Identification and characterization of metallodrug binding proteins by (metallo)proteomics

Abstract

The success of cisplatin in clinic has stimulated great interest in the development and application of metal-based drugs for therapeutic and diagnostic purposes. However, the treatment efficiency of metallodrugs suffers from side-effects and drug resistance. To overcome these challenges, targets of these metal-based drugs should be identified in order to understand the molecular mechanisms of actions of these compounds and to the intrinsic or acquired drug resistance by cancer cells and infectious microbes. This review summaries some of the recent developments in the identification of binding proteins and their target sites of platinum-, ruthenium-, gold-, arsenic- and bismuth-containing agents by proteomics and metalloproteomics, which may provide a rational basis for the design of new metal-based drugs.

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1. Introduction

The use of metal compounds in medicine can be traced back to 5000 years ago, when copper was used by Egyptians to sterilize water.^1,2 Over the past decades, extensive knowledge of the coordination and redox properties of metal ions has facilitated the development of metal-based drugs, especially after the discovery of the anti-tumor activity of cisplatin.³ Some metal complexes have been successfully used therapeutically, such as platinum, gallium and arsenic complexes in cancer therapy, gold complexes as anti-arthritis and asthma agents, and bismuth complexes in anti-ulcer treatment.^1,4,5 Enormous efforts have been made on the biocoordination chemistry of metal-based agents.⁵ Structures of several clinically used metal-based drugs are shown in Fig. 1.

Fig. 1 Structures of metal-based drugs, i.e., cisplatin, carboplatin, oxaplatin, myocrisin, NAMI-A, KP1019 and colloidal bismuth subcitrate (CBS). Note that myocrisin and CBS are polymeric complexes in solution.

However, in view of the limited spectrum of curable cancers and microbial infections, drug-resistance phenomena of cancer/microbial strains as well as undesirable side-effects of the metal-based drugs, we need to find out the molecular targets associated with the disease etiology and pathology to guide our design of new metal-based drug leads which could overcome drug resistance and exhibit less side-effects.^6–8 Nowadays, both genomics and proteomics can be used to identify putative targets of metal-based drugs. Mass spectrometry- and array-based proteomics complements functional genomic approaches, and has been widely used to investigate protein expression levels, post-translational modifications, localizations and protein–protein interactions at the protein level.^9,10 Two-dimensional gel electrophoresis (2-DE) and liquid chromatography have commonly been used to separate hundreds of proteins in a complex biological system at a given time, important for the understanding of protein expression in abnormal cells or tissues and for drug development,¹¹ whereas the inductively coupled plasma mass spectrometry (ICP-MS) allows metal in proteins to be quantified with high specificity and sensitivity.^12–14

In spite of the widespread usage of 2-DE in proteomics, the limitations of the protein separation capability of this technique cannot be neglected. Therefore, metalloproteomics has been developed as a powerful approach to increase sensitivity and resolution of protein separation.¹⁵ Immobilized-metal affinity chromatography (IMAC) in combination with 2-DE and MALDI-TOF mass spectrometry is a particularly useful tool for elucidating the metabolism of intracellular metal ions (Fig. 2) This approach has enabled over 30 high-abundance Cu- and Zn-binding proteins and their putative metal binding motifs to be identified.^12,16–19 Besides capturing metalloproteins and/or their partner proteins, IMAC can also be used to enrich metal-binding proteins with low abundance. Identification of the metallome and elucidation of their biological functions is the main research aim of metalloproteomics and metallomics, which will be beneficial towards understanding metals in biology and the molecular mechanism of actions of metallodrugs.¹² The development of (metallo)proteomics and metallomics would also facilitate the design and evaluation of new metallodrugs against a specific protein and even a specific site(s) of proteins.

Fig. 2 Flow chart of metallopoteomics in identification of metal-binding proteins and motifs.

2. Anticancer activities of platinum, gold and arsenic and ruthenium compounds

Numerous metal complexes, including classical inorganic compounds, complexes with organic ligands and organometallic species,^20,21 have been examined for their antitumor activities both in vitro and in vivo. This section focuses on the recent proteomic discoveries of target sites obtained with platinum, gold, arsenic and ruthenium complexes.

2.1 Platinum compounds

The most well-known metal complex used in the treatment of various cancers is the platinum complex cis-diamminedichloroplatinum(II) (cisplatin).³ Several other platinum complexes, including carboplatin, satraplatin, picoplatin and oxaliplatin, have also been approved subsequently for clinical use for the treatment of tumors (Fig. 1). Cisplatin enters cells by either passive diffusion or via active transport mediated by the copper transporter, Ctr1.^22–24 The metallodrug eventually inhibits DNA replication and transcription.²⁵ Cellular proteins, such as repair enzymes, transcription factors, histones and HMG-domain proteins, bind to distorted, platinated DNA structure to mediate antitumor activities.^26,27

Although for a long time scientists have focused on the characterization of platinum–nucleic acid interactions,²⁸protein-bound platinum fragments may actually represent either active anticancer species or drug-inactivation products, which are dependent on the kinetic equilibrium among distinct binding sites.^8,29,30 It was found that monoplatinated cytochrome c (cyt c) species is dominant in the ESI-MS spectra when platinum anticancer agents (cisplatin, transplatin, carboplatin and oxaliplatin) were added into the protein in a 3 : 1 ratio for 3 days at 37 °C,³¹ and Met65 was suggested to be the primary binding site for these platinum drugs due to the “soft” feature of platinum(II).³² Unexpectedly, an X-ray crystal structure of bovine superoxide dismutase (beSOD) with platinum showed that Pt²⁺ binds to a histidine residue (His19), one oxygen from a water molecule and two chloride (Fig. 3).³³ The latter (Cl⁻) has long been regarded as a leaving group. This raises the possibility that platination of this enzyme follows an unusual pathway where ammonia is released from platinum upon the inducement of the high trans-effect. For the same system with Fourier transform ion cyclotron resonance (FT-ICR) MS, it was clearly shown that Pt²⁺ is bound to imidazole Nε of a surface histidine residue (His19), two ammine and one chloride,³⁴ similar to the coordination of platinum drugs at His15 of hen egg white lysozyme (HEWL), (Fig. 3, left).³⁵ In a similar study, cisplatin and oxaliplatin were observed to bind to an N-terminal methionine-containing ubiquitin fragments, whereas transplatin is bound to a peptide with the sequence of ¹⁹Pro–Ser–Asp–Thr–Ile–Glu²⁴.³⁶ This work demonstrates that FT-ICR MS, with high resolution and mass accuracy, should pave the way for defining the coordination spheres of metal drug–protein adducts in solution.

Fig. 3 X-Ray structures of platinated hen egg white lysozyme (PDB: 2i6z; left) and beSOD (PDB: 2aeo; right). Platinum is bound to His15 and His19 in lysozyme and beSOD, respectively, with two NH₃ for the former and two chlorides for the latter bound to the Pt²⁺.

Methionine and histidine have long been speculated to play a key role in the activation of platinum drugs. It would be of interest to investigate the drug–protein interactions at the proteome level in order to fully understand the mechanism of drug resistance of platinum. Proteomic studies have been performed on both platinum-sensitive and -resistant human ovarian cancer cell lines, and by far several groups of proteins closely related to platinum resistance have been identified.^37–39 Two low mass peptides with m/z 5041 and 7324, consistently present in platinum-resistant ovarian tumor cells were identified using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS or SELDI).³⁷ Although SELDI is incapable of the exact identification of the peptides , it does provide useful information about the potential cisplatin-resistant tumor cell biomarkers. Comparative 2-DE and MALDI-MS were used to facilitate the identification of differentially expressed proteins.^38,39 Among them, nine proteins, annexin A3 and IV, cytokeratins 8 and 18, destrin, aldehyde dehydrogenase 1 (ALDH-1), cofilin 1, glutathione-S-transferase omega1 (GSTO-1), and NADP-dependent isocitrate dehydrogenase (IDHc) (Table 1), were found to be co-instantaneously significant. Clearly, further investigations are needed to understand how these proteins induce platinum resistance in ovarian cancer cells. Studies on the consequences of the over- and under-expression of these proteins could lead to the development of new protein markers and also to facilitate the development of novel therapeutic strategies.

Table 1 Binding proteins of metallodrugs identified by (metallo)proteomic studies

Function	Proteins	Metallodrug	Reference
Chaperone	Hsp70	As	55
	Hsp27	As	55
	HSPgp96	Au, Pt	48
	HspA	Bi	76
Metabolic enzyme	TPI1	Au, Pt	48
	GAPDH	As	55
	Fumarase	Bi	76
Anti oxidative stress	Peroxiredoxin 1	Au, Pt	48
	Peroxiredoxin 6	Au, Pt	48
	Thioredoxin	Au, Pt, Bi	48, 76
	AR	As	55
	TsaA	Bi	76
Translation factor	Splicing factor 17	Au, Pt	48
	PDI	Au, Pt	48
	Ef-Tu	Bi	76
Signal transduction	Annexin IV	Pt	38
	Cyclophilin A	Au, Pt	48
Cellular structure	Cytokeratin 8	Pt	38
	Cytokeratin 18	Pt	38
	Destrin	Pt	39

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Modern proteomic technologies including capillary electrophoresis-ICPMS (CE-ICPMS) and high performance liquid chromatography-ICPMS (HPLC-ICPMS) allow element-specific and high sensitivity quantitative analysis of Pt drug–protein adducts and free Pt drugs to be performed. Selected studies in the interactions between Pt drugs and proteins such as human serum albumin (HSA), hemoglobin (Hb) and holo-transferrin (holo-Tf) elucidate the pharmacokinetics of Pt drugs right after intravenous administration and, importantly, shows the potential use of these hyphenated techniques in metallodrug evaluation and clinical applications. The binding portion of cisplatin towards HSA analyzed by CE-ICPMS is 92%, indicating that the level of free cisplatin in bloodstream is fairly low after introduced intravenously.⁴⁰ The investigation of Hb-cisplatin adduct formed at sub-micromolar concentration (<μM) of cisplatin by size exclusion HPLC-ICPMS (SEC-ICPMS) gives clues in explaining the substantial decrease in functional Hb in some patients treated with cisplatin.⁴¹ Interaction of oxaliplatin with holo-Tf was also observed by SEC-ICPMS, in which both ⁵⁶Fe and ¹⁹⁵Pt signals were monitored, and the results suggest that holo-Tf may act as a drug carrier without altering its original binding with ferric iron (Fe³⁺).⁴² On the other hand, laser ablation-ICPMS (LA-ICPMS) demonstrates a good compatibility with traditionally gel-based proteomics for identifying metal-containing proteins. For instance, Pt-bound outer membrane protein A (OmpA) from cisplatin-treated E. coli cell lysate was localized in 1D-PAGE, detected by LA-ICPMS and further identified by MS.⁴³

2.2 Gold compounds

The success story of platinum drugs in cancer treatment motivated the search for other transition metal-based drug leads. As gold(III) exhibits the same isoelectronic configuration (d⁸) and structural requirement (square–planar) as platinum(II), research has increasingly focused on the potential use of gold(III) complexes as anticancer agents.⁴⁴ However, in comparison with platinum(II) compounds, gold(III) analogues turned out to be relatively unstable and light-sensitive. Gold(III) compounds are expected to be reduced to gold(I) or metallic gold in vivo, considering the intracellular reducing environment.⁴⁵ Therefore, it is crucial to develop a ligand which can stabilize the electrophilic gold(III) ions by avoiding demetalation through strong chelation and increasing the kinetic barrier for reduction to gold(I).⁴⁶

Gold(III) porphyrins were found to be stable under physiologically-relevant conditions, even in the presence of glutathione.⁴⁷ One of these complexes, [Au^III(TPP)]⁺ (H₂TPP = tetraphenylporphyrin) was a promising new chemotherapeutic drug lead and exhibited in vitro potency against a panel of cancer cell lines, including cisplatin-resistant cell lines.⁴⁷ Comparative proteomic approaches were used to investigate the anticancer activitites of [Au^III(TPP)]⁺.^48,49 It was found that several cytoplasmic proteins, 3-phosphoglycerate dehydrogenase (PHGDH), glutamate dehydrogenase 1 (GDH), actin-related protein 1 homologue A (ARP1), sorbitol dehydrogenase (SDH), and glucose-6-phosphate dehydrogenase (G6PD) (Table 1), were significantly regulated upon the treatment of the drug.⁴⁹ These proteins are mainly involved in energy production and cellular redox balance. Au(III) complex [Au^III(TPP)]⁺ treatment resulted in quick attenuation of mitochondrial membrane potential (ΔΨ_m) with the concurrent regulation of Bcl-2 family proteins, the release of cytochrome c and apoptosis-inducing factor (AIF). Therefore, the primary target of gold(III) complex [Au^III(TPP)]⁺ is the mitochondria, which certainly involved in cell death.

2.3 Arsenic compounds

Arsenic is a semi-metal or metalloid with two biologically important oxidation states, arsenic(III) and arsenic(V).⁵⁰ As a soft metal ion, arsenic(III) reacts with closely spaced proteinthiols , forming stable cyclic dithioarsinite complexes in which both sulfur atoms are bound to arsenic, which is responsible for the cytotoxicity of arsenic. Arsenic is a well-known double-edged sword: on one hand it is a carcinogen that likely promotes tumors at low concentration, and on the other hand it is a chemotherapeutic agent, e.g.arsenic trioxide (Tirisenox®) and GSAO⁵⁰ to treat acute promyelocytic leukemia and induce differentiation and apoptosis of malignant cells.^51,52

It was reported that arsenite at a high concentration induced apoptosisviac-Jun N-terminal kinase (JNK) signaling pathway, whereas at a low concentration arsenite is carcinogenic and stimulates the extracellular signal-regulated kinase signaling pathway to enhance cell proliferation.⁵³ Several differentially-regulated peptides in lung cancer cells were identified when the cells were treated with low concentration of arsenite comparing to untreated cells.⁵⁴ Identification and characterization of these proteins may reveal the molecular basis of arsenic-induced cell transformation and provide some insights into the mechanisms by which arsenic induces carcinogenesis. A proteomic analysis of rat lung epithelial cells (LECs) treated with arsenite at a high concentration identified several up-regulated proteins, including heat-shock proteins Hsp70 and Hsp27, anti-oxidative stress proteins aldose reductase (AR), heme oxygenase-1 (HO-1), ferritin light chain (FLC) and αB-crystallin (αB–C), which may play a role in the fighting against oxidative stress induced by arsenite.⁵⁵

2.4 Ruthenium compounds

Two ruthenium(III) complexes, NAMI-A and KP1019 (Fig. 1), which showed activities in cisplatin-resistant cells, are now investigated in clinical trials.⁵⁶ Ru(III) complexes are generally more specific to tumors and less toxic compared with their Pt(II) counterparts.^56,57 This has been rationalized by the proposed “activation by reduction” of Ru(III) to more reactive Ru(II) mode of action and the similarity of Ru(III) to resemble Fe(III) in biological system, e.g., rapid binding to transferrin.^58–60 Although the target(s) of Ru agents in tumor cells is still unrevealed, interactions with both DNA and proteins are believed to be responsible for their anticancer activities.^57,59

Bloodstream circumstances serve as the first battlefield where drug transformation takes place after intravenous injection and therefore much of the work has been done with monitoring the binding of Ru(III) drug candidates and human serum proteins such as serum albumin (HSA) and transferrin (Tf).⁶¹ESI-MS study of the stoichiometry of KP1019 and Tf gives a 2 : 1 ratio, same as that of Fe(III), and the corresponding mass shift reveals the loss of one chloride ligand and the hydrolysis of another chloride ligand.⁶² Comparative binding of KP1019 to HSA and Tf under physiologically relevant condition (molar ratio of HSA : Tf = 10 : 1) was quantified by CE-ICPMS.⁶³ The result demonstrated that over 98% KP1019 bound to HSA even though binding to Tf is more kinetically-favored, which strongly supports the view that HSA serves as a Ru(III) complexe “reservoir” while Tf transports the complexes into the tumor cells, as evidenced by previous similar CE-UV study and in vivo experiments.^62–64

Chromatography- or gel-based multi-dimensional separation techniques provide sufficient resolving power for proteomic studies. Online SEC-IXC-ICPMS equipped with dynamic reaction cell was developed for measuring metal-to-protein stoichiometry from the signal of ¹⁰²Ru/⁴⁸SO and applied for in vivo studies by monitoring the binding ratio of KP1019/HSA in blood during the treatment.⁶⁵ Offline 1D- and 2D-PAGE combined with LA-ICPMS identified three NAMI-A- and cisplatin-binding proteins in albumin-depleted human plasma matrix including human serum albumin precursor, macroglobulin α2 and transferrin.⁶⁶ Moreover, the drug-binding profiles in depleted plasma co-incubated with NAMI-A and cisplatin did not change compared with their individual studies, indicative of different mode of binding and the potential of their uses in combination therapy.⁶⁶ Automated triphasic RP-SCX-RP capillary column coupled with ESI-MS demonstrate the suitability for identifying drug-binding proteins and their binding motif in E. coli treated with Ru(II) complex by shotgun approach.⁶⁷ Ru-Peptide of four stress-regulated proteins and one helicase were identified by tandem MS and characteristic isotopic distribution of Ru.⁶⁷

3. Antimicrobial activities of bismuth compounds

Heavy metals are notorious for their toxic effects. However, the heaviest stable element, bismuth, is considered to be almost non-toxic.⁶⁸ Bismuth(III) is highly acidic in water (pK_a1 of ca 1.5) and easily forms stable hydroxo- and oxo-bridged clusters. Polymeric structures are usually observed for bismuth complexes as the ligand engages more than one bismuth center and behaves as a bridging ligand. The coordination number of Bi³⁺ varies from 3 to 10 and the coordination geometry is often irregular.⁶⁹ The biological effects of bismuth compounds are probably originated from their binding to proteins and enzymes.^70–72

Helicobacter pylori (H. pylori) infects about half of the people in the world and causes chronic inflammation of the stomach and peptic ulcer.⁷³ Bismuth therapy (colloidal bismuth subcitrate and ranitidine bismuth citrate) has often been recommended for eradicating H. pylori together with antibiotics.⁷⁴ Although acquisition of antibiotic resistance by H. pylori is the main reason for treatment failure, there are no reports of H. pylori developing resistance to bismuth compounds. However, the precise molecular mechanisms underlying the antimicrobial activity of bismuth is still not clear. It has been demonstrated that bismuth drugs inhibited several enzymes from H. pylori, for example, cytosolic alcohol dehydrogenase (ADH), ATPase, and urease.⁷⁵ A detailed comparative proteomic study of H. pylori cells before and after treatment of colloidal bismuth subcitrate (CBS) was carried out.⁷⁶ Eight proteins were found to be significantly regulated by CBS (Fig. 4A). These proteins are involved in either cellular processes (HspA, HspB, and neutrophil-activating protein (NapA)), or oxidative stress resistance (putative alkyl hydroperoxide reductase (TsaA), thioredoxin) and hemoglobin. The ability of pathogenic bacteria to resist oxidative stress is essential for their colonization in the host.⁷⁷ Thioredoxin is a low redox potential reductant, and facilitates electron transfer to TsaA.⁷⁸ The increased level of TsaA fragment upon bismuth treatment indicated that this enzyme is one of the chief targets of bismuth in vivo. The upregulated expression of thioredoxin may reflect the response of H. pylori to the higher level of oxidative stress.

Fig. 4 Two-dimensional gel electrophoresis (2-DE) images of total cell extracts (A) and bismuth binding fractions eluted from a Bi-nitrilotriacetate column (B), from untreated and CBS treated H. pylori 11637 cells (adapted from ref. 76 with kind permission from Springer Science + Business Media: R. Ge, X. Sun, Q. Gu, R. M. Watt, J. A. Tanner, B. C. Wong, H. H. Xia, J. D. Huang, Q. Y. He and H. Sun, JBIC, J. Biol. Inorg. Chem., 2007, 12, 831–842).

Using bismuth-affinity chromatography (Bi-IMAC), seven proteins from H. polyri, including HspA, HspB, NapA, TsaA, translation factor (Ef-Tu) and two enzymes (fumarase and UreB) (Fig. 4B), were identified.⁷⁶ Interestingly, four proteins (HspA, HspB, NapA and TsaA) almost disappeared in the bismuth binding profile of bismuth-treated cells, suggesting they may directly interact with bismuth in vivo. HspA was then overexpressed using recombinant DNA techniques, and purified. The protein binds both Ni²⁺ and Bi³⁺ at its C-terminal histidine- and cysteine-rich domain,⁷⁹ similar to some histidine- and cysteine-rich proteins/motifs.^17,80–83 Importantly, Bi³⁺ induces the quaternary structural changes of the protein, i.e. from a heptamer (its native form) to a dimer. The histidine- and cysteine-rich domain may play a critical role in nickel homeostasis and bismuth susceptibility in vivo.^79,84

4. Conclusion

It is clear that metal-based drugs will play a much more important role in medicine in future. The development of metallodrugs requires clearer understanding of the physiological processing of metal complexes and molecular basis of actions of these metallodrugs. Traditional 2-DE-MS and LC-MS based metalloproteomic approaches make it straightforward and quickly analyze/identify the molecular targets of metallodrugs at a proteome level. This review summarizes several target proteins of platinum-, gold-, arsenic- and bismuth-containing metal compounds, belonging to different functional groups (Table 1). The metallodrug may act directly or indirectly viaprotein-mediated pathways in cells. The technical advances in metallo-proteomics and metallomics will be further facilitated with the development of mass spectrometry, novel nuclear analytical techniques,⁸⁵protein separation and purification techniques (Table 2). The development of metallo-proteomics will be beneficial for the improved design of new efficient metallodrugs.

Table 2 Analytical techniques used for identification of metallodrug binding proteins

Method^a
Application
(Ref.)	Strength	Limitation
a XAS, X-ray absorption spectroscopy; XRF, X-ray fluorescence.
2-DE	• Visualization of thousands of protein spots in a single gel	• Not applicable to highly hydrophobic proteins and those with extreme pI and MW
Separation	• Compatible with MS identification	• Fractionation is required for detecting low-abundance proteins.
(38, 39, 48, 49, 55, 76, 86)	• Proteins are separated according to pI (3–10) and MW (10–120 kDa)
	• Proteins expressed differentially can be judged by software or naked eye.
CE	• For CE-ICPMS studies of drug–protein interaction, no prior step in removing excess metal ions.	• Possible loss of drugs or proteins
Separation	• Electrolyte can be selected at pH close to physiological condition.
(40, 63, 87, 88)
HPLC	• Proteins are separated by pI (SCX or IXC), MW (SEC), etc.	• Longer analysis time compared with CE and therefore not ideal for liable drugs, e.g. Ru(III)-based drugs
Separation	• RP is frequently used for desalting and pre-concentration prior to MS.
(41, 42, 88)
IMAC	• Proteins with specific metal-binding affinity are selected with columns loaded with different metals.	• Limited vacant coordination sites for protein binding
Selection		• Only suitable for drugs with no ligand
(18, 19, 76)
ESI-MS and FT-ICR-MS	• Samples can be analyzed in their solution	• Desalting is required prior to analysis. Usually coupled with RP capillary column (LC-MS)
Detection	• Soft ionization allows detection of drug-intact protein adduct(s)	• Intensity of signal depends on ionization efficiency, not necessary concentration
(31, 34, 36)	• Biotransformation of drug is showed by m/z shift
	• ESI-FT-ICR-MS provides high resolution and mass accuracy
ICPMS	• Quantification of metal species in a sample according to m/z	• No molecular characterization
Detection	• Multi-element detection	• Interference of Ar and O adducts, e.g.^32O2 and ^56ArO
(14 ,40–42, 63, 65, 89)	• Selectively detect metal-bound proteins from their non-metal-bound forms in CE-ICPMS.	• Determination of ^32S and ^56Fe not applicable unless equipped with dynamic reaction cell (or measures other isotopes instead, e.g., ^57Fe)
	• High sensitivity (nM-μM level)
	• Metal containing species can be identified by specific isotope distribution, e.g. Ru and Pt
LA-ICPMS	• Detect metal-containing proteins in 1D- or 2D-PAGE	• Preconcentration of low abundant proteins is required since only a small part of the gel band or spot is ablated by laser.
Detection	• Non-destructive. Metal-tagged proteins on gel can further be identified by MS.
(14,43,66)
MALDI-MS	• Higher salt tolerance than ESI-MS	• Highly acidic matrix weaken drug–protein interaction
Detection	• MALDI-TOF-MS provides rapid protein identification with high mass accuracy
SELDI-MS	• High throughput	• Suitable for low MW (<100 kDa) proteins or peptides only.
Detection	• Selective to proteins that can bind on the functionalized SELDI surface, e.g. metal-affinity surface.	• Lower resolution and mass accuracy than MALDI-MS
(37,90)
XAS and XRF	• Multi-element detection	• Possible damage of sample due to radiation. Maintaining sample at low temperature is required.
Detection	• Identification of metal ions with different oxidation states and even local coordination environments of metals
(15,85)	• Determination of metal-containing proteins in 1D- or 2D-PAGE

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List of abbreviations

2-DE	Two-dimensional gel electrophoresis
CE	Capillary electrophoresis
ESI	Electrospray ionization
HPLC	High-performance liquid chromatography
IXC	Ion exchange chromatography
ICP	Inductively coupled plasma
IMAC	Immobilized-metal affinity chromatography
LA	Laser ablation
MALDI	Matrix assisted laser desorption ionization
MS	Mass spectrometry
PAGE	Polyacrylamide gel electrophoresis
RP	Reverse phase chromatography
SCX	Strong cation exchange chromatography
SEC	Size exclusion chromatography

Acknowledgements

This work was supported by the Research Grants Council of Hong Kong (HKU7039/04P, HKU7043/06P, HKU7042/07P, HKU1/07C), the Area of Excellence Scheme of the University Grants Committee, Institute of molecular technology for drug discovery and synthesis (AoE/P-10/01) and The University of Hong Kong.

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Footnote

† Presented at the International Symposium on Metallomics 2007, Nagoya, Japan, November 28–December 1, 2007.

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