plasma profiling reveals human fibulin-1 as candidate ...mining, pathway analysis, gene expression...

rXXXX American Chemical Society A dx.doi.org/10.1021/pr200286c | J. Proteome Res. XXXX, XXX, 000–000

ARTICLE

pubs.acs.org/jpr

Plasma Profiling Reveals Human Fibulin-1 as Candidate Marker forRenal ImpairmentMaja Neiman,† Jesper J. Hedberg,‡ Pierre R. D€onnes,‡ Ina Schuppe-Koistinen,‡ Stephan Hanschke,§

Ralf Schindler,§ Mathias Uhl�en,† Jochen M. Schwenk,† and Peter Nilsson*,†

†Science for Life Laboratory Stockholm, KTH - Royal Institute of Technology, Box 1031, SE-17121 Solna, Sweden‡Department of Molecular Toxicology, Safety Assessment, AstraZeneca R&D S€odert€alje, SE-15185 S€odert€alje, Sweden§Nephrology and Internal Intensive Care Medicine, Charit�e-Virchow Clinic, Augustenburger Platz 1, DE-13353 Berlin, Germany

bS Supporting Information

’ INTRODUCTION

Impaired kidney function is an important factor leading toincreased morbidity and mortality, and early detection as well ascorrect and rapid diagnosis is a prerequisite for adequate treat-ment of patients. The current lack of markers is affecting thepossibilities both for detecting chronic kidney disease and acutekidney injury, underlining the need to identify refined andreliable biomarkers that will enable faster and more accuratediagnosis of kidney disease and injury in the clinic.1 Due to itssystemic function in filtrating the blood, the kidney is also amajorsite for drug-induced toxicity, andmonitoring its functionality is akey parameter in clinical trials during drug development pro-cesses. Moreover, kidney biomarkers could also serve a prog-nostic value in predicting acute and chronic alteration in renalfunction in patients already affected by chronic nephropathies.Bothmarkers that link to specific anatomic locations of injury, forexample tubular and glomerular damages as well as markers forgeneral organ-related malfunction, are needed and sought.2

Today, noninvasive standard measurements to support diag-nosis and therapeutic decisions of renal injury include serum

creatinine and blood urea nitrogen. However, these biomarkershave been reported to be insensitive, providing limited informa-tion to the type or characteristics of the disease or injury.1 Inpatients with a large renal reserve, the level of creatinine in serumis changed only after a substantial renal injury has occurred.3

Concentration of blood urea nitrogen is affected bymany factors,including protein intake, infections, and readsorption by parts ofthe nephron in addition to filtration by the glomerulus.1 Inaddition, the limitation of serum creatinine and blood ureanitrogen as indicators of kidney disease or injury is furtheremphasized by the fact that kidney patients often experiencedehydration and loss of muscle mass, both of which affects thecreatinine and blood urea nitrogen levels.

A series on preclinical biomarkers was recently presented bythe Predictive Safety Testing Consortium’s Nephrotoxicity Work-ing Group, a united effort from academia, industry and regula-tory authorities.1,2,4�7 Here, 23 urinary proteins discovered as

Received: March 30, 2011

ABSTRACT: There is a need for reliable and sensitive biomarkers forrenal impairments to detect early signs of kidney toxicity and to monitorprogression of disease. Here, antibody suspension bead arrays wereapplied to profile plasma samples from patients with four types of kidneydisorders: glomerulonephritis, diabetic nephropathy, obstructive uropa-thy, and analgesic abuse. In total, 200 clinical renal-associated cases andcontrol plasma samples from different cohorts were profiled. Parallelplasma protein profiles were obtained using biotinylated and nonfractio-nated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins,human fibulin-1 was detected at significantly higher levels in theglomerulonephritis patient group compared to the controls and withelevated levels in patient samples for all other renal disorders investi-gated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed thesame trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed thetrends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in thecontext of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discoverycohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidneydamage.

KEYWORDS: affinity proteomics, plasma profiling, antibody microarray, kidney disorders, biomarker, fibulin-1, glomerulonephritis

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Journal of Proteome Research ARTICLE

promising biomarkers for kidney injury within rat toxicologystudies were evaluated for their ability to outperform or add valueto conventional blood urea nitrogen and serum creatinine incomparison to histopathological outcome. Seven of these candi-dates were approved by the Food and Drug Administrationand the European Medicines Agency as markers to support thesafety assessment of acute renal tubule and glomerular injuryin rat toxicology studies.8 These proteins are urinary albumin,β2-microglobulin, clusterin, kidney injurymolecule-1, trefoil factor 3,total urinary protein and serum cystatin-C.

The discovery of these markers for preclinical toxicity assess-ment in rat models offer directions for the exploration of diseaserelated markers in humans. Proteomic studies of human urine andrenal tissue for better understanding of the kidney, and themechanisms of kidney disorders are growing. A majority ofthese studies aim at biomarker discovery in urine samplesusing masspectrometric analysis of peptides. Several specificpatterns of urinary peptide profiles have been obtained forpatient samples in relation to controls with potential diagnosticand prognostic values.9 Blood-derived samples have also beeninvestigated within human renal injury. Serum from patients hasbeen profiled using 2D-gel electrophoresis10 and SELDI-TOFMS,11 both resulting in disease-specific profiles. However, theproteomic workspace is still awaiting its full exploration for blood-derived samples within directed biomarker discovery approachesfor renal toxicology.

In the presented work, we aimed at the discovery of markersfor kidney malfunction in blood plasma by the use of an affinityproteomics method. Utilizing selected antibodies in relation tothe kidney, exploratory multiplexed assays were performed andidentified targets were subsequently verified across differentdisease categories and independent sample collections.

’MATERIAL AND METHODS

Sample Collection and Study DesignFor an exploratory screening of kidney disorders, plasma

samples were purchased from The Binding Site Inc. Lithiumheparin plasma samples were collected from consenting indivi-duals representing four patient groups with impaired renalfunction to various degrees (for glomerular filtration rate, seeSupplemental Figure 1, Supporting Information). The diseasegroups included patients representing renal glomerulonephritis(GN), diabetic nephropaty (DN) and obstructive uropathy(OU) as well as patients experiencing renal dysfunction ordamage due to abuse of analgesic substances (AA). All patientswere diagnosed based on ultrasound and/or X-ray imaging,supported with a CT scan for a smaller portion of the patients.For each of the four sample categories, plasma samples werecollected from healthy individuals matched in age, gender andbody mass index. Twenty subjects were included in each case andcontrol group resulting in a total number of 160 plasma samples.For information on each sample regarding age, gender, bodymass index, diagnosis, medication and measurements of anumber of clinical pathology end points see Supplemental Table1A (Supporting Information). Samples for a subsequent verifica-tion of discovered candidate markers were collected at theCharite Hospital in Berlin (Germany) from 26 glomerulone-phritis patients and 11 healthy controls. In the group withglomerulonephritis, diagnosis was made by renal biopsy in allcases. Specific renal diseases were: minimal change GN (n = 3);focal segmental glomerulosclerosis (n = 11); M. Wegener

(n = 4); membranous GN (n = 5); postinfectious GN (n = 2);myeloma kidney (n = 2) and tubulointerstitial nephritis (n = 1),see Supplemental Table 1B (Supporting Information). Allsamples were stored at �80 �C until use.

Antibody SelectionA list of candidate targets was compiled using literature

mining, pathway analysis, gene expression and proteomics data.A set of genes associated with glomerulonephritis, obstructivenephropathy and diabetic nephropathy was extracted using theIngenuity Pathway analysis system (Ingenuity Systems). Addi-tional genes expressed in normal adult kidney were added from alarge-scale gene expression study,12 as well as genes associatedwith IgA nephropathy identified by a proteomics study.13 In total,a list of 335 target proteins was deemed relevant for an initialscreen. The collection of antibodies generated by the HumanProtein Atlas project14 was mined for the target list. Followingcriteria for antibody specificity and concentration from a pre-vious study15 a total of 129 monospecific polyclonal antibodieswere used. For technical verification studies, one monoclonalantibody targeting fibulin-1 was acquired (sc-25281, Santa CruzBiotechnology). Protein targets were scored as secreted proteinsif two out of three prediction methods (SignalP, Spoctopusand Phobius) predicted the presence of a signal peptide. For acomplete list of target proteins, signal peptide prediction andantibody identities, see Supplemental Table 2 (SupportingInformation).

Preanalytical Sample PreparationPlasma samples where aliquoted into 96-well plates in a

randomized order including triplicates of 6 samples. To evaluateeffects of repeated thawing and freezing of the samples, 3 samplesthat had been subjected to 3, 6, and 9 repeated freeze�thawcycles were also included in the sample layout. For this purpose,samples had been thawed at room temperature from�80 �C forabout 30 min before being frozen again at�80 �C. The sampleswere labeled with biotin, as previously described,16 whichinvolved the following: Samples were heated at 56 �C for 30 min(PCR-block, BioRad) before diluted 1:10 in phosphate bufferedsaline (PBS) and labeled with 20 μg NHS-PEG4-biotin (Pierce)per 3 μL crude plasma for 2 h at 4 �C. The labeling reaction wasstopped by the addition of 12.5 μL of 0.5 M Tris-HCl (pH 8.0,Sigma) and the samples were stored in�20 �C until further usage.

Suspension Bead Array GenerationAntibody coupled beads were prepared as previously described17

with minor modifications. In short, 5 � 105 magnetic beads perbead-ID (MagPlex microspheres, Luminex Corp.) where dis-tributed into flat bottomed 96-well plates (Greiner BioOne) andwashed with 0.1 M NaH2PO4 buffer (pH 6.2). Beads wereactivated by 0.5 mg N-hydroxysuccinimide (Pierce) and 0.5 mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Pierce) in100 μL phosphate buffer. Upon a 20 min activation andsubsequent washing in 0.05 M MES pH 5.0, 1.6 μg of antibodyin 100 μL MES-buffer was added and incubated for 2 h in roomtemperature. Additional bead identities were functionalizedeither with 5 μg of recombinant albumin binding protein18

produced in-house as previously described,19 with 1.6 μg ofrabbit IgG (Bethyl) or without addition of protein. After 2 hincubation, the beads where washed twice in PBST (PBS, 0.05%Tween20) and stored in plates in 100 μL of a gelatin based buffer(Blocking Reagent for ELISA, Roche) with the addition of NaN3.Suspension bead arrays (SBAs) were then prepared by combining

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equal volumes of each bead identity. After volume adjustmentand sonicating for 5 min (Branson Ultrasonic Corp.) the beadarrays were stored at 4 �C. The coupling efficiency of theantibodies was evaluated by an antirabbit IgG conjugated toR-phycoerythrin (Jackson Immunoresearch). As the number ofantibodies exceeded the number of available bead identities, twodifferent SBAs were created for the screening and denotedSBA1.1 and SBA1.2. Replicate experiments were conducted withsamples and beads stored over four months with SBA2.1 andSBA2.2, which were created by combining the antibody coupledbeads in a new composition. For verification studies, SBA3consisting of the antibodies selected as potential markers wascreated. For information about the composition of each SBA, seeSupplemental Table 2 (Supporting Information).

Assay ProcedureUpon analysis, the 1:10-diluted biotinylated plasma samples

were thawed at room temperature, diluted 1:50 in assay buffer(0.5% w/v polyvinylalcohol (Sigma), 0.8% w/v polyvinylpyrrili-done (Sigma) and 0.1% w/v casein (Sigma) supplemented with10% v/v rabbit IgG (Bethyl)) yielding a total sample dilution of1:500. A heat treatment of 56 �C for 30 min was performed and45 μL sample where transferred to a 96-well assay plate (GreinerBioOne), mixed with 5 μL bead mixture and incubated overnightat room temperature on gentle shaking (Thermomixer, Eppendorf).The beads were then washed in 3� 100 μL PBST using a liquidhandler system (PlateMate 2 � 2, Matrix) and incubated in50 μL 0.4 M paraformaldehyde in PBS. After 10 min, the beadswere washed as noted above prior the addition of 50 μLstreptavidin conjugated R-phycoerythrin (0.5 μg/mL, Invitrogen)and incubated for 20 min. After washing as above, 100 μL PBSTwas added for the read out performed by a Luminex Lx200instrument, set to count at least 50 beads per ID and well. Everydata point acquired was reported as the median fluorescenceintensity (MFI) in arbitrary units (AU) of all beads of eachidentity within one reaction well.

Statistical AnalysisData analysis was performed within the free software environ-

ment for statistical computing and graphics; R.20 For each of thetwo screening experiments, data was generated as four distinctdata matrices and MFI values were processed by probabilityquotient normalization21 as in accordance to previous efforts,22

followed by an antibody based scaling. All subsequent analyseswere performed on raw data in parallel to investigate possiblenormalization introduced artifacts. Variance within and betweenanalyses was evaluated by calculating Spearman’s rank correla-tion. For evaluating freeze�thaw effects and variance withintechnical replicates, coefficients of variations was calculated foreach antibody over all replicate samples. Wilcoxon rank sum testswere applied to log2-transformed normalized data to determinep-values and confidence intervals for the identification of anti-bodies with the power to separate cases from control groups,where outlier samples detected by principal component analysesand replicates had been removed. Findings were denoted sig-nificant if they concordantly showed p-values below 0.01 in bothreplicate experiments. All data shown in statistics and figures arereported from the probability quotient normalized data from thefirst of the discovery experiments unless stated otherwise.

Western BlotPlasma samples were diluted 1:60 into PBS-glycerol and

incubated with SDS reducing buffer in 70 �C for 10 min.

Gel separation was performed in a 4�12% BisTris gel(Invitrogen) in MOPS-based buffer (2.5 mM MOPS, 2.5 mMTris Base, 0.005% SDS, 0.05 mM EDTA at 200 V for 1 h and15 min in room temperature followed by electroblotting ontoPVDF membranes (0.45 μm pore size, Invitrogen) in a transferbuffer (25 mM bicine, 25 mM BisTris, 1 mM EDTA, 10% v/vEtOH) at 30 V for 3 h in 4 �C. Transfers were confirmed withPonceau stain (Pierce) and membranes were stored dry. Uponusage, membranes were soaked in 95% ethanol and blocked in5%w/vmilk powder and 1% v/v Tween20 in Tris-buffered saline(TBS) for 1 h at room temperature. The blocking buffer wasexchanged and membranes were incubated with antifibulin-1antibody (0.5 μg/mL) overnight at 4 �C. The blots were washed4� 5 min in TBS-Tween20 and horse radish peroxidase-con-jugated goat antirabbit IgG antibody (DAKO) diluted 1:3000 inblocking buffer was applied and incubated for 1 h at RT. Afterwashing as above, the detection was performed with TMBMsubstrate (MOSS inc.) and 15 min development. The blots werefinally rinsed in dH20, and scanned with a table top scanner.Band intensities were translated to relative intensities usingthe publicly available image analysis tool ImageJ (http://rsbweb.nih.gov/ij/).

’RESULTS

Study DesignThis study aims to expand the list of potential serum or plasma

biomarkers for kidney impairment, whether based on druginduced toxicity or renal disease. Here, a multiparallel antibodysuspension bead array approach has been utilized for the screen-ing of plasma samples from controls and patients representingvarious kidney disorders; glomerulonephritis, diabetic nephro-pathy, obstructive uropathy and analgesic abuse. By this ap-proach, minute amounts of biotinylated samples were profiledusing antibody-coupled beads targeting proteins of potential

Figure 1. Antibody suspension bead array procedure. (A) Plasmasamples from cases and controls are distributed in 96-well plates,positioning the different samples in randomized locations. The proteinsof the plasma samples are then labeled with biotin and (B) antibodies areselected and covalently coupled to color-coded beads. (C) Beads andsamples are combined and during this incubation, the antibodies bindtheir respective targets. (D) These interactions are detected by theaddition of a reporter dye, a streptavidin-conjugated fluorophore,followed by read-out (E) where one laser detects the internal fluores-cence code of the bead to assign an antibody identity and a second laserdetects the amount of reporter dye as a measure of the antibody-targetinteraction.

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interest.16 A dual multiplexing capacity in both the dimension ofsamples and antibodies is enabled through that individuallyantibody coated color coded beads are combined into asuspension bead array. This array is subsequently applied to amicrotiter plate sample format enabling a high multiplexingcapacity and high sample throughput. The procedure is de-scribed in Figure 1.

Selection of AntibodiesA prerequisite for microarray based proteomic profiling is the

availability of large numbers of affinity reagents.23 We have hereutilized the resources within the Human Protein Atlas (www.proteinatlas.org) for the access to validated antibodies. A list of

potential target proteins was generated from various sources withhundreds of proteins that had any a priori known or speculativeconnection to function of or expression in kidneys. Based on thecoincidental availability of antibodies for these proteins withinthe Human Protein Atlas project, a final list was generated with129 antibodies targeting 94 different proteins (SupplementalTable 2, Supporting Information). With the addition of beadstargeting human serum albumin and beads to monitor unspecificinteractions, a total of 135 beads were generated.

Suspension Bead Array (SBA) AssayDisease-specific protein profiles were identified from profil-

ing four discovery cohorts consisting of 160 plasma samples

Figure 2. Intensity overview and variance analysis. (A) Signal intensities from 135 antibody coupled beads profiling 160 plasma samples were found torange from 50 to 22 000 AU. Antibodies targeting predicted plasma proteins (red) were found at a higher frequency among the antibodies associatedto the highest signals. (B) In hierarchical cluster analysis of protein profiles from all replicate samples using all 135 antibodies, samples cluster accordingto originating individual. Replicates subjected to repeated freeze�thawing three, six and nine times (FT3, FT6 and FT9) still cluster according toindividuals and not according to freeze�thaw cycle. The overall variance of the freeze�thaw samples was only moderately greater than for the replicatesamples, indicating that repeated thawing and freezing does not introduce any systematic effect that overrules the individual sample profiles obtained inthis measurement. Samples are named after their corresponding disease group: DN = diabetic nepropathy, OU = obstructive uropathy, AA = analgesicabuse, GN = glomerulonephritis, NL = healthy controls.

Figure 3. Comparative protein profiling. Protein profiles from 129 antibodies were used to compare 20 glomerulonephritis patients to 20 matchedcontrols. All antibodies are presented in volcano plots, in which the x-axis shows the differences in median intensity levels between the compared groupsfor each antibody and the y-axis displays the negative logarithm of the p-value, the probability for the acquired data under the hypothesis that there is nodifference between cases and controls. In the first set of screening analysis (left), several disease specific differences were discovered as statisticallysignificant (p-value < 0.01). In the replicated experiment (right), all three antibodies targeting fibulin-1 (FBLN1) were consistently significant(black points).

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from four patient groups and matched controls. Two replicateexperiments were performed four months apart using the samesamples and antibodies to enhance the confidence of the datagenerated. In order to exclude the potential influence of a uniqueset of antibodies being combined in an SBA, the compositions ofthe array employed in the second analysis were different to thefirst round analysis by interchanging every second antibody-coupled bead from the SBA list. As a result, the differentantibodies targeting the same protein no longer were analyzedwithin the same suspension bead array (see Supplemental Table2 for SBA composition, Supporting Information). From the 129antibodies analyzed, the signal intensities ranged over 2.6 ordersof magnitude (50�22 000 AU) and Figure 2A shows thedistribution of the obtained intensity levels in average over allplasma samples. In general, there was a trend that highest signalintensities were obtained from antibodies targeting proteins witha predicted signal sequence suggesting that they are possiblysecreted out of the cells into the bloodstream. Of the 22antibodies of highest signal intensities (>4000 AU), 17 targetedknown plasma proteins. At the low intensity end, beads that hadundergone the coupling procedure without addition of antibodyrevealed the degree of interactions to the beads (<200 AU). Toexemplify potential interactions to antibody coupled beads, rabbitIgGmoleculeswithout a defined specificity froma commercial sourcewere included which revealed similar intensity values (<300 AU).

Sixteen antibodies were found to have signal intensity distributions inthe same range as the rabbit IgG.

Technical Aspects and VariabilityBetween two replicate experiments using bead arrays of

different compositions, sample profiles were concordant with amedian Spearman correlation of 0.83 (total range 0.74�0.90).Within the two experiments the correlation of experimentaltriplicates ranged from 0.95�0.98 in the first experiment andfrom 0.98�0.99 in the second experiment. Within these experi-mental triplicates, the median coefficient of variation (CV) forantibodies was 11% (4�41%) in the first experiment and wasindependent of absolute signal intensities. It was observed that115 out of 129 antibodies had a CV lower than 20%. In thesecond experiment, the median CV was 8% (3�28%) and 125antibodies showed a coefficient of variation lower than 20%.

Freeze�Thaw EffectsIn the identification of plasma biomarkers, a potential con-

founder is the effect of repeated freezing and thawing of samples.In order to characterize such effects in the set of targetedproteins, three samples that had been subjected to repeatedthawing and freezing three, six and nine times were included intothe experimental setup. A hierarchical cluster analysis of theprotein profiles from processed samples and the rest of thetechnical replicates (Figure 2B) showed that regardless of freeze

Figure 4. Fibulin-1 as a candidate marker for glomerulonephritis. (A) Fibulin-1 has been reported with several isoforms (P23142, UniProt). Isoform Ais a shorter splice variant, and isoform B and C have alternative sequences in the C-terminal part compared to the canonical isoform D. All antibodiesemployed were raised against antigens that cover the part of the protein which is shared by all isoforms. (B) Besides the three antibodies present in thescreening, one monoclonal antibody targeting fibulin-1 (mAb) was added in complement analyses. All four antibodies separated the GN patients (grayboxes) from their matched controls (white boxes) in the suspension bead array assay. (C) In a receiver operator characteristics analysis, the HPAantibodies showed a superior discriminatory power compared to the monoclonal binder and no separation was observed for an albumin (ALB) bindingprotein.

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thaw cycle, the samples show protein profiles that clusteraccording to individual. The median CV obtained across freeze�thawed replicates was 14% for the first experiment and 12% forthe second experiment. In the presented setting, the overallvariation in the freeze�thawed samples was found to be in thesame range as for the technical replicates.

Comparative Protein Profiling of Renal ImpairmentsIn a comparison of sample protein profiles, 20 patients and 20

matched controls per renal disorder were screened. In total fourdisease categories and 160 samples were profiled using 129antibodies. The majority of proteins that were identified andscored as significantly different between cases and controls werediscovered within the glomerulonephritis (GN) cohort (Figure 3).Among these, fibulin-1 (FBLN1) was targeted by several anti-bodies that all revealed differences with statistical significance. Inthe second experiment, it was found that the discriminatorypowers of the fibulin-1 antibodies were maintained. For the otherdisease cohorts—diabetic nephropathy, obstructive uropathyand analgesic abuse—a few proteins appeared with disease specificprofiles in the first experiment but could not be confirmed atsimilar significant levels in replicate experiments. Since antibo-dies targeting fibulin-1 were concordantly significant in separat-ing GN patients from controls in both experiments regardless ofbead array composition, this protein was selected for furtherstudies.

The relations between the detected fibulin 1 profiles to serumcreatinine (CR) and to blood urea nitrogen (BUN) levels wereinvestigated for theGNgroup (shown in Supplementary Figure 2,Supporting Information). A trend was observed that sampleswith the highest BUN and CR values were also detected withhigher fibulin 1 profile values. Besides fibulin, further proteinswere identified as potentially interesting in the context of kidneydisorders. Examples of those being supported by both experi-ments of this initial screening were GC, SLC13A3, GDA, MACF1,MAPK3, RBP4, SFXN1, and BBOX1. Due to the significance andconfidence of having foundmultiple antibodies supporting fibulin-1,we will focus on the description of FBLN1, while the additionalprotein targets will need more extended verification efforts, whichwill be subject of coming studies.

Fibulin-1 Target Verification via Additional AntibodiesFibulin-1 was targeted by three antibodies in the screening:

HPA001612 (HPA1), HPA001642 (HPA2) and HPA001613(HPA3). While HPA1 and HPA2 had been raised toward thesame antigen, corresponding to residues 266�402 in the nativeprotein, HPA3 had been raised toward an antigen correspondingto residues 417�559 (Figure 4A). All three HPA antibodiescould separate the GN patients from matched controls bymeasuring fibulin-1 in the direct labeling assay (Figure 4B).Mean values and 95% confidence intervals of the ratios betweenthe sample group medians are presented with correspondingp-values in Table 1. To exclude the possibility that the finding offibulin-1 as a discriminatory marker was associated to HPAantibodies rather than the target protein, a monoclonal antibodytargeting fibulin-1 was also investigated. This antibody was raisedtoward an antigen corresponding to residues 1�190 of the full-length protein (Figure 4A). Coupled to a bead and employed inthe direct labeling assay, it was found to reveal a concordantgroup dependent difference as the HPA antibodies (Figure 4B).The mean of the ratio obtained here was 1.4 with a 95%confidence interval of 1.1 to 1.6 with an associated p-value of0.007. Receiver operator characteristics of the results from the

direct labeling assay showed a diagnostic potential of the HPAantibodies targeting fibulin-1 (Figure 4C), with diagnostic accu-racy based on the same samples that were used for selectionof fibulin-1.

Technical Verification by Western Blot AnalysisTo verify the profiles for the proteins targeted by HPA anti-

bodies, Western blots with plasma samples were performed withfibulin-1 antibody HPA1. Four samples from the GN categoryshowing high signal intensity profiles for fibulin-1 as well as theirrespective matched controls were chosen for Western blot detec-tion. As shown in Figure 5A, HPA1 detected a target protein ofabout 70�80 kDa in plasma, which corresponds to a predictedsize for fibulin-1 of 77 kDa, (according to Uniprot P23142). Theintensity profile determined in the Western blot bands werecongruent with those obtained from the antibody array analysis(Figure 5B). This confirms that the difference between cases and

Figure 5. Technical verification of fibulin-1 profiles. Protein profiles forglomerulo-nephritis patients obtained in the suspension bead assay werereproduced in a Western blot using antifibulin-1 antibody HPA1 fordetection. (A) Most prominent bands on the blot (dashed square)correspond to a protein size of about 77 kDa which is the predicted sizeof fibulin-1. (B) Intensities of the Western blot bands were translatedinto relative intensities (line chart) where the differences between thesamples correspond to signals obtained from suspension bead array(bars). Here, signals from Western blot and antibody arrays are shownfor representative patients with glomerulonephritis (GN, gray bars) andmatched controls (NL, white bars).

Table 1. Discriminatory Capacity of Fibulin-1 in Glomeru-lonephritis Case Control Comparisons

experiment 1 experiment 2

antibody p-value

mean of ratio GN/ctrl

(95% conf. interval) p-value

mean of ratio GN/ctrl

(95% conf. interval)

HPA1 0.0003 1.6 (1.3�1.9) 0.0003 1.6 (1.3�2.0)

HPA2 0.00001 1.7 (1.3�2.2) 0.000005 1.8 (1.5�2.3)

HPA3 0.004 1.3 (1.1�1.5) 0.002 1.2 (1.1�1.4)

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controls in the discovery screening was associated to relativeabundance of fibulin-1.

Biological Verification of Fibulin-1 as a Biomarker in anIndependent Cohort

To disconnect current findings from a specific sample source,an independent cohort of plasma from GN patients and controlswas profiled with all four fibulin-1 antibodies. In this third set ofexperiments, coprofiling of a subset of previously analyzeddiscovery samples (8 cases + 8 controls) and the verificationcohort (26 cases + 11 controls) confirmed fibulin-1 as a markerdiscriminating GN cases from controls in each cohort separatelyand in a statistically significant manner for the HPA antibodies(Figure 6A). Receiver operator characteristics of the results fromthe verification cohort confirmed the diagnostic potential offibulin-1 (Figure 6B). The direct labeling analysis revealed elevatedintensity profiles for the verification cohort in comparison to thediscovery cohort. Western blot analyses of both cohorts did notreveal apparent differences in abundance of fibulin-1 between thetwo sample sources (data not shown).

Fibulin-1 Across Other Kidney DisordersFibulin-1 was furthermore found to be able to clearly separate

the other kidney disease categories investigated herein; diabeticnephropathy and obstructive uropathy, from their respectivecontrols although with slightly reduced significances. Such

differences were also observed but less pronounced for theanalgesic abuse group (Table 2). One possible reason for thiswas that this control group was found to be more heterogeneousfor fibulin-1 than the other, see Figure 7, where 8 individuals (allmen) displayed higher detected levels of fibulin-1 using HPA1.The trends shown in Figure 7 and Table 2 indicate that fibulin-1is not exclusively a marker specific for glomerulonephritis alone,but generic across the investigated types of renal impairment.

’DISCUSSION

The presented study shows the use of antibody suspensionbead arrays defined by lists of organ related targets for thediscovery and verification of potential disease markers. Profil-ing plasma from different categories of kidney disorders and

Figure 6. Biological confirmation of fibulin-1 association to glomerulonephritis in a separate cohort. (A) When profiling an independent cohort ofglomerulonephritis patients, the potential of fibulin-1 in separating 26 cases (gray boxes) from 11 controls (white boxes) was confirmed. (B) Here, thediagnostic potential of fibulin-1 is confirmed as indicated by the AUC-values for the different antibodies. In this cohort, HPA3 showed the greatestdiscriminatory power.

Table 2. Discriminatory Capacity in the Form of p-Values ofFibulin-1 in Case Control Comparisons Across Four Types ofKidney Disorders

HPA1 HPA2 HPA3

disorder exp 1 exp 2 exp 1 exp 2 exp 1 exp 2

Diabetic nephropathy 0.004 0.007 0.003 0.006 0.1 0.1

Obstructive uropathy 0.0002 0.0005 0.003 0.0008 0.02 0.01

Analgesic abuse 0.05 0.09 0.09 0.1 0.01 0.2

Glomerulonephritis 0.0003 0.0003 0.00001 0.000005 0.004 0.002

Figure 7. Fibulin-1 across other kidney diseases. The profiles of fibulin-1as separator between cases and controls in renal impairment wassimilar to GN for the other diseases investigated; diabetic nephro-pathy (DN), obstructive uropathy (OU) and analgesic abuse (AA).Shown here are the profiles for fibulin-1 antibody HPA1 obtained inexperiment 1.

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validating the indentified targets by multiple affinity reagents,orthogonal methods as well as samples collected at a differentclinical location, lead to the discovery of fibulin-1 as a marker forrenal impairment.

The identified kidney related biomarker candidate fibulin-1has been reported in various isoforms and as a secreted plasmaprotein with numerous interaction partners in the circulation,such as fibrinogen. It is also a structural component of theextracellular matrix incorporated into fibrillin containing fibersin the basal membranes of several tissues.24�26 Fibulin-1 is highlyexpressed in placenta and has been shown to have a functionalrole in placentation.27 Homozygotic knockout mice die at birthof ruptured small vessels, disrupted alveolar in the lungs andmalfunctioning glomeruli in the kidneys, while the clottingcascade was unaffected.28 This indicates that fibulin-1 is astructural protein essential for formation of small vessel structuresin circulation, kidneys and lungs. Fibulin-1 has been reportedwith a differential expression mainly on a transcriptome level inrelation to various cancers, cardiovascular diseases and multiplesclerosis.29�33 In the presented context it is speculated that areduced renal function could be associated to disrupted struc-tures in the kidney, which could result in an elevated level of freefibulin-1 accumulated in circulation. This finding would be ofinterest to put into a larger context involving other proteins of theextracellular matrix in relation to kidney malfunction, such as, forexample, elastin and collagen, which have been reported as potentialmarkers and therapeutic targets for renal disorders.9,34,35

Naturally, the mechanism behind an increased plasma fibulin-1 level and the potential relation to insult at specific anatomicalregions in the kidney remain to be elucidated. Furthermore,longitudinal clinical studies where levels can be monitored overtime would yield valuable information on fibulin-1 as early or latemarker and the potential to use of fibilin-1 for monitoring anearly kidney insult such as drug induced kidney injury or a latechange in morphology such as disease progression or regression.

The applied method uses a single capture reagent to deter-mine the presence of a biotinylated protein in a sample. Thisstrategy is a prerequisite to enable the high multiplexicity in abroad screening of protein profiles. The analysis of proteincompositions in complex samples with a single immunocaptur-ing agent will generally not match the same sensitivity andspecificity of the dual detection enabled by sandwich assays.Nevertheless, the degree of multiplexing is limited in sandwichassay,36 and the composition of the different sandwich affinitypairs is of high importance. In contrast to this, the flexibility of thedirect labeling approach on a bead-based system did not influ-ence the performance of the assay in general, as shown here bythat bead arrays of shuffled composition gave a high interassaycorrelation. The study further shows that confirmed findingswere independent of the array composition, which suggests thatbuilding up arrays of validated and functional capture agents ispossible and that the performance of individual markers is likelynot affected.

The obtained differences in fibulin-1 profiles between patientswith glomerulonephritis compared to controls were shown to beconsistent regardless of sample storage time, suspension beadarray composition, data processing and cohort origin. Theconfidence for fibulin-1 being detected as a potential biomarkerwas increased by several independent binding events from bothpolyclonal and monoclonal antibodies raised toward differentregions of the protein. All these antibodies gave rise to con-cordant profiles and Western blot analyses showed bands of a

predicted molecular mass. However, absolute signal intensitiesfor fibulin-1 obtained in the direct labeling assays were observedto be different for separately collected GN-cohorts. This was notfound to be the case in the denaturing conditions of Westernblot. A possible reason for these differences could be a result fromdifferent collection protocols including different collection tubesand centrifugation speed as well as time elapsed betweencollection and freezing and final storage conditions. On amolecular level, such effects could alter the sample matrix andinteracting proteins could differentially enhance or reduce thesignal obtained by direct labeling. A current assumption is thatthe degree of interacting fibrinogen or other binding partners offibulin-1 could be dependent on plasma preparation protocolsgiving rise to different absolute signal intensity levels. Suchinteractions would also not be detectible under denaturingconditions as in aWestern blots analysis. Tests evaluating sampleheat treatments of 56 �C prior incubation with the bead arraysshowed no effect on fibulin-1 detection. This could indicate thatpotential fibulin-1-containing complexes withstand heat untilheated under reducing conditions, such as required for SDS-gelpreparation. In spite of these observations, the difference be-tween cases and controls was consistent regardless of theinvestigated sample cohort, and there is supporting evidencefrom different binding agents that the observed differencesresulted from increased levels of fibulin-1 protein in the circula-tion of patients.

In conclusion, antibody suspension bead arrays were appliedfor the exploration of directly labeled human plasma samples inthe quest for biomarkers for renal impairment. The current statusreveals human fibulin-1 as a candidate marker for renal impair-ment. To evaluate the full potential of this protein, the develop-ment of clinically more suitable assay formats such as sandwichimmunoassays is now required. With such a test system, multipleand larger clinical collections from various origins and contextsare to be screened to reveal the capacity of this protein as abiomarker for renal impairment.

’ASSOCIATED CONTENT

bS Supporting InformationSupplementary Figure 1. Estimated glomerular filtration rate.

Supplementary Figure 2. Relations between fibulin 1 profiles andserum creatinine (CR) and to blood urea nitrogen (BUN) levels.Supplementary Table 1A+1B. Complete sample tables withmetadata. Supplementary Table 2. Complete antibody table.This material is available free of charge via the Internet athttp://pubs.acs.org.

’AUTHOR INFORMATION

Corresponding Author*Tel: +46 8 5248 1418. E-mail: [email protected].

’ACKNOWLEDGMENT

We thank Linn Fagerberg (KTH) for help with signal peptidepredictions of protein targets. We also thank Per-Åke Nygren(KTH) for fruitful discussions and the entire staff of the HumanProteinAtlas for their efforts. This studywas funded by the ProNovaVINN Excellence Centre for Protein Technology (VINNOVA,Swedish Governmental Agency for Innovation Systems) and bygrants from the Knut and Alice Wallenberg Foundation.

I dx.doi.org/10.1021/pr200286c |J. Proteome Res. XXXX, XXX, 000–000


’ABBREVATIONS

SBA, suspension bead array;GN, glomerulonephritis; DN, diabeticnephropathy;OU, obstructive uropathy; AA, analgesic abuse; AU,arbitrary units

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plasma profiling reveals human fibulin-1 as candidate ...mining, pathway analysis, gene expression...

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