Proteomic Signature of Endothelial Dysfunction Identified inthe Serum of Acute Ischemic Stroke Patients by the iTRAQ-BasedLC−MS ApproachRakesh Sharma,†,▲,● Harsha Gowda,‡ Sandip Chavan,‡,§ Jayshree Advani,‡,§ Dhanashree Kelkar,‡,∥

G. S. Sameer Kumar,‡ Mitali Bhattacharjee,‡ Raghothama Chaerkady,‡,§ T. S. Keshava Prasad,‡,§

Akhilesh Pandey,‡,∥,⊥,#,∇ Dindagur Nagaraja,*,○ and Rita Christopher*,†

†Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore 560029, India▲Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, Hong Kong●Department of Biology and Chemistry, City University of Hong Kong, Hong Kong‡Institute of Bioinformatics, International Technology Park, Bangalore 560 066, India§Manipal University, Manipal 576 104, India∥McKusick-Nathans Institute of Genetic Medicine and ⊥Departments of Biological Chemistry, #Pathology, and ∇Oncology,Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States

○Department of Neurology, Dharwad Institute of Mental Health and Neuro Sciences, Dharwad 580001, India

*S Supporting Information

ABSTRACT: Acute ischemic stroke (AIS) is a devastating cerebrovascular disorder that leads topermanent physical and neurological disabilities in adults worldwide. Proteins associated withstroke pathogenesis may appear in the serum of AIS patients due to blood−brain barrierdysfunction, thus permitting the development of blood-based biomarkers for early diagnosis ofstroke. These biomarkers could perhaps be an adjunct to the existing imaging modalities and aidin better management and therapeutic intervention during the course of the disease. For thisexploratory study, a combination of multiplexed isobaric tagging using iTRAQ reagents and highresolution tandem mass spectrometry was used to identify differentially expressed proteins inserum samples from AIS patients. The quantitative proteomic analysis of serum from both AISand control subjects revealed 389 high confidence protein identifications and their relative levels.Among them, 60 proteins showed a ≥1.5-fold change in the AIS subjects. We verified the alteredserum levels of candidate proteins such as vWF, ADAMTS13, S100A7, and DLG4 throughELISA, and the results also corroborate with the experimental findings. vWF and ADAMTS13are key players that regulate blood hemostasis, and their altered concentration may contribute toendothelial dysfunction. S100A7 is a novel candidate protein identified in this study that is also known to mediate inflammation,endothelial proliferation, and angiogenesis. The current study provided a potential and novel biomarker panel that may in turnprovide diagnostic aid to the existing imaging modalities for the rapid diagnosis of ischemic stroke

KEYWORDS: hemostasis, thrombus, inflammation, LTQ-Orbitrap mass spectrometer, diagnostic marker, ischemic stroke,high density lipoprotein (HDL), low density lipoprotein (LDL)

■ INTRODUCTION

Stroke is one of the leading causes of morbidity and mortality inboth developed and developing countries. Despite being a majorpublic health concern, effective disease management strategiesfor the treatment of acute ischemic stroke (AIS) are limited.Thrombolytic therapy with an intravenous tissue plasminogenactivator should be administered within the limited therapeutictime window of 4.5 h poststroke; hence, only <10% of patientsreceive this therapy. An attempt to extend the therapeuticwindow up to 9 h using recombinant desmoteplase, a novelplasminogen activator, has recently failed,1 and a trial using thedefibrinogenating agent ancrod has not been successful.2

Currently, the identification of the cause of stroke is primarily

based on a neurological assessment and on various imagingtechniques, such as cranial computed tomography (CT), mag-netic resonance imaging (MRI), and arteriography. Clinicianshave to depend solely on these imaging techniques to identify anischemic stroke and rule out a hemorrhagic event. However, CTand MRI scans are not absolutely reliable methods, as CT hasonly 16% sensitivity for ischemic stroke and 89% sensitivity forhemorrhagic stroke in the early hours of diagnosis. Moreover,20% of ischemic stroke cases are not detected with the MRI scantechnique. Furthermore, imaging diagnostic facilities are not

Received: January 1, 2015

Article

pubs.acs.org/jpr

© XXXX American Chemical Society A DOI: 10.1021/pr501324nJ. Proteome Res. XXXX, XXX, XXX−XXX

available in all treatment centers, and this hinders the rapiddiagnosis of stroke and thus the administration of the drug.Hence, there is an urgent requirement for novel molecularmarkers with which to diagnose AIS. Relative quantitation usingmultiplexed isobaric tags, such as iTRAQ, and high resolutiontandem mass spectrometry is a promising technology for proteinbiomarker identification.The identification of stroke biomarkers in biological fluids

could have implications as an adjuvant to imaging modalities ina paramedical setup. Numerous approaches to finding a strokebiomarker for early diagnosis have been tried with hardly anypositive outcomes.3 Ethical issues prevent the collection ofcerebrospinal fluid (CSF) from stroke patients, which limits itsapplication in biomarker studies. Blood may be an ideal fluid forbiomarker studies in stroke as it is a vascular complication andprotein level variations associated with stroke are likely to appearin the blood.4 Although numerous efforts are in progress toidentify protein biomarkers in blood for the prognosis and theearly diagnosis of stroke, there has been limited success usingblood as a result of comorbidity and its associated complexity.Recent advances in sample preparation for proteomics and highresolution mass spectrometry have once again emphasized theimportance of serum and plasma as sources of diagnostic markersthrough proteomics.5−12 Several targeted approaches to validatepotential biochemical markers in stroke animal models usingCSF/blood have been reported.13−16 However, literatureon CSF/serum biomarkers using animal stroke models by pro-teomics approaches is limited17. Hence, our study using advancedproteomics approach demonstrates a new avenue for biomarkerdiscovery in stroke.The present study was aimed at the protein profiling of

serum to identify potential candidate markers of AIS through aniTRAQ-based LC−MS/MS approach and to further validate theexperimental findings of the discovery phase of the study.

■ EXPERIMENTAL DETAILS

Study Subjects, Sample Collection, and Storage

This study was approved by the Institute Ethics Review Board ofthe National Institute of Mental Health and Neuro Sciences(NIMHANS), Bangalore, India, and written informed consentwas obtained from all participants. We recruited patients whopresented at the Neurological Services of NIMHANS Hospitalwith a clinical diagnosis of ischemic stroke confirmed by cranialCT scans or MRI studies. Patients with hemorrhagic strokeor stroke secondary to neuroinfections, trauma, malignancy,renal or liver dysfunction, or any other terminal illnesses wereexcluded. Our control group was composed of healthy volunteerswith no prior history of cerebrovascular diseases. Controls werematched with patients for age and gender. Table 1 providesdemographic details of the study participants. A total of 5 mL ofvenous blood was collected from the median cubital vein ofeach patient. The serum was separated, centrifuged at 1500g for10min, and stored at−80 °C. Blood samples were collected fromall patients for both discovery and validation experiments within24 h of the onset of the first symptom of ischemic stroke.Removal of High Abundant Proteins for Sample Preparation

A total of 20 AIS or control serum samples were pooled andimmunodepleted of the 14 most abundant proteins using aHuman 14 MARS Liquid Chromatography affinity column(4.6 × 100 mm; Agilent Technologies, Santa Clara, CA) as perthe vendor instructions. Unbound depleted serum was collectedand desalted using 3 kDa MWCO filters (Millipore, San Diego).

The total protein amount was determined by Lowry’s assay,and protein normalization was confirmed using 10% SDS-PAGE(Figure S1, Supporting Information).iTRAQ Labeling

To label each condition, we used 200 μg of proteins.18 Briefly,proteins were denaturated by 2% SDS followed by reductionwith reducing agent (TCEP) and alkylation by cysteine blockingagent (MMTS). Protein samples were then digested with trypsin(Promega, Madison, WI) at 37 °C for 12 h. The tryptic peptidesfrom each condition were divided into two equal halves and thedivision followed by iTRAQ labeling that was carried out inreplicates as per vendor instructions (iTRAQ Multiplex kit;Applied Biosystems/MDS Sciex, Foster City, CA). Furthermore,the peptides from the control samples were labeled with reagentscontaining 114 and 115 reporter tags, and the peptides from theAIS samples were labeled with reporter tags 116 and 117,respectively. Labeled peptides from each condition were pooled,dried, and reconstituted in 10 mM KH2PO4 and 20% ACN(pH 2.8) for further fractionation by strong cation exchangechromatography.SCX-Based Fractionation

SCX chromatography was carried out as described previously.19

The peptides were fractionated on a PolySULFOETHYL Acolumn (200 Å, 5 μm, 200 × 2.1 mm; PolyLC, Columbia, MD)using an Agilent 1200 LC system (Agilent Technologies).Peptide fractions were collected using a linear gradient of solventB (350 mM KCl in solvent A, pH 2.8) over 70 min at a flow rateof 300 μL/min. On the basis of the UV absorbance at 214 nm,the least complex fractions were pooled to generate a total of34 fractions. Subsequently, the pooled fractions were desaltedusing C18 stage tips (3 M Empore, SDB-XC, product number2240/2340).LC−MS/MS Analysis

The analysis of the labeled peptides from each fraction wascarried out on an LTQ-Orbitrap Velos ETD mass spectrometer(Thermo Scientific, Bremen) interfaced with an Agilent 1200nanoLC system. The online reversed-phase chromatographyincluded a trap column (75 μm × 2 cm, C18 material, 5 μm,100 Å) at a flow rate of 4 μL/min and an analytical column(75 μm × 10 cm, Magic C18 AQ, 3 μm particle size, pore size

Table 1. Demographic Characteristics of Acute IschemicStroke Patients and Control Subjectsa

variablesAIS patients(n = 50)

controls(n = 35) p value

age 51.3 (17.34) 50.1. (16.78) >0.05male:female 31:19 21:14 >0.05smokers 24 (48%) 8 (22.9%) <0.05alcoholics 21 (42%) 10 (28.5%) <0.001diabetes 21 (40.2%) 11 (31.4%) <0.001hypertension 29 (58%) 9 (25.7%) <0.001systolic BP (mmHg) 142 (17.2) 128 (16.2) <0.001diastolic BP (mmHg) 88.4 (20.2) 79 (15.3) <0.001random glucose (mg/dL) 128 (7) 118.73 (23) <0.001total cholesterol (mg/dL) 198 (40) 195 (47) >0.05HDL cholesterol (mg/dL) 53 (20) 59 (22) <0.001triglycerides (mg/dL) 178 (40) 138 (43) <0.001LDL (mg/dL) 109 (12) 108 (5) >0.05aPatient and control results are expressed as mean (SD) for eachparameter. Comparisons were made by unpaired t test. Values ofp <0.05 are statistically significant.

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100 Å) at a flow rate of 350 nL/min. The peptides were elutedusing a linear gradient of 5−45% acetonitrile over 75 min.The electrospray source consisted of a 10 ± 2 μm emitter tip(New Objective, Woburn, MA) maintained at 2.4 kV. Data wereindependently acquired in a data-dependent manner with full-scanMS acquired using the FTmass analyzer at amass resolutionof 60 000 and MS/MS scans at a mass resolution of 15 000. Foreach survey scan in a MS cycle, the 20 most intense precursorions were selected for MS/MS. HCD fragmentation wasperformed at a 42% normalized collision energy. To avoid therepeated selection of ions for MS/MS, we set the dynamicexclusion window to 30 s. The AGC for full FT MS and FTMS/MS was set to 1 million and 0.1 million ions with a maximumaccumulation time of 300 ms. The lock mass was enabled foraccurate mass measurements using polydimethylcyclosiloxane(m/z, 445.120 002 5) ions.

Data Analysis

The MS data obtained were processed using ProteomeDiscoverer (Version 1.4.0.288, Thermo Fisher Scientific,Germany). To maximize the number of protein identifications,we searched the data using the SEQUEST algorithm against theNCBI Human RefSeq database 52 containing 33 985 proteinentries (including known contaminants). Parameters for thesearches were trypsin as the enzyme, an allowance of one missedcleavage, the oxidation of methionine as the dynamic modifi-cation, static modifications that included alkylation at cysteine,and iTRAQ modification at the N-terminus of the peptide andlysine. The precursor and fragment mass tolerance were set to20 ppm and 0.1 Da, respectively, with a signal-to-noise ratio of1.5 and a precursor mass range of 350−7000 Da. Peptide andprotein data were fetched using high peptide confidence (1%FDR) and top one peptide rank filters. The relative expressionpatterns of proteins were determined on the basis of the relativeintensities of the reporter ions of the corresponding peptides.Furthermore, p values were computed using an online opensource quantitative proteomics q value calculator (QPPC) tool.20

The results file containing the unique peptide ratios was exportedfrom Proteome Discoverer, and the data were considered asinput to calculate SD and p values. The criteria for selecting aconfident list of differential proteins were set to ≥2 peptides, asingle peptide with multiple PSMs, and a p value of ≤0.05.Bioinformatics Analysis

Proteins were categorized on the basis of biological process,cellular component, and molecular function classificationthrough annotations in the Human Protein Reference Database(HPRD; http://www.hprd.org),21 which is compliant with theGene Ontology (GO) Consortium. The differentially expressedproteins were further analyzed for pathway enrichment analysisby using the pathway architect module in GeneSpring (version12.6).

Data Availability

The mass spectrometry data were submitted to the HumanProteinpedia and can be visualized using the following link:http://www.humanproteinpedia.org/data_display?exp_id=00703.

Enzyme-Linked Immunosorbant Assay

We measured the concentrations of vWF, ADAMTS13(Assay Pro, Saint Charles, MO), DLG4, and S100A7 (USCNLife Sciences, Wuhan, China) in serum using commerciallyavailable ELISA kits. The serum antigen levels were measured in

the replicates from 50 AIS and 35 control cases. The kit protocolwas implemented as per the instructions from the vendors.

Statistical Analysis

The statistical analysis of the ELISA results was carried outusing GraphPad Prism version 5.04 (San Diego, CA). The datawere reported as means ± SD, SE. The statistically significantdifference among the control and disease groups was calcu-lated by Student t test. A p value of 0.05 or less was consideredsignificant. The Receiver Operating Characteristic (ROC)analysis was used to evaluate the performance of the candidatebiomarkers to identify the AIS cases from the controls. Youden’sindex was calculated from the sensitivity and specificity values ofvarious candidate markers of stroke from the ROC test.22

■ RESULTS AND DISCUSSION

Quantitative LC−MS/MS Analysis of AIS and Control SerumProteins

The present study was carried out to identify differentiallyregulated serum proteins in AIS. The experimental plan for thisstudy is shown in Figure 1. A total of 20 pooled serum samplesfrom AIS patients and healthy controls were analyzed in repli-cates for the estimation of the relative abundance of proteinsusing 4-plex iTRAQ. A total of 1 98 000 MS/MS spectra wereacquired from a total of 34 LC−MS/MS runs, of which 54 899PSMs were assigned to 3458 peptides after 1% FDR was applied.Through this strategy, 389 proteins were identified, of which191 proteins were identified with at least two or more peptidesand 198 proteins were identified based on single peptideevidence. Of these proteins, 60 showed a 1.5-fold or greaterdifference in their abundance in AIS serum when compared tothat of the control samples, of which 33 contained a signal peptidesequence. Detailed lists of proteins and peptides, along with theassociated experimental information, are provided in Tables S1and S2 in the Supporting Information, respectively. Among thedifferentially expressed proteins, 25 proteins were more abundantand 35 were less abundant in the serum of AIS subjects comparedto that of control healthy subjects. Furthermore, we arrived atsignificant protein hits on the basis of the p values as shown inTable 2. A partial list of these proteins with altered levels in AIS isprovided in Table 3. Representative MS/MS spectra of twoexamples of differentially expressed proteins von Willebrandfactor (vWF) and S100 calcium-binding protein A7 (S100A7) areshown in Figures 2 and 3, respectively. A schematic of the processby which these altered serum proteins bring about endothelialdysfunction and blood homeostasis is shown in Figure 4.

Bioinformatics Analysis of Serum Proteins of AIS

Blood serum is blood plasma devoid of clotting factors.Serum harbors distinct proteins of different origins, eithersecreted from tissues/cells or leaked from tissues/blood cells,which are involved in many different molecular events andassociated disease processes. Serum proteins identified in thisstudy were compared to those in the Plasma ProteomeDatabase23 (http://www.plasmaproteomedatabase.org/) to de-termine the known proteins in plasma. Out of the 389 serumproteins identified, 337 had been previously reported in plasma.The current study explores proteins such as DLG4, MPO, andCRP, which had been reported to have amajor role in endothelialdysfunction and inflammation. We have also examined thesecretory potential of the proteins using the SignalP tool,24

which predicts the presence of signal peptide sequences. A totalof 213 serum proteins were found to have signal peptides.

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A similar comparison using HPRD resulted in an overlap of215 serum proteins that were known to be secreted through the

classical pathway. Of the two comparisons, 196 proteins werecommon to both HPRD and SignalP.

Figure 1. Outline of the workflow employed to study altered levels of the serum proteome in AIS using a 4-plex iTRAQ strategy.

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Categorization of Differentially Regulated Proteins Basedon GO Enrichment Analysis

We categorized the differentially expressed proteins on the basisof their biological and molecular functions and cellular processesby using annotations in HPRD. The proteins identified werefound to be involved in important biological processes suchas cell signaling, adhesion, and signal transduction (22%);metabolism and energy pathways (27%); and cell function andmaintenance (19%), immune response (10%), and transport(7%) (Figure S2, Supporting Information). The classificationof the differentially expressed proteins on the basis of theirmolecular functions was enriched for proteins with complementactivity (12%), catalytic activity (10%), chemokine/cytokineactivity (7%), transporter activity (5%), channel regulator andintracellular ligand-gated ion channel activity (4%), and celladhesion activity (4%).Functional Annotation of Differentially Expressed Proteinsin AIS

The pathway analysis of the differentially expressed proteins in AISserum samples using the Pathway Architectmodule inGeneSpring(version 12.6) revealed that the enrichment of several proteinsinvolved in inflammatory pathways such as IL-1, IL-6, IL-17, andinterferon γ, as well as pathways of complement activation and

blood coagulation, cascade along with platelet adhesion to theexposed collagen (C3, C7, C9, platelet glycoprotein V, plateletbasic protein, vWF), folate and vitamin B12 metabolism (MPO,SAA1, ACT, CRP, sICAM-1), focal and cell adhesion (BCAM,ICAM-1, TSP-4), oxidative stress, and apoptotic pathways.

Biological Roles of Differentially Expressed Proteins

The proteins that were found to be more abundant in the serumof AIS patients were confined to various biological functions(shown in Table 2) including acute-phase reactants, inflamma-tory markers, oxidative stress markers, involvement in neuro-protection, endothelial dysfunction, neuromodulatory molecules,focal adhesion molecules, and several others.

Potential Markers of Endothelial Dysfunction

In this study, we identified proteins that are known to be in-volved in regulating blood hemostasis and endothelial function.These include vWF, ADAMTS13, ICAM1, and TSP4. Alteredlevels of these proteins could indicate endothelial dysfunction.To further validate the mass spectrometry-derived data, weverified the differential abundance of three selected proteins(vWF, ADAMTS13, and S100 calcium-binding protein A7)using ELISA. The vWF is synthesized in endothelial cells andmegakaryocytes and circulates in blood.25 It has a key role inthrombus formation at sites of vascular damage. The associa-tion between vWF and coronary heart disease has been wellestablished; however, there are very few published studiesutilizing other proteomics approaches to determine themechanism and involvement of vWF in AIS.26−28 Nevertheless,various investigations in the recent years have provided clinicalevidence that reveals a vital association of vWF in strokeetiopathogenesis.29,30 In vitro experiments demonstrated thatvWF promotes leukocyte adhesion by acting as a ligand for theleukocyte receptors P-selectin and integrins.31 vWF-boundplatelets have been shown to support leukocyte tethering androlling under high shear stress.32 Recently, it has been establishedthat vWF promotes the extravasation of leukocytes from bloodvessels in a strictly platelet- and GPIbα-dependent way.33

We observed serum vWF as being 1.8-fold increased in AIScompared to the amount in the control. An ELISA test showedvWF levels of 28.79 ± 7.55 μg/mL in AIS patients as comparedto the levels of 16.9 ± 5.83 μg/mL shown in control subjects(p value of <0.05), which verifies the iTRAQ data (Figure 2B).ADAMTS13 is a vWF cleaving protease that regulates plasma

vWF levels. It is also a major determinant of platelet adhesionafter vessel injury. A proper balance between ADAMTS13 andvWF is essential for normal blood hemostasis, and a change intheir serum levels significantly reflects stroke pathophysiology inthe patients. High vWF and low ADAMTS13 has been shown toincrease the risk of ischemic stroke.30 The initial increase in vWFfollowed by the decrease in ADAMTS13 activity in blood may bedue to the alterations in the coagulation cascade, which togetherfacilitate the thrombus formation and breakdown during acutestroke. A recent study by Anderson and coworkers30 has foundsignificant association between altered serum levels ADAMTS13and vWF. Therefore, to examine the clinical implications ofADAMTS13 and vWF in thrombosis formation and breakdownafter ischemic stroke, we selected ADAMTS13 for quantifica-tion by ELISA in this study. ADAMTS13 level in serum wassignificantly reduced in AIS patients compared to the levels inhealthy subjects (5.33± 0.7 versus 3.38± 0.59 ng/mL; p < 0.05)(Figure 2C), corroborating the findings of Anderson andcoworkers.

Table 2. Classification of Candidate Proteins Identified inAcute Ischemic Strokes, Based on Function and BiologicalRelevance

biomarker category candidate proteinsfold change(AIS/control) p value

inflammations lipopolysaccharide-binding protein

2.0 <0.05

serum amyloid A 2.5 <0.05C-reactive protein 6.4 <0.05α-1 acid glycoprotein-1 1.9 <0.05myeloperoxidase 2.1leucine-richα-2-glycoprotein

1.9 <0.05

myoglobin 1.6 0.045α-1-antichymotrypsin 1.7 <0.05β-2-microglobulin 1.6 0.002inter-α-trypsininhibitor heavy chain

1.5 <0.05

H3neuroprotective insulin-like growth-

factor-bindingprotein 1

1.7 0.031

adiponectin 1.9 0.007endothelial dysfunction C3 1.7 <0.05

C7 1.3 <0.05C9 1.3 <0.05von Willebrand factor 1.8 <0.05

oxidative stress glutathione-S-transferase κ 1

1.6

neuromodulatory/braininjury

discs, large homolog 4 2.3

extracellular matrix andfocal adhesion molecule

thrombospondin-4 1.5 0.035

intercellular adhesionmolecule 1

1.7 0.019

basal cell adhesionmolecule

1.7

other (angiogenesis) S100 calcium-bindingprotein A7

−5.0a <0.05

aDifferentials discussed in this table correspond to the fold changes ofall upregulated proteins except for S100A7, which is downregulated.

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Table 3. Partial List of Differential Proteins Identified in Acute Ischemic Strokes Compared to Those Identified in Controls

gene symbol protein namefold

changenormalCSF

normalserum/plasma signal/TM localization

CRP C-reactive protein 6.4 + + SP extracellularSAA1 serum amyloid A protein 2.5 + + SP extracellularDLG4 discs, large homolog 4 isoform 2 2.3 − + − plasma membraneMPO myeloperoxidase 2.1 − + SP extracellularLBP lipopolysaccharide-binding protein 2.0 + + SP extracellularORM1 α-1-acid glycoprotein 1 1.9 + + SP extracellularLRG1 leucine-rich alpha-2-glycoprotein 1.9 + + SP mitochondrial membrane,

extracellular, plasma membraneADIPOQ adiponectin 1.9 − + SP extracellularVWF von Willebrand factor 1.8 + + SP extracellularC3 complement C3 1.7 + + SP extracellularBCAM basal cell adhesion molecule 2 1.7 − + TM plasma membraneSERPINA3 α-1-antichymotrypsin 1.7 + + SP extracellularIGFBP1 insulin-like growth-factor-binding protein 1 1.7 − + SP extracellularICAM1 intercellular adhesion molecule 1 1.7 − + TM plasma membraneLCN2 neutrophil gelatinase-associated lipocalin 1.7 + + SP extracellularELANE neutrophil elastase 1.7 − + SP cytoplasmKIFC1 kinesin-like protein KIFC1 1.7 − + − microtubuleB2M β-2-microglobulin 1.6 + + SP extracellularSHBG sex-hormone-binding globulin isoform 4 1.6 + + SP extracellularMB myoglobin 1.6 + + − sarcoplasmHLA-A HLA class I histocompatibility antigen,

A-1 α chain1.6 + + SP plasma membrane

FCGBP IgGFc-binding protein 1.5 + + SP extracellularDBH dopamine β-hydroxylase 1.5 − + SP extracellularLGALS3BP galectin-3-binding protein 1.5 + + SP extracellularITIH3 inter-α-trypsin inhibitor heavy chain H3 1.5 + + SP extracellularPCLO protein piccolo isoform-2 1.7 + −GSTK1 glutathione S-transferase κ 1 isoform d 1.6 + +THBS4 thrombospondin-4 1.5 + + SP

Downregulated ProteinsPROZ vitamin-K-dependent protein Z isoform 2 0.73 − + SPCCDC138 coiled-coil domain-containing protein 138 0.75 − − − −C4B complement C4−B 0.75 + + − extracellularPPBP platelet basic protein 0.75 − + SP extracellularLTF lactotransferrin isoform 2 0.7 + + SP secretory granuleSLC38A10 putative sodium-coupled neutral amino acid

transporter 10 isoform a0.7 − + TM integral to membrane

PI4KA phosphatidylinositol 4-kinase α isoform 1 0.7 − + − Golgi apparatusTBC1D1 TBC1 domain family member 1 isoform 4 0.7 − + − endoplasmic reticulumDCD dermcidin 0.7 + + SP extracellularSLC30A9 zinc transporter 9 0.7 − − TM eytoplasmAPOC1 apolipoprotein C−I 0.7 + + SP extracellularSS18L2 SS18-like protein 2 0.7 − − − −B3GNT2 UDP−GlcNAc:β-Gal β-1,3-N-

acetylglucosaminyltransferase 20.7 − + TM integral to membrane

IL17RC interleukin-17 receptor C isoform 6 0.7 − − SP plasma membraneGSN gelsolin isoform b 0.7 + + SP extracellularAPOA4 apolipoprotein A-IV 0.6 + + SP extracellularGADD45GIP1 growth arrest and DNA damage-inducible-proteins-

interacting protein 10.6 − − − cytoplasm, nucleus

KCP Kielin/chordin-like protein isoform 1 0.6 − + SP −SYCP2L synaptonemal complex protein 2-like 0.6 − + − −S100A9 protein S100-A9 0.6 − + − cytoplasmCASP14 caspase-14 0.5 − + − cytoplasmUNC79 protein unc-79 homologue 0.5 − − −PITPNM3 membrane-associated phosphatidylinositol transfer

protein 3 isoform 20.5 − + TM nucleus

KCNN3 small conductance calcium-activated potassiumchannel protein 3 isoform b

0.2 − − TM plasma membrane

S100A7 protein S100-A7 0.2 − + − cytoplasm

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We also assessed the diagnostic efficiency of the endothelialdysfunction markers by ROC analysis and obtained ROC curveareas of 0.91 for vWF and 0.96 for ADAMTS13. The specificityand sensitivity were 83% and 90% for vWF and 90% and 98% forADAMTS13 with significant Youden Index scores of 0.7 and0.8, respectively. Therefore, vWF and ADAMTS13, measuredtogether, could contribute to the understanding of the etio-pathogenesis of stroke and could allow clinicians to monitor theextent of endothelial dysfunction among AIS patients.Thrombospondin (TSP4) is expressed by the endothelial

cells and the smooth muscle cells of large blood vessels andis abundantly expressed in capillaries.34 In vivo studies byMustonen and others have shown increased TSP4 expression inresponse to pressure overload,35 in heart failure, hypertrophy,36

and after ischemia.37 Frolova et al. demonstrated the role of TSP4

in macrophage adhesion,38 migration, and the pro-inflammatorycascade of events. These effects of TSP4 on macrophageenrollment in atherosclerotic lesions indicates that it has afunctional role in the development of lesions and in the proneareas of the subendothelial matrix of the blood capillaries.34 TSP4is a regulator of inflammation. There are no studies that reportTSP4 antigen serum levels in AIS. We found an increase in serumlevels (1.5-fold) of TSP4 in AIS. We hypothesize that the elevatedTSP4 may perhaps be associated with the formation of atheromaand at some stage in thombosis, and it may possibly have aregulatory role in the vasculature during stroke etiopathogenesis.Soluble cell adhesion molecules (sCAMs) are known inflam-

matory mediators and play a key role in the development ofischemic lesions. Several studies reported elevated serum andplasma levels of sICAM1 in ischemic stroke39 and heart disease40

Figure 2. Representative MS/MS spectra of peptides with reporter ions identified for vonWillebrand factor protein (vWF) (A). Insets show the relativeintensities of reporter ions, wherein iTRAQ labels 114 and 115 represent control samples and 116 and 117 represent AIS samples. The vWF wasupregulated 1.8-fold. Panels B and C represent box-and-whisker plots for VWF and ADAMTS13, in which the concentrations of vWF (μg/mL) andADAMTS13 (ng/mL) were measured using ELISA in serum samples in healthy individuals (n = 35) and in AIS cases (n = 50). Themean serum levels ofvWF were significantly elevated, whereas the ADAMTS13 levels were deregulated in AIS compared to the those of controls (p value <0.05). Thestatistical analyses were performed with the Student t test. Values of p <0.05 are statistically significant.

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patients as a suggestive marker of endothelial dysfunction. Wefound the sICAM1 levels to be increased 1.7-fold in AIS serum,which is in concordance with the previous studies. ICAM1 is apotential candidate that needs to be validated in a larger cohort ofserum samples in AIS.Inflammatory Mediators

We found several proteins involved in inflammation to be over-expressed, including C-reactive protein (CRP), myeloperoxidase

(MPO), lipopolysaccharide-binding protein (LBP), and serumamyloid A (SAA). CRP is a peripheral marker of inflammationand atherosclerosis. CRP has been found to be a predictivemarker for ischemic events in patients with intracranial largeartery occlusive disease or transient ischemic attack41 and for therisk of ischemic stroke in the elderly.42 CRP concentration wasalso found to be an independent predictor of survival and earlyneurological deterioration43 after ischemic stroke. Elevated CRP

Figure 3. Representative MS/MS spectra of peptides with reporter ions identified for the S100 calcium-binding protein A7 (S100A7) (A). Panel Bdepicts box and whisker plots of S100A7, wherein serum antigen levels of S100A7 (μg/mL) were estimated by ELISA in control subjects (n = 35) and inacute ischemic stroke subjects (n = 50). The mean serum levels of S100A7 were significantly decreased in AIS cases as compared to those of healthycontrols (p value <0.05).

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levels have been reported to be a related outcome of cerebraltissue injury,44 and, as it is an inflammatory marker, higher CRPlevels were associated with severe stroke. Our study reports CRPto be increased 6.4-fold in AIS. MPO is a peroxidase enzyme thathas been found to be associated with human plaques45,46 andexerts potent proatherogenic effects. MPO−platelet interactionsin the vascular inflammation have been shown to be ofparamount significance in platelet and neutrophil granulocyteactivation and in early episodes of inflammation. Burdess andothers demonstrated evidence of elevated serum MPO levels asearly predictors of coronary artery complications.47,48 MPO-mediated endothelial dysfunction may be a key mechanistic linkbetween inflammation, oxidation, and AIS, and it is regarded asan attractive prognostic biomarker and a potential target fortherapeutic interventions. The current study has shown anincrease in serum MPO levels of 2.8-fold in AIS and requiresfurther verification to establish its clinical implications in AIS(Supplementary Figure S4-A in the Supporting Information).The LBP protein has a high affinity for the LPS receptor CD14,which has a significant role in promoting inflammatory cascadesthrough TLRs.49 The role of LBP in innate immune systemactivation has been previously implicated in patients sufferingfrom atherosclerosis.50 Hence, LBP may have a significant role toplay in innate immune system activation and atherosclerosis

formation leading to AIS. Our experimental results suggest LBPto be increased by 1.9-fold in AIS cases. SAA is an acute-phaseprotein expressed by endothelial cells and is known to modulateplatelet aggregation or adhesion at the endothelial cell surfaceand to promote thrombus formation.51 It is suggested as a usefulbiomarker for the confirmation of atherothrombotic ischemicstroke diagnosis.52 The serum level of SAA in this study wasfound to be 2.5-fold overexpressed in AIS patients.

Neuroprotective Mediators

Adiponectin (APN), a hormone secreted from adipose tissues,contributes to anti-inflammatory activities53 and protectiveeffects.54 APN can significantly increase the cerebral blood flowduring ischemia55 and consequently reduce the cerebral infarctsize through an endothelial nitric oxide synthase (eNOS)-dependent mechanism. It is found to be induced by ischemicand reperfusion insults.56 APN has a cerebroprotective functionthrough its anti-inflammatory action andNF-κB (p65), which is akey component in this process, thereby preventing ischemicinjury through eNOS-dependent mechanisms. Interestingly, theresults of our study provide evidence wherein the serum APNlevels were found to be increased in AIS. APN and its receptorsmight serve as potential molecular targets for ischemic stroketreatment. Insulin-like growth-factor-binding protein 1 (IGFBP1)

Figure 4. Schematic representation of the altered serum proteins that bring about endothelial dysfunction and blood homeostasis in AIS.

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is a circulatory plasma protein belonging to the IGFBP family withan IGFBP and a type I thyroglobulin domain. Decreased levels ofIGFBP1 have been shown to be associated with vascular riskin mice models and cause atherosclerosis by decreasing thebioavailability of nitric oxide (NO) in the aortic endothelium.57

Hence, increased IGFBP1 is known to have a protective functionin CVD patients. We identified it to be overexpressed (1.7-fold) inAIS, which might have a significant role in vascular homeostasispoststroke.Mass spec results clearly illustrate APN and IGFBP1 proteins

to be overexpressed. Therefore, validation of candidate markersAPN and IGFBP1 will further potentiate findings of the recentstudy in AIS. A representative MS/MS spectrum of a peptideidentified from IGFBP1 is shown in Supplementary Figure S4-Bin the Supporting Information.

Membrane Neuromodulatory Proteins

Discs, large homolog 4 (DLG4), also known as postsynapticdensity protein 95 (PSD95), is a member of the MAGUK(membrane-associated guanylate kinases) protein family. PSD-95 interacts with a wide range of cytoplasmic signaling moleculesthat are implicated in excitotoxicity and neurotransmissionregulation. It also promotes the formation of the NMDA-receptor/PSD-95/nNOS complex that further stimulates theproduction of reactive oxygen species (ROS) and higher NOlevels. PSD-95 inhibitors prevent the formation of this NMDA-receptor/PSD-95/nNOS complex and thus reduce NO levels.Hence, PSD-95 inhibitors are promising neuroprotectants withtherapeutic importance in stroke therapy.58 In the current study,PSD-95 was found to be increased by 2.3-fold in AIS patientscompared to healthy controls, as shown in SupplementaryFigure S3-A in the Supporting Information. Validation resultswere not in concordance with the experimental findings(Supplementary Figure S3-B in the Supporting Information),which may be due to several factors including cross-reactingantibodies. It must be emphasized (supported by MS/MSspectrum with reporter ions) that mass spectrometry data clearlyprovide evidence of a DLG4 protein identified with higherabundance in AIS as compared with control serum samples.

Other Secretory Proteins

The S100 calcium-binding protein A7 (S100A7) belongs to theS100 family of proteins. It is a pro-inflammatory protein59

produced by immune cells surrounding the wound area60 and isexpressed in neurons, glia, and astrocytes in the brain.61 Frompublished literature, it is evident that S100A7 has a pivotal role ininflammation in various diseases. Our study reports S100A7 inischemic stroke to be 5-fold decreased. Upon further validation, asignificant decrease in the serum antigen levels of S100A7 wasobserved in patients compared to the levels in controls (0.254 ±0.047 versus 0.130 ± 0.014; p < 0.05) (Figure 3B). Furthermore,the area under the curve was calculated from the ROC analysis,and the ROCAUC was found to be 0.912 (95% CI 0.98−1.00)with a sensitivity of 97% and a specificity of 91%. Youden’s indexfor S100A7, calculated from the sensitivity and specificity valuesobtained from ROC analysis, was 0.88. Recent findings by Qinet al.62 in Alzheimer’s patients showed that the binding of S100A7to its receptors on human platelets increases the expression ofα-secretase activity andADAM10 in platelet cells. S100A7 increasesthe expression of ROS and VEGF and acts through RAGE topromote endothelial cell proliferation.63 Chen et al. has previouslyreported the role of S100A7 in angiogenesis by enhancing VEGFlevels in the rat model of stroke.64 Increased VEGF levels in serumhave been correlated to recovery in AIS poststroke.65 Although the

exact mechanism of action and function of S100A7 in stroke is stillunclear, it may possibly have a role in poststroke recovery.

Known and Novel Downregulated Proteins of AIS

We identified downregulated proteins that are known to beassociated with ischemic stroke and neurodegenerative disorderssuch as gelsolin (GSN) (1.5-fold), S100 calcium binding proteinA9 (S100A9) (1.9-fold), and lactotransferrin isoform-2 (LTF)(1.4-fold). Novel proteins with low serum levels identified in AISinclude caspase-14 (CASP14) (5.9-fold), platelet basic protein(PPBP) (1.4-fold), synaptonemal complex protein 2-like(SYCP2L) (1.9-fold), kielin/chordin-like protein isoform 1(KCP) (1.8-fold), beta-Ala-His dipeptidase (CNDP1) (1.3-fold), insulin-like growth factor-binding protein 3 isoform b(IGFBP3) (1.3-fold), and platelet glycoprotein V (GP5) (1.3-fold). Novel downregulated proteins that are components ofeither ion channels or transporters were also identified, includingprotein unc-79 (UNC79) (1.9-fold), small conductance calcium-activated potassium channel protein 3 isoform b KCNN3 (4.5-fold), and putative sodium-coupled neutral amino acid trans-porter 10 isoform (SLC38A10) (1.3-fold). These proteins mayserve as modulators mediating neuron excitotoxicity followingbrain injury. Gelsolin (GSN), is an actin-binding and calcium-binding protein mediating the disassembly of actin filamentsand activity of calcium channels.66 It exists in both forms−intracellular cytoplasmic (cGSN) and extracellular secreted(pGSN). pGSN is reported to be secreted in blood through aninterstitial fluid of the extracellular matrix from muscle tissue andalso known to be present in CSF. GSN is an inflammatorybiomarker with decreased serum/plasma levels associated invarious diseased pathologies including various animal modelsof stroke.66,67 Recently, GSN knock-out mice studies haveestablished its neuroprotective effects following stroke.68 Ourresults have shown decreased serum levels (1.5-fold) of GSN inAIS, which further confirm findings of previous studies in theliterature. Therefore, GSN may modulate the inflammatoryresponse in acute phase of stroke, thereby offering protectionagainst the inflammation-associated degeneration near theinfarct region post-stroke and perhaps may serve as a potentialdrug target for protection against neurodegeneration after AIS.Monitoring serum levels along with other inflammatorymarkers could have implications for predicting better prognosisof disease.Protein Unc-79 (UNC79) is an associated component of

the NALCN sodium leak channel complex, a cation channelactivated by both neurotensin (NTS) and neuropeptide,substance P, which further modulates neuronal excitability.A report from Boxun et al. further substantiates the involvementof the UNC79-UNC80-NALCN cation channel complex formaintaining extracellular calcium levels in the neurons, whichhelp to sustain background current and neuronal excitability.69

UNC-79 is also known to be a vital component of ion channel forvigorous circadian locomotor rhythms in Drosophila;70 however,its clinical implication in ischemic stroke remains elusive.Our study has shown significantly reduced levels (1.9-fold) ofUNC79 protein in pooled serum samples of AIS patients(Supplementary Figure S4-C in the Supporting Information).The role of UNC79 in stroke pathogenesis needs to beelucidated, and further validation in larger clinical samples iswarranted.

Apolipoproteins as Risk Markers of AIS

Recent targeted approaches using mass spectrometry to profileapolipoproteins and their isoforms in various pathologies such as

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neurological, cerebrovascular, and cardiac disorders have gainedsubstantial importance. Additionally, increased levels of theseproteins may be associated with a higher risk for cardiovasculardisease and stroke. Our study identified altered levels of severalapolipoproteins that include ApoA4 (1.6-fold), ApoC1 (1.4-fold), ApoC2 (1.4-fold), ApoC4 (1.3-fold), and ApoE (1.5-fold).Apoliopoprotein IV (ApoA4) is a key component of HDL and

chlyomicrons. It has a major role in chylomicrons and VLDLsecretion and catabolism. It is a potential activator of lipoproteinlipase and lecithin-cholesterol acyltransferase. Altered levels ofApoA4 and its associated lipoprotein abnormalities in AIS are notwell understood.We identified reduced serum levels (1.6-fold) ofApoA4 in AIS from LC−MS/MS analysis. Hence, it is a novelcandidate marker that requires further validation to elucidate itsrole in disease progression post stroke. We suggest that it may behelpful in predicting risk of stroke along with other clinicalobservations such as lipid profile and risk factors. ApolipoproteinE (ApoE) is an abundant protein in blood, which participatesin the transportation of lipids and cholesterol by interactingthrough LDL receptors and other proteins. Recent studieshave revealed that altered binding capability of APOE isoformsto lipids and receptors resulted in high risk for pathologiesassociated with neurological, cerebrovascular and cardiovasculardisorders.71−75 Low serum levels of ApoE have been found inAlzheimer’s disease patients.76 Limited studies report alteredApoE levels in body fluids of subarachnoid hemorrhage77 andAIS patients,78 but its association with stroke requires furtherinvestigation. The current study observed elevated serum levels(1.5-fold) of ApoE in AIS. The utility of ApoE as a biomarker forAIS needs to be validated in a larger cohort. Altered plasma levelsof lipoproteins may have serious implications in prognosis, riskstratification, effective treatment, and management of stroke.

■ CONCLUSIONS

The present effort is aimed at identifying differential proteins ina high throughput manner to encompass a deep serum profileof stroke and correlate it with stroke pathophysiology in asystematic manner. The identification of differentially expressedproteins in serum samples of AIS patients provides a platform fordeveloping diagnostic assays as alternatives to imaging. To thisend, we propose a panel of candidate protein markers in serumfor stroke diagnosis, as a single proteinmarker may not have suffi-cient diagnostic sensitivity. We suggest that vWF, ADAMTS13,and S100A7 could be established as candidate markers for thediagnosis of AIS that, together with other clinical parameters,could assist in prompt diagnosis along with existing imagingmodalities at the bedside. Although we validated a few candidateproteins in a relatively small sample size, validation on a largecohort across different ethnic groups is warranted. Candidateprotein markers that need further validation include adiponectin,insulin-like growth factor binding protein 1, myeloperoxidase,intercellular adhesion molecule-1, apolipoprotein A-1V, apoli-poprotein E, gelsolin, and protein unc-79 homolog. The currentstudy provides a resource of potential candidate protein bio-markers to understand the role of various pathways in strokepathophysiology and to identify therapeutic targets, therebyimproving the acute management of stroke patients.

■ ASSOCIATED CONTENT

*S Supporting Information

Supplementary Table 1: a complete list of proteins identifiedfrom this study. Supplementary Table 2: list of all the peptides

identified from acute ischemic stroke. Supplementary Figure S1:normalization of serum proteins (unbound depleted fraction)from AIS and control (replicates) after Agilent MARS-14 serumdepletion, confirmed by 10% SDS-PAGE. SupplementaryFigure S2: gene-ontology-based classification of proteins.Cellular-process- (A) and subcellular-localization- (B) basedclassification-identified proteins used in this study. Supplemen-tary Figure S3: representative MS/MS spectra for discs, largehomolog 4. Supplementary Figure S4: representative MS/MSspectra for myeloperoxidase, insulin-like growth-factor-bindingprotein 1, and protein unc-79 homolog. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors

*D. Nagaraja E-mail: dr.dnagaraja@gmail.com. Tel: 91-9448359959. Fax: 0836-2748400.*R. Christopher E-mail: rita.nimhans@yahoo.com. Tel: 91-080-26995162. Fax: 91-080-26564830.Funding

This work was funded by the Department of Biotechnology(DBT), the Government of India (grant reference no. BT/01/COE/08/05).Notes

The authors declare no competing financial interest.

■ ABBREVIATIONSAIS, acute ischemic stroke; MARS, multiple affinity removalsystem; LC−MS, liquid chromatography mass spectrometry;SCX, strong cation exchange chromatography; LVD, large vesselstroke

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