfide interchain disulfide and HXCH interchain disulfide (Cys51-Cys172) none none

70.9/5.4

36

-assists protein folding

none

57.8/5.1 85.0/4.9 18.7/4.5

55 39 68

-mitochondrial protein import -molecular chaperone -hsp90 co-chaperone

2 HH, HXXH HX4H none

63.2/6.4

50

-molecular chaperone and stress response nucleic acid binding proteins 32.9/4.8 46 -component of 40S ribosomal subunit 43.3/9.1 24 -polypeptide chain elongation at the ribosome 27.1/4.6 39 -regulates the association of the 40S and 60S ribosomal subunits 46.4/5.3 31 -assists mRNA binding to ribosomes 17.3/5.8 70 -binds DNA nonspecifically glycolytic enzymes 36.1/8.6 25 -glycolysis (2nd phase, 1st step) -glycolysis (2nd step) and gluconeogenesis 36.8/8.7 66 -anaerobic glycolysis (final step) 45.0/8.6 62 -glycolysis (2nd phase, 2nd step) and is a polymerase R cofactor cytoskeletal proteins 50.0/4.9 52 -major component of microtubules 49.8/4.7 38 -major component of microtubules 18.7/8.5 46 -major component of actin cytoplasmic rods 42.05.3 40 -cell motility miscellaneous 29.3/4.6 44 -multifunctional regulator of cell signaling 46.3/6.8 53 -converts aspartate to glutamate 52.4/5.7 33 -glutathione biosynthesis 26.4/4.7 40 -heparin-binding protein with mitogenic activity 46.9/6.5 48 -converts isocitrate to 2-oxoglutarate 26.9/5.1 52 -acts as a Cl- ion channel in nucleus, cytoplasm and plasma membrane 28.9/5.8 63 -implicated in immuno-proteasome assembly 21.1/7.0 78 -inhibits Raf-1 63.1/8.4

28.8/6.7 23.3/5.2 55.7/6.8

24

45 44 68

-phosphoglycerate mutase family -inhibits GTP exchange of Ran -biosynthesis of glycosaminoglycans

HXH, CXXC, CX3C none HH, HXXH CXXC, CX3C CXXC none HX3H, CX3C CXXC, HH, HXH, HX4H HX4H HXXH CX4C, HX4H none none HH none HX3H HXH, HX3H none HX5H none CX4C HH, HXXH none HXXH HXH

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Figure 3. 2DE map of Hep G2 cytoplasmic proteins displaying copper-binding ability. After elution from a copper-IMAC column the proteins were separated on a pH 3-10 IPG strip and resolved on a vertical 12% SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue R-250 and protein spots were analyzed by MALDI QqTOF mass spectrometry. The identified proteins are listed in Table 2.

calcium-, zinc-, magnesium- and copper-binding proteins). Of the 48 identified cytosolic proteins, 36 contain putative metalbinding domains (H(X)nH (n ) 0-5) or C(X)mC (m ) 2-4)) or known metal cation-binding motifs. The proteins without putative metal-binding domains may be present as a result of protein-protein interactions with copper-binding proteins. Functional Distribution of Identified Proteins. The components of the Hep G2 copper metalloproteome outlined here include both known metal-binding proteins and proteins not previously known to bind metal. The 67 identified proteins have a wide range of cellular functions (Figure 4) and represent mostly high abundance proteins. As expected, the Hep G2 copper metalloproteome is distinct from the general Hep G2 proteome previously described.8 Although it is unlikely that all of the identified proteins bind copper under physiological conditions we feel that some of the proteins that do not bind copper physiologically could be targets of copper during copper toxicity, such as in Wilson disease patients. A number of functional clusters of proteins emerged from this study. Nucleic acid binding proteins, particularly hnRNPs (ASF2, hnRNP A2/B2, H, H3, K, L), are prevalent in the 19 microsomal proteins identified. Twenty different components of hnRNP complexes have been identified in humans where they form large and diverse mRNA-protein complexes.13 Certain hnRNPs are extremely abundant.14 Other microsomal proteins identified are ribosomal and splicing proteins. All of these proteins are localized in the microsomal fraction due to their association with the ribosomal complex. The role of transition metals during ribosomal assembly has not been explored though a number of ribosomal proteins have been shown to display novel zinc-binding ability, suggesting a possible role for zinc in ribosomal function.15 Other nucleic acid binding proteins were identified in the cytoplasmic fraction and include translation initiation and elongation factors (eIF-3A, eIF-4A, 838

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Figure 4. Distribution of identified proteins in the Hep G2 (A) microsomal and (B) cytosolic copper metalloproteome. Known metal cation-binding proteins consist of calcium-, zinc-, magnesium-, iron- and copper-binding proteins.

eEF-1A). Copper interaction with eEF-1A has been previously noted where cellular copper depletion led to a decrease in glycerylphosphorylation at Glu 301 in the copper-binding region.10 Other functional clusters of proteins identified here include redox proteins, protein chaperones, glycolytic enzymes and cytoskeletal proteins. Copper-Binding via Cysteine Residues. We identified a large number of redox proteins and protein chaperones. We have recently shown that the chaperone protein disulfide isomerase possesses novel copper-binding ability, binding copper via its two catalytic CXXC motifs.6 CXXC motifs are used by the Wilson and Menkes ATPases (ATP7B and ATP7A), Atox1 and other copper chaperones to bind copper.16-19 In addition to copperbinding, the CXXC motif is one of the best characterized redox motifs and is used for the generation, isomerization, and reduction of disulfide bonds.20,21 Interestingly, a number of proteins containing CXXC motifs were identified in this study. These include protein disulfide isomerase, hnRNP L, SAP 62, stress-induced phosphoprotein 1, eIF-3A, eIF-4A, GPI, and cell growth regulating nucleolar protein. Our study indicates that a copper(II)-loaded-IMAC column has significant affinity for cysteine-containing motifs. Although copper-IMAC columns have been used for the purification of cysteine-containing peptides,22 the effectiveness of copper-IMAC for selectively capturing proteins with cysteine-containing motifs is not widely appreciated. Peroxiredoxin (Prx) 1 and Prx2, members of the typical 2-Cys family of peroxiredoxins, were also identified here. Prxs are multifunctional thiol-specific antioxidant proteins that are highly abundant in the cytoplasm of mammalian cells23 where they regulate antioxidant defense and tumor suppression24 and control hydrogen peroxide signaling.25 The structures of Prx1 and Prx2 reveal that the redox-active cysteines form a nonrigid intermolecular disulfide bridge.26,27 Initial metal-binding studies in our laboratory indicate that typical 2-Cys Prx family members

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Copper Metalloproteomics

have novel metal-binding ability that influences the protein’s oligomeric state and might involve binding by a novel mechanism, via the intermolecular disulfide bridge. Prxs have been shown to bind to the protein chaperone cyclophilin A (CyPA), a protein implicated in the reduction and activation of human Prxs.28 Since CyP-A contains no putative metal-binding motifs, it may have been identified due to association with a Prx. Indeed, it is possible that other proteins identified here are due to indirect association to the IMAC column via protein-protein interactions or other nonspecific interactions. However, these represent a small fraction of the identified proteins. Studies are ongoing to determine the extent of this phenomenon. There is a striking overlap between the metal-binding proteins identified in our study and those identified in studies using redox proteomics to identify proteins glutathionylated at cysteine residues.29,30 To identify glutathionylated proteins these researchers radiolabeled glutathione and oxidatively stressed cells. Heat shock cognate 71 kD protein, stress-induced phosphoprotein 1, heat shock protein 60 kD, protein disulfide isomerase, R-enolase, Prx1, CyP-A, actin, cofilin, hepatomaderived growth factor, and a translation elongation factor were all identified in our study, as well as glutathionylated in human T lymphocytes.29 Furthermore, in Hep G2 cells heat shock cognate 71 kD protein, CyP-A, actin, and Ran specific GTPase activating protein (RanBP1) were found to bind glutathione.31 The intersection of these proteomes can be attributed to metalbinding sites and glutathione-binding sites both containing surface accessible cysteine residues. This gives rise to the paradox that glutathione, a copper transporter itself, likely competes for access to metal-binding sites of other copper transporters. Effectiveness of the Cu(II)-IMAC Strategy. A major challenge of proteomics is the selective isolation of new and functionally significant groups of proteins for analysis. Several effective isolation methods have been described for the proteomic analysis of metal-binding proteins.10,15,32 To selectively isolate hepatic copper-binding proteins we employ a copper(II)-loaded IMAC column. Our copper-IMAC strategy for isolating copperbinding proteins utilizes approximate in vivo buffer conditions and is ideal for the selective identification of high abundance proteins with affinity for copper. Chromatography with IMAC columns has long been used in protein separation and purification protocols.33 Ren et al. analyzed the effectiveness of a Cu(II)-IMAC column for selecting histidine-containing peptides for comparative proteomics.34 They found it to be highly selective for histidine-containing peptides with a low degree of nonspecific binding. Our results suggest that Cu(II)-IMAC also has significant affinity for cysteine-containing proteins. Here, we employ the IMAC technology in a novel fashion, to isolate a biologically related subset of proteins. The presence of known copper-binding proteins (albumin, transferrin, protein disulfide isomerase) validates the IMAC approach. Although advances in 2DE and MS have facilitated the rapid analysis of large groups of proteins,35-37 resolving membrane proteins by 2DE remains a major challenge of proteomic research.38 We expect that a number of low abundance and/ or highly hydrophobic proteins remain unidentified in our study due to the technological limitations of 2DE. Conspicuously absent are a number of known copper transporters (Atox1, Ccs, ATP7A, ATP7B, and hCTR1). We suspect that several of these proteins were present in the IMAC eluate but

were below the detection level of 2DE. All of these absences are easily explained. ATP7A is not expressed in hepatocytes.39-41 ATP7B and hCTR1 are both membrane bound and thus incompatible with 2DE separation. Furthermore, hCTR1 is a plasma membrane protein and absent from the cytosolic and microsomal fractions used for this analysis. The copper chaperones, Atox1 and Ccs, are present at low abundance and are below the detection limit of this study. The detection limit of this study is limited by the use of Coomassie Blue stain which lies between 50 and 100 ng of protein. In the future, we plan to use silver staining to achieve greater sensitivity. Another copper-protein missing from our identification was Cu/Zn superoxide dismutase (SOD1). This essential antioxidant is an abundant cytosolic protein that coordinates copper with four histidine residues.42 However, with a molecular weight of 15.8 kDa, superoxide dismutase might be too small to be visible on the 12% SDS-PAGE gels used for our 2DE analysis. Additionally, metalloproteins that bind copper with a high affinity at integral sites, such as SOD1, likely pass through the Cu(II)IMAC column due to occupation of binding sites by physiological copper. The strength of the IMAC strategy is instead its ability to capture proteins involved in intracellular copper transport, that is, proteins that bind exchangeable copper. This is demonstrated in this study by the identification of albumin and transferrin. The isolation of metalloproteins such as SOD1 and ceruloplasmin is a challenge yet to be solved.

Conclusions We have identified numerous components of the human hepatic copper metalloproteome. This unique metalloproteome consists of known copper-binding proteins and proteins not previously known to bind copper. Some of these bind copper with short metal-binding motifs, whereas others possess spatial copper-binding sites due to their tertiary structure. We have shown that the copper(II)-IMAC system has a significant affinity for cysteine-containing motifs. Increasing the sensitivity of our proteomic analysis will improve our knowledge of hepatic copper transport and the disease mechanisms of copperassociated diseases. We are currently characterizing the copperbinding functions of the identified proteins. This characterization will determine to what extent the detected copper-binding ability of these proteins is physiological. Work in our laboratory indicates that when Hep G2 lysate is applied to copper-, ironand zinc-IMAC columns unique proteomes are generated upon elution. This indicates that the metalloproteomic strategy described here is readily transferable to the study of other biologically important metals. Abbreviations: IMAC, immobilized metal-affinity chromatography; 1D SDS-PAGE, 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; MALDI QqTOF, matrix assisted laser desorption/ionization quadrupole time-of-flight; 2DE, 2-dimensional electrophoresis; MS, mass spectrometry; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; IEF, iso-electric focusing; IPG, immobilized pH gradient; DTT, dithiothreitol; hnRNP, heterogeneous nuclear ribonucleoprotein; ER, endoplasmic reticulum; Prx, peroxiredoxin; CyP-A, cyclophilin A.

Acknowledgment. This research is supported by a grant from the Canadian Institutes of Health Research Grant MOP1800 and by the Coady Family Fund for Hepatic Research. We thank Suyun Yang for expert technical assistance. Journal of Proteome Research • Vol. 3, No. 4, 2004 839

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