COMMONICATION

* E-mail: jingwu.kang@sioc.ac.cn; Tel.: 0086-021-54925385; Fax: 0086-021-54925481 Received February 27, 2013; accepted April 9, 2013; published online April 29, 2013. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201300145 or from the author. † Dedicated to Professor Qingyu Ou on the occasion of her 80th birthday. Chin. J. Chem. 2013, 31, 715—720 © 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 715

DOI: 10.1002/cjoc.201300145

Drug Target Identification Using Affinity Core-Shell Magnetic Nanoparticles and Mass Spectrometry†

Yunhuan Wei,a Tongdan Wang,b Chao Liu,a Qianqian Zhang,a Lishun Wang,b Gongli Tang,a,c and Jingwu Kang*,a

a Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China b Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital,

Shanghai Jiao Tong University, School of Medicine, Shanghai 200025, China c National Key Laboratory of Organic Biochemistry Opening Foundations, Shanghai 200032, China

Affinity core-shell magnetic nanoparticles (MNPs) were prepared for identifying the target proteins of drugs in the cell lysate when used in combination with nano-high-performance liquid chromatography tandem mass spec-trometry (HPLC-MS/MS)-based shotgun proteomic analysis. A number of new potential targets of cyclosporine A (CsA) could be identified, owing to the high efficacy of the affinity MNPs in drug target identification. To the best of our knowledge, this is the first time to reveal such an abundant target spectrum of CsA.

Keywords drug target identification, core-shell magnetic nanoparticle, mass spectrometry, proteomics, cyc-losporine A, dasaninib

Introduction A large number of therapeutic drugs are currently

used with the limited knowledge of their targets.[1] In addition, the paradigm “one drug, one target” is no longer considered to be true.[2] Occasionally, hitting multiple targets often results in complicated side ef-fects;[2,3] while, sometimes, it can result in the discovery of new therapeutic uses for old drugs in the treatment of unrelated diseases.[2,3] Therefore, there is a strong need for carefully identifying the spectrum of targets of a particular drug as thoroughly as possible, so as to pre-dict its full therapeutic potency and its potential side effects.[2-4] Affinity chromatography has long been used for drug target identification.[5,6] However, the conven-tional separation matrix used in affinity chromatography, such as agarose beads, is not stable in the organic sol-vents in the process when small-molecule drugs were immobilized on the bead. Moreover, the highly porous structure of agarose beads can result in steric hindrance, which can prevent the target proteins from being able to access the immobilized drugs.[7] Therefore, it is essen-tial to develop new kinds of affinity matrices that ex-hibit improved efficiency with respect to drug target discovery.

Recently, magnetic nanoparticles (MNPs) have at-tracted extensive attention as a promising platform for biological sample separation, owing to their superpara-magnetism.[8-12] MNPs should also act as an ideal affin-

ity separation matrix owing to the following advantages: (i) they can be dispersed homogeneously in the sample solution; (ii) the size of MNPs is closer to that of pro-teins, allowing the target proteins to access the immobi-lized drug molecules more readily; (iii) their high sur-face-to-volume ratio allows for an increase in the im-mobilization density of small-molecule drugs; and (iv) their magnetic response makes the separation process much easier and simpler. Only recently, Nishio et al. reported the development of a method for preparing MNPs coated with poly(styrene-co-glycidyl methacry-late) (GMA) for use in the affinity purification of drug targets.[11]

Here, we report a novel approach for the preparation of affinity MNPs that can be employed for identifying drug targets when used in combination with a nano- high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS)-based proteomic analysis technique. In contrast to previously reported organic polymer-coated MNPs[11] the silica-encapsulated MNPs investigated in this study could be prepared more easily using sol-gel processes. The efficacy of the silica-en-capsulated MNPs in drug target identification was demonstrated using two small-molecule drugs, the im-munosuppressant cyclosporine A (CsA) and the anti-cancer drug dasatinib. Five target proteins of CsA in the cell lysate have been identified by our method. To the best of our knowledge, this is the first time such a wide spectrum of targets of CsA has been revealed.[13-15]

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Experimental Synthesis of affinity MNPs

The affinity MNPs were prepared with a procedure as shown in Scheme 1. Firstly, the MNPs were prepared by the co-precipitation method. Briefly, a solution con-taining 1 mol/L Fe3＋ and 0.5 mol/L Fe2＋ was prepared by dissolving 2.703 g FeCl3•6H2O and 0.994 g FeCl2• 4H2O in 10 mL aqueous HCl (0.4 mol/L). After de-gassing by purging with helium, 100 mL aqueous NaOH (0.5 mol/L) was slowly added in dropwise under nitrogen with strong mechanically stirring at room tem-perature. The mixture was heated in an oil bath at 80 ℃ for 30 min. After cooling down to room temperature, the resulting MNPs were collected by a magnet, and were rinsed with 0.1 mol/L aqueous hydrochloric acid and water.

The MNPs were then coated with silica through the sol-gel process by using a reverse microemulsion (wa-ter-in-oil) system to produce spherical silica-coated MNPs (Fe3O4@SiO2). The reversed microemulsion so-lution was prepared by mixing 123.2 mL cyclohexane, 25.6 mL 1-hexanol, and 28.32 g Triton X-100, followed by vigorous stirring for 15 min. After that, 1.6 mL TEOS was injected, followed by stirring for another 15 min. To this solution, 3.28 mL MNPs supernatant (100 mg/mL) was added and dispersed into the microemul-sion solution by sonication. Thereafter, 0.96 mL ammo-nium hydroxide was added to catalyze the sol-gel proc-ess. After stirring for 24 h, 200 mL ethanol was added

to break the microemulsion, then the MNPs were col-lected by centrifuging at 7830 r/min for 3 min, and washed with ethanol for at least four times to remove surfactant. After drying under vacuum, 90 mg silica- coating nanoparticles were dispersed in 10 mL anhy-dride ethanol followed by addition of 1 mL 3-meth-acryloxypropyl-trimethoxysilane (MPS) for reaction 5 h at room temperature. The nanoparticles were washed with ethanol for three times and dried under vacuum.

Synthesis of Fe3O4@SiO2@PSD nanoparticles The MPS modified MNPs (Fe3O4@SiO2) were fur-

ther coated with styrene (ST)-glycidyl methacrylate (GMA) copolymer through an emulsion polymerization followed by a seeded polymerization. Briefly, 300 mg MPS modified Fe3O4@SiO2 nanoparticles was dis-persed in monomer solution consisting of 10 mL ethanol, 150 mL deionized water, 30 mg SDS, 10 mL ST, 0.4 mL GMA, and 0.4 mL divinylbenzene (DVB). The mixture was degassed by purging with helium for 10 min, and heated in an oil bath. The polymerization was initiated by addition of 0.5 mL potassium persulfate (KPS) solution (20 mg/mL) keeping the temperature at 75 ℃. The polymerization was kept for 12 h. The final product was collected using a magnetic field, followed by washing three times with ethanol and two times with water to remove excess monomer and surfactant. The seed polymerization of GMA was carried on in an aqueous solution. The resulting MNPs were mixed with 1 mL GMA in 20 mL water. After stirring at 80 ℃ for 15

Scheme 1 Procedure for the preparation and functional modification of the core-shell Fe3O4@SiO2@PSDG MNPs. Compound 4: NHS-activated CsA derivative; Compound 7: c-dasatinib (see Figure S2)

Drug Target Identification Using Affinity Core-Shell Magnetic Nanoparticles and Mass Spectrometry

Chin. J. Chem. 2013, 31, 715—720 © 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 717

min, 0.1 mL aqueous solution of KPS (0.1 g/mL) was injected to initiate the seed polymerization. The reaction was kept for 12 h, the final product was collected using a magnet and washed with ethanol and water.

The detailed experimental procedures for modifica-tion and immobilization of the small molecular drugs on the surface of MNPS, cell culture and drug target en-richment were described in the Supporting Information.

Protein analysis by mass spectra analysis A hybrid linear ion trap (LTQ) Orbitrap mass spec-

trometer (Thermo, CA) equipped with an ADVANCE Spray Source (Michrom Bioresources, CA) was used to identify the proteins. The isolated protein sample to be identified was evaporated in a vacuum centrifuge till dry and reconstituted in 50 mmol/L NH4HCO3. After alky-lation with 10 mmol/L DTT and 55 mmol/L iodo-acetamide, the protein sample was digested with trypsin overnight at 37 ℃. The resulting peptide mixture was desalted and concentrated using a Peptide Microtrap (MW 0.5－50 kDa, 0.5×2 mm, Michrom BioResources, CA). The eluent was evaporated in a vacuum centrifuge to dryness and reconstituted in a solution of 2% acetoni-trile in 0.1% formic acid.

The thus-prepared protein sample (10 μL) was in-troduced at a flow rate of 1 μL/min for 15 min in a re-verse-phase capillary column [0.1×150 mm, packed with Magic C18 resin (5 μm, 100 Å, Michrom Biore-sources, CA)] equipped with an autosampler (HTS-PAL, CTC Analytics, Zwingen, Switzerland). The separation was performed using an 80-min gradient (from 0 to 35% B for 60 min, to 80% B for 8 min, to 95% B for 12 min, and back to 0% B for 20 min). Buffer A comprised 2% acetonitrile in 0.1% formic acid, and buffer B 98% ace-tonitrile in 0.1% formic acid. The MS/MS spectra were acquired from the ten most intense ions having charge states ≥2, while using the data-dependent scan mode. Only the MS signals that exceeded 500 ion counts trig-gered an MS/MS attempt, and 5000 ions were acquired for each MS/MS scan. The dynamic mass exclusion window was set to 27 s. Single charged ions and ions with unassigned charge states were excluded from the triggering MS/MS scans. The normalized collision en-ergy was set to 35%. The acquired MS/MS data were searched against the International Protein Index (IPI) human database using the search engine Sage-N Sor-cerer 2 (Thermo, CA). The search parameters were set to a mass tolerance of 10 ppm for the peptide parent ions and 1 Da for the fragment ions. Three missed cleavages sites were allowed. Fixed modification of carbamidomethylation of cysteine residues (157 Da) and differential modification of oxidation of methionine (16 Da) were used. The cut-off cross correlation (XCorr) scores for each charge state were as follows: 2.5 (＋2), 3.0 (＋3), and 3.5 (＋4). The results were further ana-lyzed using the Scaffold 3 program (Proteome Software, Portland, OR), which integrates the tools Protein Prophet and Peptide Prophet. It was essential that two

unique peptides be identified independently for each protein, and the peptide and the protein probabilities be 95 % or higher.

Results and Discussion The procedure for preparing the core-shell-structured

Fe3O4@SiO2@PSDG MNPs is shown in Scheme 1. TEM image of the Fe3O4 MNPs (Figure 1A) shows that the thus-prepared Fe3O4 cores had an average size of about 8 nm. The Fe3O4 MNPs were then encapsulated within silica through sol-gel processes using a reverse microemulsion (water-in-oil) system to produce spheri-cal silica-coated MNPs (Fe3O4@SiO2).

Fe3O4@SiO2 MNPs contained several dark Fe3O4 cores. The advantage of using the reverse microemul-sion system for preparing the Fe3O4@SiO2 MNPs was that the repeatability and overall yield of the synthesis procedure could be improved, allowing for the prepara-tion of the MNPs on a large scale.

The Fe3O4@SiO2 MNPs were modified using 3-methacryloxypropyltrimethoxysilane (MPS), and sub-sequently created a thin layer of poly(styrene-divinyl styrene-glycidyl methacrylate) (PSDG) on them via emulsion polymerization. This was followed by seeded polymerization with glycidyl methacrylate to introduce more epoxy groups on the surfaces of the Fe3O4@ SiO2@PSDG MNPs. The PSDG layer formed was about 10 nm in thickness (the light-grey-colored layer in Figure 1D). The thus-prepared Fe3O4@SiO2@PSDG MNPs were characterized using Fourier transform in-frared (FTIR) spectroscopy (see Figure S1 in Support-ing Information).

The epoxy groups on the surfaces of the Fe3O4@ SiO2@PSDG MNPs were opened with 4,7,10-trioxa- 1,13-tridecanediamine (TTDA) in an aqueous solution at 100 ℃ (Scheme 1) in order to introduce hydrophilic linkers that allowed small-molecule drugs to be an-chored on the surfaces of the MNPs. The TTDA linkers also made the surfaces of the MNPs highly hydrophilic, so as to reduce the nonspecific adsorption of proteins.

CsA was first converted into N-hydroxysuccinimide ac-tivated 4-pentenoate (compound 4 in Figure S2A),[13] so that it could be immobilized on the surfaces of the MNPs. After the reaction of immobilization, the unre-acted amino groups of the linkers were blocked with 0.3 mol/L acetic anhydride for 24 h. The amount of the drug immobilized on the surfaces of the MNPs, determined using HPLC by comparing the amounts of the drug de-rivative in the reaction solution before and after the immobilization reaction, was found about 10−6－10−5 mmol/mg. This was the case for both the drugs tested. To immobilize the dasatinib derivative (compound 7 in Figure S2B), the amino groups of the TTDA linkers was converted into carboxyl groups using succinic anhydride (Scheme 1). Even after being modified with CsA and dasatinib, the Fe3O4@SiO2@PSDG MNPs remained well dispersed in an aqueous solution for 7 d without

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Figure 1 TEM images of the (A) Fe3O4 MNPs, (B) Fe3O4@SiO2 MNPs and (C) and (D) Fe3O4@SiO2@PSDG MNPs.

aggregation or precipitation (Figure S3). The efficacy of the CsA-modified Fe3O4@SiO2@-

PSDG MNPs in capturing CsA-binding proteins was demonstrated using recombinant peptidyl prolyl cis- trans isomerase A (PPIA, human cyclophilin A), which is overexpressed in E. coli BL21 (DE3) containing the PPIA-expressing plasmid. A histidine tag was also ex-pressed so that it could be purified using immobilized metal ion affinity chromatography. As can be seen in Figure S4 (Supporting Information), lane 1 of the gel was used for the resolution of PPIA from the whole-cell lysate of E. coli BL21, purified using immobilized metal ion affinity chromatography, and lane 2 was used for the resolution of the proteins from the whole-cell lysate of E. coli BL21, captured by the CsA-modified MNPs. Both approaches gave the same protein, which had a molecular weight of about 19 kDa. To capture the CsA-binding proteins using the CsA-modified MNPs, all nonspecifically bound proteins were washed away with a washing buffer. The MNPs were then boiled in a sodium dodecyl sulfate polyacrylamide gel electropho-resis (SDS-PAGE) sampling buffer for 10 min to release the bound proteins. Lane 2 of the gel was used for the control experiment, during which no proteins could be captured on the MNPs when 0.1 mmol/L CsA was added to the cell lysate before the addition of the CsA-modified MNPs. This implied that the immobilized CsA and free CsA competitively bind with PPIA. Moreover, it also implied that the nonspecific adsorp-tion exhibited by the MNPs was minimal. Afterward, the CsA-modified MNPs were used to capture the CsA-binding proteins in the lysate of Jurkat T cells. The captured proteins were resolved using SDS-PAGE and visualized using silver staining (shown in Figure 2A).

As can be seen from the left lane of the gel shown in Figure 2A, the captured proteins exhibited a band that corresponded to a molecular weight of about 19 kDa. As can be seen from the right lane, almost no proteins were captured by the CsA-modified MNPs when a sample of 0.1 mmol/L CsA was added to the cell lysate before the addition of the affinity MNPs. This suggested that the nonspecific adsorption of proteins on the surfaces of the MNPs could be controlled effectively.

Each protein band in the left lane of the gel shown in Figure 2A was excised, digested with trypsin, and ana-lyzed using nano-HPLC-MS/MS. The proteins identi-fied in the band near the 19 kDa region are listed in Ta-ble 1. It is interesting to note that a total of five PPIs were identified using our method, whereas only PPI-A (recombinant human cyclophilin A) has been previously reported as being the primary target of CsA.[14,15] The cellular role and the enzymatic function of most PPI shave not been fully understood.[16]

To the best of our knowledge, this is the first time such a wide spectrum of targets of CsA has been re-vealed. Three batches of CsA-modified MNPs were prepared and used to capture proteins, resulting in the capturing of the same binding proteins in each case. This implied that the approach was reliable and robust. Moreover, the protein bands in the gel were identified as corresponding to various ribosomal proteins (listed in Table S1). These ribosomal proteins did not appear in the right lane of the gel, shown in Figure 2A. This is probable because the ribosomal proteins also bound strongly with CsA.

To further explore the efficacy of the affinity MNPs in capturing drug targets in cell lysates, they were tested using dasatinib, a small-molecule kinase inhibitor used

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Chin. J. Chem. 2013, 31, 715—720 © 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.cjc.wiley-vch.de 719

Table 1 The identified potential target proteins of CsA

Proteinname IPI-ID UPN SC/%

Peptidyl-prolyl cis-trans isomerase A (PPIA) 00419585 32 82 Peptidyl-prolyl cis-trans isomerase B (PPIB) 00646304 7 30 Peptidyl-prolyl cis-trans isomerase F, mitochondrial (PPIF) 00026519 4 28 Peptidyl-prolyl cis-trans isomerase-like 1 (PPIL1) 00007019 3 27 Peptidyl-prolyl cis-trans isomerase-like 3 (PPIL3) 00300952 5 41 IPI-ID: International Protein Index; UPN: unique peptides number; SC: sequence coverage.

Figure 2 SDS-PAGE-based resolution of the proteins captured from (A) a lysate of Jurkat T cells using the CsA-modified Fe3O4@SiO2@PSDG MNPs and (B) from the lysate of K562 cells using the dasatinib-modified Fe3O4@SiO2@PSDG MNPs.

clinically for treating imatinib-resistant chronic mye-logenousleukemia.[17,18] Thus far, its cellular targets have not yet been completely uncovered.[18-21] Dasatinib was immobilized on the surfaces of the MNPs through a reaction between the amino group of a dasatinib deriva-tive (compound 7 in Figure S2B) and the carboxylate group present on the surfaces of the MNPs. Unreacted carboxyl groups were blocked using a 1 mol/L ethanol-amine solution. After being incubated with the cell lys-ate of K562, the bound proteins were resolved using SDS-PAGE (shown in Figure 2B). A control experiment was performed in which 0.1 μmol/L free dasatinib was added to the cell lysate before the introduction of the dasatinib-modified MNPs. Proteins with molecular weights ranging from 45 kDa to 100 kDa were bound to the dasatinib-modified MNPs. These bound proteins were analyzed using nano-LC-MS/MS, and the results are given in Table S2. Of all the bound proteins, a total of 25 protein kinases could be identified. The proteins identified included the tyrosine-protein kinase BTK, and CDK2, CDK5, CDK9, CSNK2A2, and MAPK14; these proteins have been reported previously in litera-ture.[18,21,22] The other identified protein kinases have not been reported yet in literature. The work for valida-tion is in progress in our group.

Conclusions In conclusion, we developed a novel approach for

the preparation of an MNP-based affinity matrix, which can be used for drug target identification and isolation in cell lysates. The prepared MNPs were uniform in size, highly stable, and highly dispersible in organic solvents and buffer solutions without showing aggregation or precipitation. These features allow for the surfaces of the MNPs to be readily modified and drugs to be immo-bilized on them in both aqueous and organic solutions. Because of the smooth and hydrophilic nature of their polymer shells, the MNPs exhibited high efficacy in the capturing of drug target proteins. In addition, they also exhibited very low nonspecific adsorption of proteins. This MNPs-based affinity matrix may be attractive for use in the fields of chemical biology and pharmacology.

Acknowledgement This work was supported by the National Natural

Science Foundations of China (Nos. 21175146, 20975109, 90713021) and the National Key Laboratory of Organic Biochemistry Opening Foundations.

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