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Journal of Chromatography A, 1359 (2014) 84–90

Contents lists available at ScienceDirect

Journal of Chromatography A

jo ur nal ho me pag e: www.elsev ier .com/ locate /chroma

rofiling of drug binding proteins by monolithic affinityhromatography in combination with liquidhromatography–tandem mass spectrometry�

uepei Zhanga, Tongdan Wangb, Hanzhi Zhanga, Bing Hanb,ishun Wangb, Jingwu Kanga,c,∗

Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, State Key Laboratory of Organic and Natural Products Chemistry,ingling Road 345, Shanghai 200032, ChinaKey Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,outh Chongqing Road 227, Shanghai 200025, ChinaShanghaiTech University, Yueyang Road 319, Shanghai 200031, China

r t i c l e i n f o

rticle history:vailable online 15 July 2014

eywords:onolithic column

ffinity chromatographyrug target identificationhemical proteomics

a b s t r a c t

A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinitychromatography in combination with HPLC–MS/MS is reported. Two immunosuppresive drugs, namelyFK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithiccapillary affinity columns were prepared through a single-step copolymerization of the drug derivativeswith glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinitymonolithic column facilitates the purification of the drug binding proteins from the cell lysate. By com-bining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506-

and 32 CsA-binding proteins including all the literature reported target proteins of these two drugswere identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins.The result supports that the monolithic capillary affinity chromatography is likely to become a valuabletool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

A large number of small molecular drugs possess multiple tar-ets (polypharmacology), although they were designed to aim at

single target [1]. The “off-target” effect may lead to complicateddverse effects; on the other hands, that may provide an oppor-unity to repurpose existing drugs [1,2]. Therefore, it is necessaryo profile targets of the small molecule drugs at the proteomicevel. However, up to date, target identification of small-molecule

rugs or small-molecule probes remains a vital and daunting task

n the community of pharmacology as well as chemical biology

� Invited paper for the Honor Issue of Professor Peichang Lu’s 90th birthday.∗ Corresponding author at: Shanghai Institute of Organic Chemistry, Chinesecademy of Sciences, Lingling Road 345, Shanghai 200032, China.el.: +86 21 54925385; fax: +86 21 54925481.

E-mail address: jingwu.kang@sioc.ac.cn (J. Kang).

ttp://dx.doi.org/10.1016/j.chroma.2014.07.020021-9673/© 2014 Elsevier B.V. All rights reserved.

[3,4]. Generally applicable and robust methods for the drug targetidentification are urgently needed [5].

Affinity chromatography is the most direct approach used topurify target proteins of small molecule drugs [5–8]. The target pro-teins can be captured by the agarose-based affinity beads on whichthe studied drug molecules are immobilized covalently throughthe linker, and subsequently eluted for identification (pull-downexperiment) [9]. Up to date, almost all the pull-down experimentswere performed manually with the following procedure: Cell lysatewas incubated with affinity matrices for a certain period of timeat 4 ◦C, centrifuged, and transferred to spin columns for elution ofthe target proteins. The columns were then drained and washedwith wash buffer. Retained proteins were eluted by SDS–PAGE sam-ple buffer [7]. Subsequently, the bound proteins were subjected toSDS–PAGE separation. The protein bands were excised for an in-gel

trypsin digestion followed by the mass spectrometry (MS)-basedshotgun proteomic analysis [9]. One disadvantage of such a pro-cedure is that a large amount of starting protein should be usedand the stringent wash conditions are required [10]. This is a very

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edious process. Moreover, the moderate affinity binding proteinsay be lost in the stringent wash process.Alternatively, capillary liquid chromatography with the mono-

ithic column can be an attractive approach for the purificationf target proteins. The monolithic bed with high permeabilitynd a relative high surface area which are favorable for a chro-atographic separation [11–14]. Most importantly, the monolithic

olumns can be easily amenable to capillary-HPLC to allow a fastnd high performance purification of drug target proteins frominute biological samples. Thus far, a few affinity monolithic

olumns have been reported[15,16]. However, no one concerninghe drug target identification.

FK506 and CsA are immunosuppressant drugs used after organransplantation. The action of these 2 drugs are due to their inter-ction with the immunosuppressant-immunophilin complexes6]. CsA binds the immunophilin cyclophilin and inhibits thealcium/calmodulin dependent protein phosphatase calcineurin,hile FK506 interacts with the immunophilin FK506 binding pro-

ein (FKBP12) [6,17]. Although their immunosuppressive actionechanism is clear, however, the action mechanisms of acute and

hronic nephrotoxicity[18], apoptosis [19], nerve regeneration [20]emain unclear. Profiling of their binding proteins in the proteomeevel might lead to a better understanding on their “off target” effectnd pave the way to get safer immunosuppressant or develop newse of them.

In this study, we reported a new approach for a proteome-widerofiling of drug target proteins by using monolithic capil-

ary affinity chromatography in conjunction with HPLC–MS/MS.he monolithic affinity columns were prepared in a single-tep copolymerization of FK506 or CsA derivatives with glycidylethacrylate and ethylene dimethacrylate inside a fused silica

apillary column. With our approach, totally 33 FK506- and 32 CsA-inding proteins were identified. Among them voltage-dependentnion-selective channel protein 1 and serine/threonine-proteinhosphatase PGAM5 were verified by using the recombinant pro-eins.

. Experimental

.1. Chemical and materials

Glycidyl methacrylate (GMA), ethylene dimethacrylate [6],imethyl formamide (DMF), Grubbs 2nd generation catalyst,-hydroxysuccinamide (NHS), acrylamide, thiourea, toluene,,7,10-trioxa-1,13-tridecanediamine, tris (hydroxymethyl)minomethane (Tris) and bovine serum albumin (BSA) wereurchased from Sigma–Aldrich (St. Louis, MO). Triethylamine,hosphoric acid, azodiisobutyronitrile (AIBN) and recombinantuman FKBP12 were purchased from Sinopharm (Shanghai,hina). Trypsin was from Promega (Madison, WI, USA). Voltage-ependent anion-selective channel protein 1 (VDAC1) was frombnova (Taiwan, China). Acetonitrile (HPLC grade) was fromerck (Darmstadt, Germany). Vinyltrimethoxysilane (VTMOS)as purchased from Trustchemsilanes (Nanjing, China). FK506

nd CsA were purchased from Shanghai Boyle (Shanghai, China).used silica capillary with a dimension of 370 �m o.d. and 200 �m.d. was obtained from Polymicro Technologies (Phoenix, AZ, USA).

.2. Capillary liquid chromatography

All separations were performed on a homemade capillary liq-

id chromatography system consisting of a Pu-2080 HPLC pump, aE 970 UV detector with a capillary flow-cell holder (Jasco, Tokyo,

apan) and a Rheodyne 7725i HPLC injection valve equipped with 100 �L loop (Rohnert Park, CA, USA). The mobile phase delivered

. A 1359 (2014) 84–90 85

by the HPLC pump at flow rate of 200 �L/min was split with a zero-dead-volume T (VICI, Schenkon, Switzerland). The majority of themobile phase was split to waste, while the remainder for drivingthe separation was about 1–5 �L/min depending on the split ratiocontrolled by the length of the capillary tubing (50 �m i.d.). Oneend of the monolithic column was directly connected to the injec-tion valve, and another end was connected to the capillary flow cell(50 cm, 100 �m i.d., 365 �m o.d.) by a zero dead volume PCU-360Picoclear union (New Objective, MA, USA). Data were processedwith a HS2000 chromatography workstation (Yingpu, Hangzhou,China). All separations were carried out at ambient temperature(20–25 ◦C).

2.3. Synthesis of the FK506 and CsA derivatives

The procedure for synthesis of FK506 and CsA derivativesis shown in Supporting Information Fig. S1. Briefly, FK506 waschemically modified by NHS-activated 4-pentenoate (3 molarexcess) through a reaction catalyzed by Grubbs 2nd gener-ation catalyst (10% molar) in dry 1,2-dichloroethane. Afterstirring the reaction mixture overnight at 70 ◦C, the solvent wasevaporated and the residue was purified by flash chromatog-raphy on silica gel with a mixture of solvents consisting ofdichloromethane/ethylacetate. The resulting FK506 derivative wasdissolved in dry dichloromethane, then equal molar 4,7,10-trioxa-1,13-tridecanediamine was added. After stirring for 5 h at roomtemperature, equal molar NHS-activated 4-pentenoate was addedand the solution was stirred for another 5 h. Finally, the FK506derivative was purified with a RP-C18 semi-preparation column(10 mm × 250 mm, packed with Hypesil ODS 5 �m 100 A, ThermoFisher Scientific, Waltham, MA, USA). The purity of the final productwas determined as 99% by HPLC analysis. The CsA derivative wassynthesized following the identical synthetic procedure (Fig. S1).

2.4. Preparation of the monolithic affinity capillary columns

FK506 monolithic column was prepared in a single step (seeFig. 1). Briefly, the fused silica capillaries were etched with 0.1 MNaOH for 30 min at room temperature, then flushed with 0.1 MHCl, water and methanol for 30 min, respectively. After drying thecapillary at 120 ◦C for 2 h, solution of vinyltrimethoxysilane in drytoluene 10% (v/v) was charged into the capillaries and kept at 45 ◦Cfor overnight to functionalize the inner surface. The capillaries wereflushed with methanol for 30 min, and then purged with dry nitro-gen gas for 30 min for dryness.

The composition of the polymerization mixture was optimizedas follows: 60 wt% porogen solvents (54 wt% 1-dodecanol, 6 wt%cyclohexanol), 16 wt% cross-linker EDMA, 23 wt% monomer GMAand 1 wt% affinity ligand (FK506- or CsA derivative), and AIBN(1 wt% with respect to total monomers). The rationale for selecting1 wt% affinity ligand is that it is their maximum solubility in thepolymerization mixture. The polymerization mixture was ultra-sonicated in ice bath for 20 min, then degassed by purging withnitrogen gas for another 10 min. After fully filling the vinylizedcapillary with the polymerization mixture, both ends of the capil-lary were sealed with rubber septum, and the capillary was placedin an electric heating oven to allow polymerization reaction at60 ◦C for 24 h. Finally, the resulting monolithic column was flushedwith methanol to remove the porogenic solvent and the unreactedreagents. A control column was also prepared under the identical

conditions except in the absence of the affinity ligand. The epoxygroups on the surface of the monolith probably were opened bythe treatment with formic acid solution to increase the hydrophilicproperty of the monolithic bed.

86 X. Zhang et al. / J. Chromatogr. A 1359 (2014) 84–90

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ig. 1. Scheme for synthesis of FK506 modified affinity monolithic column in a singlolumn.

.5. Purification of the binding proteins of FK506 and CsA inurkat T-cell extract

Cell extract of Jurkat T cells were prepared according to the Dig-am method (Supporting information). The concentration of totalroteins was determined as 2 mg/ml by using the protein assay kitsBio-Rad, Hercules, CA, USA). After loading 100 �L cell extract ontohe monolithic column, the column was flushed with the wash-ng buffer consisting of 50 mM Tris–HCl (pH 7.5) and 150 mM NaClor 30 min to remove the unbound proteins. The retained proteinsere then eluted with 40% acetonitrile in water. The eluted sample

olution was collected, vacuum dried and reconstituted in 30 �Lris buffer (50 mM Tris–HCl, pH 7.5). Affinity-purified proteinsere separated by sodium dodecyl sulfate–polyacrylamide gel

lectrophoresis (SDS–PAGE) and subsequently silver or Coomassierillant Blue stained.

.6. Competition binding assay

To 4 aliquots of 100 �L cell lysate, various amount of free FK506r CsA was added giving a final drug concentration of 0 nM, 10 nM,00 nM and 1000 nM. After incubation at 4 ◦C for 30 min, they were

oaded on the monolithic affinity column and flushed with theashing buffer to remove the unbound proteins. The bound pro-

eins were eluted and subjected to SDS–PAGE separation and silvertaining.

.7. Nano LC-MS/MS analysis

A hybrid linear ion trap (LTQ) Orbitrap mass spectrometerThermo Fisher Scientific, Waltham, MA, USA) equipped withDVANCE Spray Source (Michrom Bioresources, CA) was used forrotein identification. Each protein band was excised and washed.fter alkylation with 10 mM DTT and 55 mM iodoacetamide, therotein samples were digested with trypsin overnight at 37 ◦C.he resulting peptide mixture was desalted and concentrated with

Peptide Microtrap (MW 0.5–50 kDa, 0.5 mm × 2 mm, MichromioResources, CA, USA). The eluent was evaporated in a vacuumentrifuge to dryness, and reconstituted in a solution consist-ng of 2% acetonitrile in 0.1% formic acid. The prepared sample

merization process and SEM images from the cross section of the FK506 monolithic

(10 �L) was introduced onto a reversed-phase capillary column(0.1 mm × 150 mm, packed with 5 �m 100 A Magic C18 resin,Michrom Bioresources, CA, USA) with an auto sampler (HTS-PAL,CTC Analytics, Zwingen, Switzerland) at a flow rate of 1 �L/minfor 15 min. The separation was performed using an 80 min gra-dient (from 0 to 35% B for 60 min, to 80% B for 8 min, to 95% Bfor 12 min, and back to 0% B for 20 min). Solution A was consistedof 2% acetonitrile in 0.1% formic acid, solution B was consisted of98% acetonitrile in 0.1% formic acid. MS/MS spectra were acquiredin a data-dependent scan mode from the ten most intense ionswith charge states ≥2. Only MS signals exceeding 500 ion countstriggered a MS/MS attempt and 5000 ions were acquired for aMS/MS scan. Dynamic mass exclusion window was set to 27 s.Single charged ions and ions with unassigned charge states wereexcluded from triggering MS/MS scans. The normalized collisionenergy was set to 35%. Acquired MS/MS data were searched againstthe human IPI database with the search engine Sage-N Sorcerer2 (Thermo Fisher Scientific, Waltham, MA, USA). Search param-eters were set to a mass tolerance of 10 ppm for peptide parentions and 1 Da for the fragment ions. Three missed cleavages siteswere allowed. Fixed modification of carbamidomethylation on Cys(157 Da) and differential modification of oxidation on Met (16 Da)were used. The XCorr scores cutoff for each charge state were asfollows: 2.5 (+2), 3.0 (+3), 3.5 (+4). The results were further ana-lyzed by Scaffold 3 proteome software (Portland, OR, USA) whichintegrates both Protein Prophet and Peptide Prophet. Two uniquepeptides must be identified independently for each protein, thepeptide probability and the protein probability must be 95% orhigher.

3. Results and discussion

3.1. Preparation and characterization of the monolithic affinitycolumns

FK506 and CsA were chemically modified to introduce a

hydrophilic spacer terminated in a double bond for the purposeof immobilization. The schematic representation for the prepara-tion of FK506- and CsA monolithic columns is shown in Fig. 1A.The prepared monolithic columns were primarily characterized by

X. Zhang et al. / J. Chromatogr

Fig. 2. Chromatograms showing the retention of FKBP12 on the control monolithiccolumn (A) and on the FK506 monolithic column (B), retention of BSA on the FK506monolithic column (C). Conditions: column dimensions, 20 cm × 200 �m i.d. Sam-ples were loaded onto the column and washed with the washing buffer, 50 mMTris–HCl (pH 7.5) containing 150 mM NaCl; eluent consisting of 40% acetonitrile inwl

safaett(cT5amFoocca

Ftwct

FK506 binding proteins, including the known FK506 target pro-

ater was applied at 10 min to elute the highly retained FKBP12; detection wave-ength, 214 nm.

canning electron microscopy. As shown in Fig. 1B, the pore-sizend skeleton of the FK506 monolithic bed are uniform. The sur-ace area was measured as 11 m2/g by using the BET surface areanalyzer. The mechanical strength of the monolithic column wasvaluated by flushing the column with a solvent of 40% acetoni-rile under pressure up to 23 MPa. A linear relationship betweenhe flow rate (ranging from 2 to 22 �L/min) and the back pressurefrom 2.9 to 23 MPa) was obtained with R2 of 0.9998 (Fig. S9) indi-ating a high mechanical strength under the pressure up to 23 MPa.he permeability (K) of the monolithic columns was measured as.24 × 10−14 m2 indicating a good permeability [21]. Moreover, thepproximate loading capacity of the affinity column was deter-ined by injection various amount of FKBP12 solution. Exceeded

KBP12 was no more retained on the column and eluted as a markerf injection overloading. The injected amount of FKBP12 nearlyverloading was considered to be the loading capacity of the affinityolumn. By this way, the loading capacity of the FK506 monolithicolumn with a dimension of 200 �m i.d. × 200 mm was measureds 6 �g FKBP12 (see Supporting information and Fig. S10).

Most importantly, the affinity specificity of the FK506 column toKBP12 was confirmed with pure recombinant FK506 target pro-ein FKBP12. As shown in Fig. 2, FKBP12 was retained strongly,

hile bovine serum albumin (BSA) did not at all on the FK506

olumn. On the other hand, FKBP12 was not retained at all onhe control column where in the absence of FK506. The retention

Fig. 3. Chromatograms (A) and picture of SDS–PAGE (B) showing a competitive bi

. A 1359 (2014) 84–90 87

of FKBP12 on the FK506 column was so strong that it can onlybe eluted by a denaturing eluent consisting of 40% acetonitrile inwater.

3.2. Competition binding assays

A competition binding assay was performed to convince thatbound proteins purified by the monolithic affinity column aredirectly related to the native drugs. As shown in Fig. 3A, the peakarea of the bound proteins (migration time is around 50 min)decreased obviously with increasing the concentration of com-petitor FK506. The bound proteins obtained under each FK506concentration were collected and subjected to SDS–PAGE and sil-ver staining. It is clearly shown in Fig. 3B that the presence of freeFK506 in the cell lysate attenuated most of the protein bands inthe gel. A competition binding assay for CsA was also performedto verify that the identified proteins are directly associated to freeCsA (shown in Fig. S12).

3.3. Profiling of FK506 and CsA binding proteins in cell lysate

A chromatogram for the purification of FK506 binding pro-teins in the extract of Jurkat cell is shown in Fig. 4A. It clearlyshows that the whole target purification process can be moni-tored via the chromatogram. First, numerous unbound proteinswere removed by flushing the column with the washing buffer.Afterwards, the bound proteins were eluted by 40% acetonitrileaqueous solution, and three fractions denoted as F1, F2 and F3,respectively were collected. The same procedure was also per-formed on the control column. The eluent from 40 to 50 min onthe control column was collected as a control sample to discrimi-nate the nonspecific proteins (see Fig. S11). The 4 collected sampleswere separated by SDS–PAGE and stained by Coomassie BrillantBlue or silver. As shown in Fig. 4B, F1 and F2 contained minorand major FK506 binding proteins, respectively; while F3 con-tained almost no protein implying the binding proteins had beeneluted completely before 50 min. The gel lanes of SDS–PAGE forF1, F2, and the control sample were excised for an in-gel trypsindigestion according to the standard procedures [22]. The result-ing peptide mixtures were analyzed with nano-HPLC–MS/MS toidentify the binding proteins. All the identified proteins elutedfrom the control column are listed in Table S1. They are all con-sidered as the nonspecific proteins which should be subtractedfrom the identified binding proteins of FK506 or CsA. Totally 33

teins FKBP1A (FKBP12), calmodulin and calcineurin were identified(Table 1). Calcineurin is a heterodimer consisting of subunits A andB. FK506 only forms complex with calcineurin B subunit [23]. In

nding of proteins in cell lysate between immobilized FK506 and free FK506.

88 X. Zhang et al. / J. Chromatogr. A 1359 (2014) 84–90

Fig. 4. A typic chromatogram (A) and picture of SDS–PAGE (B) for the purification of FK506-binding proteins from Jurkat T cell lextract with the FK506 monolithic column.Unbound proteins were removed by the washing buffer consisting of 50 mM Tris–HCl buffer (pH 7.5) containing 150 mM NaCl; FK506 binding proteins were eluted with 40%a 8 to 45

obtipcpapt

TI

cetonitrile in water; detection wavelength, 280 nm. Fraction (F1) was eluted from 30 to 55 min.

ur experiment, only calcineurin B subunit was identified proba-ly because the calcineurin heterodimer is easily dissociated duringhe preparation of cell lysate [24]. Meanwhile, totally 32 bind-ng proteins of CsA were identified (Table 2). The known targetroteins, cyclophilin A (peptidyl-prolyl cis–trans isomerase A),alcineurin were identified. Moreover, other members of peptidyl-

rolyl cis–trans isomerase (PPIase) family, including PPIase B, F, H,nd PPIase-like 1 were also identified. This result consists with ourrevious work, where the binding proteins of CsA were also iden-ified by using the affinity magnetic nanoparticles [25]. However,

able 1dentified proteins purified by FK506 monolithic affinity column.

Protein name Gene

45 kDa calcium-binding protein CAB460 kDa heat shock protein, mitochondrial HSPDATP synthase subunit alpha ATP5ATP synthase subunit beta ATP5ATP synthase subunit delta ATP5B-cell CLL/lymphoma 7 protein family member C BCL7Calcineurin B CNB

Calcium and integrin-binding protein 1 CIB1

Calmodulin CALMCalmodulin-like protein 5 CALLCalnexin CANXCystatin-A CSTACytochrome c oxidase subunit 5A COX5Cytochrome c oxidase subunit 5B COX5Cytochrome c1 CYC1Eukaryotic translation initiation factor 2 subunit 1 EIF2SEukaryotic translation initiation factor 2 subunit 2 EIF2SEukaryotic translation initiation factor 2 subunit 3 EIF2SFructose-bisphosphate aldolase A ALDOHeat shock protein HSP 90-beta HSP9Isoform 3 of Bcl-2-like protein 2 BCL2Macrophage migration inhibitory factor MIF

Membrane-associated progesterone receptor component 1 PGRMNADH dehydrogenase NDUFPeptidyl-prolyl cis–trans isomerase (FKBP12 or FKBP1A) FKBPPeptidyl-prolyl cis–trans isomerase E PPIE

Programmed cell death 6 PDCDReticulocalbin-2 RCN2Serine/threonine kinase receptor-associated protein STRASerine/threonine-protein phosphatase PGAM5 PGAMStress-70 protein, mitochondrial HSPAVoltage-dependent anion-selective channel protein 1 VDACVoltage-dependent anion-selective channel protein 2 VDAC

2 min; fraction (F2) was eluted from 42 to 50 min; and fraction (F3) was eluted from

only 5 CsA binding proteins were identified by using the affinitymagnetic nanoparticles. By comparing the number of identifiedbinding proteins, we draw a conclusion the monolithic columnapproach performed much more effective than the latter.

In our experiment, the literature reported target proteins ofFK506 and CsA, whitch function immunosuppressant action, were

all identified correctly. Many other binding proteins have not beenreported in literatures yet. Validation of the unreported bindingproteins are rather difficult because it is hard to get the pure pro-teins. Only two recombinant proteins, namely VDAC1 and PGAM5,

name MW(kDa) Percent coverage %

5 42 71 61 5A1 54 14B 57 50D 18 14C 33 18

22 1622 14

1 17 315 16 11

72 18 11 34A 17 21B 14 14

35 91 36 312 38 443 51 41A 39 90B 83 24L2 37 6

16 17C1 22 53A5 11 45

1A 12 8123 14

6 22 18 39 14P 40 195 32 9

9 74 71 31 242 27 29

X. Zhang et al. / J. Chromatogr. A 1359 (2014) 84–90 89

Table 2Identified proteins purified by CsA monolithic affinity column.

Protein name Gene name MW(kDa) Percent coverage %

Calcineurin B CNB 22 45Calumenin CALU 17 22Chromobox protein homolog 3 CBX3 21 44Eukaryotic translation initiation factor 1A, X EIF1AX 16 8Eukaryotic translation initiation factor 2 subunit 2 EIF2S2 38 8Fructose-bisphosphate aldolase A ALDOA 39 21Glyceraldehyde-3-phosphate dehydrogenase GAPDH 32 34l-Lactate dehydrogenase B chain LDHB 37 24Lamin-B receptor (fragment) LBR 24 9NADH dehydrogenase NDUFS8 16 22Nascent polypeptide-associated complex subunit alpha NACA 205 1Nicotinamide mononucleotide adenylyltransferase 1 NMNAT1 32 11Peptidyl-prolyl cis–trans isomerase A (cyclophilin A) PPIA 18 48Peptidyl-prolyl cis–trans isomerase B PPIB 24 47Peptidyl-prolyl cis–trans isomerase F PPIF 22 8Peptidyl-prolyl cis–trans isomerase H PPIH 19 20Peptidyl-prolyl cis–trans isomerase-like 1 PPIL1 18 16Plasminogen activator inhibitor 1 RNA-binding protein SERBP1 45 30Programmed cell death 6 PDCD6 22 30Prostaglandin E synthase 2 (fragment) PTGES2 34 13S-phase kinase-associated protein 1 SKP1 19 16Serine/threonine kinase receptor-associated protein STRAP 40 33Serine/threonine-protein kinase PRP4 homolog PRPF4B 115 3Serine/threonine-protein phosphatase PGAM5, mitochondrial PGAM5 32 13Serine/threonine-protein phosphatase PP1-alpha catalytic subunit PPP1CA 38 22Transcription factor A, mitochondrial TFAM 26 9Translocon-associated protein subunit alpha SSR1 34 9Translocon-associated protein subunit delta SSR4 16 14Translation initiation factor eIF-2B subunit alpha EIF2B1 33 54

TRMT112 12 37VDAC1 31 29VDAC3 31 19

wtc

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tRNA methyltransferase 112 homologVoltage-dependent anion-selective channel protein 1

Voltage-dependent anion-selective channel protein 3

ere obtained for the purpose of validation. As shown in Fig. 5,hese two proteins were strongly retained on the FK506 monolithicolumn implying a specific affinity interaction.

The FK506- and CsA binding proteins were compared and theenn diagram is shown in Fig. 6. Totally 8 binding proteins, includ-

ng their known common target, calcineurin, are overlapped. Thenother overlapped protein PGAM5 belongs to mitochondrial pro-ein phosphatase, which has been discovered as a convergent point

or multiple necrosis pathways [26]. Interestingly, calcineurin Bnd PGAM5 are all belong to serine/threonine-protein phosphatase27]. The work for validation of the other target proteins is on theay.

ig. 5. Chromatograms showing the retention of pure VDAC1 (containing 0.5 mMlutathione) (A) and PGAM5 (B) on the FK506 modified monolithic column. Condi-ions: column dimensions, 20 cm × 200 �m i.d.; mobile phase, 50 mM Tris–HCl (pH.5) containing 150 mM NaCl; the captured proteins were eluted by 40% acetonitrile

n water; detection wavelength, 214 nm.

Fig. 6. Comparison of the identified binding proteins of FK506 and CsA. Venn dia-

gram shows the number of overlapped proteins purified by using the monolithicaffinity columns.

4. Conclusion

Monolithic capillary affinity chromatography combinesHPLC–MS/MS has been demonstrated to be a new approachfor proteome-wide profiling of target proteins of small moleculedrugs. With our new approach, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported targetproteins of FK506 and CsA were identified. The affinity monolithiccapillary column can be prepared simply through a single-stepcopolymerization. Such a prepared affinity column displays a supe-rior chromatographic property, such as good biocompatibility,high permeability, and strong mechanical strength to allow a high

performance purification of the drug binding proteins from cellextracts. Moreover, the monolithic column can be easily amenableto the HPLC system to improve the efficiency and automation. The

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onolithic affinity chromatography is likely to be a general toolor chemical biology research and drug discovery.

cknowledgements

We thank Dr. Xiaodong Wang for providing us the purifiedGAM5. This work was financially supported by the National Natu-al Science Foundations of China (21375140, 21175146, 90713021,1071668) and the State Key Laboratory of Bio-organic and Naturalroducts Chemistry opening foundations.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.chroma.014.07.020.

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