Proteomic Analysis of Human Fetal Atria and VentricleZhen Qi Lu,† Ankit Sinha,‡ Parveen Sharma,† Thomas Kislinger,*,‡,! and Anthony O. Gramolini*,†,§,!

†Department of Physiology, ‡Medical Biophysics, Faculty of Medicine, and §Heart and Stroke/Richard Lewar Centre of Excellent forCardiovascular Research, University of Toronto, Toronto, Ontario, Canada!Princess Margaret Cancer Center, Toronto, Ontario, Canada

*S Supporting Information

ABSTRACT: In this study we carried out a mass spectrometry-basedproteome analysis of human fetal atria and ventricles. Heart protein lysateswere analyzed on the Q-Exactive mass spectrometer in biological triplicates.Protein identi!cation using MaxQuant yielded a total of 2754 atrial proteingroups (91%) and 2825 ventricular protein groups (83%) in at least 2 of the 3runs with "2 unique peptides. Statistical analyses using fold-enrichment (>2)and p-values (#0.05) selected chamber-enriched atrial (134) and ventricular(81) protein groups. Several previously characterized cardiac chamber-enriched proteins were identi!ed in this study including atrial isoform ofmyosin light chain 2 (MYL7), atrial natriuretic peptide (NPPA), connexin 40(GJA5), and peptidylglycine alpha-amidating monooxygenase (PAM) for atria,and ventricular isoforms of myosin light chains (MYL2 and MYL3), myosinheavy chain 7 (MYH7), and connexin 43 (GJA1) for ventricle. Our data wascompared to in-house generated and publicly available human microarrays,several human cardiac proteomes, and phenotype ontology databases.KEYWORDS: Bioinformatics, chamber speci!city, fetal tissue, mass spectrometry, ventricle, Q-exactive

! INTRODUCTIONCardiovascular disease is the leading cause of deaths world-wide.1 There is a continuing need for a greater understanding ofcardiovascular physiology to provide e"ective methods inimproving the diagnostic and therapeutic outcomes ofcardiovascular disease. The human heart is composed of 4chambers, with two upper atrial chambers and two lowerventricular chambers. The atria and ventricles are responsiblefor di"erent functions within the heart. The atria initiateselectrical events through its pacemaker potential, primes theventricles, and are enriched with numerous golgi andendoreticular (ER) proteins,2 whereas the ventricles providemuscular contraction for pumping blood over a large distancein the body and contain an abundance of mitochondrialproteins that provide energy for contractions.2 Limited accessto healthy human cardiac tissues has restricted previous studiesto focus on the transcriptomic and proteomic analysis of themouse heart.3,4 Although mouse studies have provided essentialinsight into chamber di"erences, the relative protein expressionpatterns and di"erences are di#cult to extrapolate and apply tohuman hearts. Initial studies examining human cardiac tissueswere mostly conducted via microarray analysis of the adulthuman heart, and studies were performed on available diseasedadult tissues, primarily in ventricles.5$8

Detailed, high accuracy global proteomic analyses of heartchambers has been made possible with advancements ininstrumentation and computational tools. The aim of this studywas to characterize the di"erences between the human cardiac

chambers. To this extent we carried out comparative proteomicand transcriptomic analysis of human fetal atria and ventricularsamples, which identi!ed chamber-enriched and chamberspeci!c proteins. We further compared the fetal microarraydata generated in this study to available adult data to identifyexpression di"erences between fetal and adult tissues. Publiclyavailable cardiac proteome and phenotype databases alsoprovided further validation of the novel chamber-enrichedproteins.

! MATERIALS AND METHODS

Tissue Collection

The human fetal samples were obtained from electiveterminations of normal pregnancies via the Health CenterBiobank Program (Hospital for Sick Children ResearchInstitute, REB protocol #1000029263). Written consent wasobtained from the patients and heart tissues were sent forbanking (REB protocol #1000011232). Further details ofbiobanking and surgical procedures were outlined in Strandberget al.9 Fetal hearts of 17 to 23 weeks of age were obtained andstored frozen in $80 °C until mass spectrometry.Sample Preparation for Mass Spectrometry

The frozen hearts were thawed and homogenized in 50% (v/v)2,2,2-tri$uoroethanol (TFE) in phosphate-bu"ered saline using

Received: July 26, 2014

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a dounce homogenizer for 25 strokes on ice. Total proteinsfrom 15 mg of tissue were denatured, reduced, alkylated, anddigested into peptides as previously described.10,11 Brie$y, afterhomogenization, the lysate was incubated for 120 min at 60 °C,and cysteines were reduced using 5 mM dithiothreitol at 60 °Cfor 30 min and subsequently alkylated using 25 mM iodoaceticacid. Tissue lysate was then diluted !ve times (v/v) using 100mM ammonium bicarbonate bu"er and digested using 5 !g ofmass spectrometry grade trypsin (Thermo). Digested peptideswere desalted using OMIX C18 pipet tips and dried undervacuum centrifugation. The lyophilized peptide pellet wasredissolved in liquid chromatography$mass spectrometry(LC$MS) grade water/0.1% formic acid, and peptideconcentration was determined using NanoDrop spectropho-tometer (Thermo Scienti!c). Peptide mass spectrometry wasperformed exactly as described previously.10 Mass spectrometerand MaxQuant parameters can be found in the Analysis andDocumentation of Peptide and Protein Identi!cations(Supporting Information).

Mass Spectrometry Data Search

Raw !les were searched using MaxQuant version 1.4.1.212 usingUniProt complete human proteome protein sequence database(version: 2012$07$19; number of sequences, 20 232). Targetdecoy was utilized to control for false discovery rate (FDR) ofthe matched peptide spectra, with FDR set to 1%. “Proteingroups” consist of proteins grouped together that cannot beseparated with commonly shared peptides. Protein groupsidenti!ed with two or more peptides were used for subsequentanalysis (razor plus unique). The un!ltered list of all identi!edprotein groups can be found in Table S1, SupportingInformation.

Statistics and Data Analysis

The gene ontology (biological processes) for each group ofinterest was computed using the DAVID BioinformaticsDatabase (http://david.abcc.ncifcrf.gov).13 The overrepre-sented GO terms (p-value # 0.05 ranked by the number ofproteins in each category) were reported to characterize large-scale biological di"erences between the chambers.Venn diagrams were generated using the Venn Diagram

Plotter v. 1.5.4798.30840 written by the Department of Energy(PNNL, Richland, WA) and VennDIS v.1.0 from Dr.Kislinger’s laboratory. Volcano plots for fold-changes and p-values as well as all bioinformatic analyses were done in R withstandard packages. Protein groups with fold-change > 2 and p-value # 0.05 by student’s t test were considered enriched in achamber, and proteins with label-free quanti!cation (LFQ)intensities of value 0 were replaced with 1 to compute the fold-changes. Heatmaps were generated using Java TreeViewv.1.1.6r2 via k-mean clustering (1000 runs) using Clusterv.3.0. The most optimal number of clusters was computed bythe Elbow Analysis in R that maximized the percent ofvariances explained with the least number of clusters. Wefurther compared the atrial and ventricular proteome from thisstudy to recently published Human Proteome Map database forhuman fetal whole heart proteome (http://www.humanproteomemap.org)8 and the ProteomicsDB for adultventricular proteome (https://www.proteomicsdb.org).14 Pro-teins reported by other studies were compared to the proteingroups from this study, and a match was reported if the proteinwas identi!ed within a protein group.

Microarray Analysis

Cardiac tissue was also analyzed by a parallel microarrayanalysis. Tissue was immediately stored in RNAlater stabiliza-tion reagent (Qiagen). Total mRNA was extracted using 1 mLof TRIzol and 200 !L of chloroform as described previously.15

Microarray analysis was carried out in triplicate by theUniversity Health Network Microarray Facility (Toronto)using the A"ymetrix Human 1.0 st v1 microarray chips. Thehuman adult atrial (samples GSM80654, GSM80655, andGSM80698) and ventricular (samples GSM657, GSM658, andGSM659) microarrays were obtained from the GeneExpression Omnibus (http://www.ncbi.nlm.nih.gov/geo)16 onthe same microarray chip. The raw data from in-house andpublicly available (series number GSE3526) microarrays wererobust multiarray (RMA) normalized, and only genes withindividual probe intensities greater than three times the globalmedian intensity were reported. The processed microarray datawas then cross-referenced with the proteome data, via genemapping.Both atrial and ventricular-enriched gene products, clustered

according to fetal proteome and transcriptome, were searchedfor known cardiac phenotypes in humans against the HumanGenetic Association Database (http://geneticassociationdb.nih.gov)17 and known phenotypes in mouse against the MouseGenome Informatics (http://www.informatics.jax.org).18

Human to mouse orthologues were generated using theBiomart database (http://central.biomart.org).Proteins with both high protein and transcript expressions

from the ventricles were compared to the Human ProteomeMap. Cardiac cell expression (in cardiomyocytes or non-cardiomyocytes) of these ventricular-enriched proteins identi-!ed in both studies was con!rmed by immunohistochemicalstaining of adult ventricular sections from the Human ProteinAtlas v.12 (http://www.proteinatlas.org).19

! RESULTS

Assessment of Proteome Data Quality

To interrogate chamber speci!c proteomes, human fetal cardiacchambers were obtained and prepared for mass spectrometryanalyses as previously described (Figure 1). Atrial andventricular biological triplicates were analyzed to observebiological variability, and subsequent analyses used LFQintensities to adjust for systematic biases between runs.Following mass spectrometry, a total of 32 441 atrial and 32816 ventricular peptides were identi!ed. For the subsequentanalysis, we selected only those proteins with at least twopeptides, resulting in a total of 2754 protein groups in the atriaand 2825 in the ventricles. To select for “chamber-enriched”protein groups, we calculated the LFQ intensity ratios as ameasure of fold-abundance (>2) and signi!cance by p-values(#0.05). Using both parameters as a prioritization strategy, weidenti!ed 134 atrial-enriched protein groups (Table S2,Supporting Information) and 81 ventricular protein groups(Table S3, Supporting Information). The key experimentalsteps are summarized in Figure 1.A comparison of the data generated by the triplicate

biological samples showed that 91% of protein groups (2518/2754) were identi!ed in at least two atrial biological replicates,whereas 83% (2343/2825) of protein groups were identi!ed inat least two ventricle replicates as shown in Figure 2A,B,respectively.

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We next compared the atrial and ventricular proteinsidenti!ed in this study to two previously published humanproteomics studies (Figure 2C); the data from Wilhelm et al.contains a human adult ventricular proteome14 and the datafrom Kim et al. contains the proteome of human fetal wholeheart.8 Wilhelm et al. identi!ed 4031 unique proteins and Kimet al. identi!ed a total of 9665 unique proteins. The gene nameswere compared between the two previously publishedproteomes and the 3012 proteins identi!ed in this study ("2unique peptides). We identi!ed 898 proteins that were notpreviously identi!ed in the adult ventricle and detected 29 fetalproteins not found in either of the other two large scale studies.The 465 proteins only identi!ed in the adult ventricles datashowed GO terms enriched in immune response (n = 49) anddefense response (n = 38). Comparison of the 2754 fetal atrialand 2825 ventricular protein groups from this study showed187 uniquely identi!ed protein groups in the atria, 258 in theventricles, and 2567 in common (Figure 2D).

Enriched Atrial and Ventricular Protein GroupsA detailed analysis of the atrial and ventricular protein groupsexpression levels by volcano plot is shown in Figure 3 for 3012unique protein groups (134 atrial enriched, 81 ventricularenriched, and 2797 common). Within the chamber-enrichedgroups we observed enrichment of the previously known atrial-enriched proteins such as peptidylglycine alpha-amidatingmonooxygenase (PAM), natriuretic peptide A (NPPA), andmyosin light chain 2, atrial isoform (MYL7),20$22 while theventricular-enriched proteins included myosin light chain 2,ventricular isoform (MYL2), myosin light chain 1, ventricularisoform (MYL3), and myosin heavy chain 7 (MYH7).20

Although connexin 40 (GJA5) and connexin 43 (GJA1)23 werealso detected in this study and were commonly known to beenriched in the atria and ventricles, respectively, they did notmake statistical signi!cance cuto"s. A complete list of chamber-enriched protein groups can be found in Tables S2 and S3,Supporting Information.Chamber Enriched ProcessesIn order to gain insight into the biological process enriched ineach chamber, we grouped the chamber-enriched proteingroups into gene ontology terms (134 atrial enriched, 81ventricular enriched, and 2797 common). Top atrial biologicalprocesses showed a signi!cant enrichment (p # 0.05) ofintracellular transport (n = 13). Enrichment of vesiculartransport may be associated with greater cellular communica-tion and signaling. Ventricular-enriched proteins showedoverrepresented biological processes of oxidation$reduction(n = 9) and muscle contraction (n = 8). Muscular contractileproteins generate the force required for systematic circulation.Oxidation reduction GO terms were mainly involved inmitochondrial processes and the electron transport chain tosupply energy to the ventricles. Lastly, the common proteinsshowed overrepresented GO terms from the atria and ventricleswith general biological processes of protein localization (n =275), translation (n = 194), and other enriched GO terms fromboth chambers. The full list of biological processes can befound in Table S4, Supporting Information.Bioinformatic Validation via TranscriptomicsTo gain a better understanding of human cardiac chambers andtranscript levels we compared the chamber-enriched proteins toan in-house human fetal heart microarray generated as part ofthis study. A total of 8853 genes were identi!ed by microarraypro!ling with a probe intensity 3-fold above the global median.Gene products from the transcriptome analyses weredetermined to be di"erentially expressed if the p-value was#0.05 between the biological triplicates. The proteome andtranscriptome expression of all 2456 gene products are shownin Figure 4A indicating di"erent patterns of transcript andactual protein expression. We then examined the transcript andprotein expressions of only chamber-enriched proteins from theproteome level (134 atrial plus 81 ventricular proteins) asshown in Figure 4B. Transcriptomic and proteomic data wereseparated into three di"erent groups, namely, high protein andtranscript expression, high protein and low transcriptexpression, and subthreshold transcript expression. We furtherranked the high protein and transcript expression group by thesum of log10 LFQ intensity ratios and log2 fetal probe intensityratios for both the atria and the ventricles, whereas the highprotein and low transcript expression group and subthresholdtranscript level group were ranked by log10 LFQ intensityratios. The complete ranked list can be found in Table S5 for

Figure 1. Overall experimental design and analysis $owchart. Shownare the key experimental steps for sample preparation, proteinidenti!cation, and statistical analysis of chamber-enriched proteins.Total cardiac tissues were lysed, proteins were digested, and peptideswere separated on the Q-Exactive mass spectrometer with threebiological repeats. Protein identi!cations were performed viaMaxQuant v. 1.4.1.2 against the UniProt complete human proteomedatabase with a false discovery rate of less than 1% at both peptide andprotein levels. The number of spectra, matched peptides, and proteinsidenti!ed for both chambers are shown above. All 2754 atrial and 2825ventricular protein groups, having at least two peptides, were subjectedto further statistical enrichment via fold-enrichment by mean LFQintensities (>2) and p-value (#0.05) that resulted in 134 atrial and 81ventricular protein groups.

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Figure 2. Human cardiac chamber proteomics. (A) The Venn diagram shows the number of overlapping protein groups between the atrial biologicaltriplicates (atrium 1, 2, and 3) with at least two peptides. The number of protein groups in each of the categories is indicated by the color matchednumbers. In total, 2754 protein groups were identi!ed in the atria. (B) The Venn diagram shows similar representation of the results, and a total of2825 protein groups were identi!ed in the ventricles. The atrial and ventricular biological triplicates showed high reproducibility. (C) The Venndiagram compares cardiovascular proteome between Wilhelm et al., Kim et al., and fetal atrial and ventricular data sets from this study. All 898proteins from this study were not present in the adult data and 465 proteins from the adult heart were absent in both fetal heart proteome. Thisstudy detected 29 proteins unidenti!ed in the two other proteome studies. (D) Comparison of fetal atrial and ventricular data showed 187 atrialunique, 258 ventricular unique, and 2567 common protein groups with more than two peptides identi!ed in this study.

Figure 3. Protein expression across the human atrial and ventricular samples. Volcano plot of the total atrial and ventricular protein groups with atleast two peptides plotted with p-value against the ratio of LFQ intensities for atria over ventricles. All 134 protein groups enriched in the atria arealong the positive x-axis, and all 81 ventricular-enriched protein groups are along the negative x-axis. Speci!cally, atrial-enriched protein groups areshown in blue and red for ventricles with fold-enrichment in a chamber > 2 and p-value # 0.05. Previously known chamber-enriched proteins aremarked by *. All 2797 protein groups with p-value > 0.05 are indicated in gray.

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the atria and Table S6 for the ventricles (SupportingInformation). The 134 atrial-enriched proteins were groupedinto 46 gene products with high protein and transcriptexpression, 69 high protein and low transcript expression, and19 subthreshold at the transcript level, whereas the 81

ventricular-enriched proteins were divided into 45 high proteinand transcript expression, 24 high protein and low transcriptexpression, and 12 subthreshold at the transcript level. The lackof correlation between transcript and protein expressions in thelow and subthreshold transcript level groups was most likely

Figure 4. Correlation of proteomic data with microarray and cardiac phenotype ontology. (A) Scatterplot of microarray log2 probe intensity ratioversus proteome log10 LFQ intensity ratio of atria over ventricles for all 2456 gene products. Each dot represents a protein identi!ed in this studywith at least two peptides. (B) Scatterplot of only chamber-enriched proteins in the atria or ventricles at the proteome level. Blue dots indicatesigni!cance at the transcript level (p-value # 0.05) in the atria, red for the ventricles, and nonsigni!cant in gray. Highly signi!cant enriched proteinsare labeled by the respective gene symbols, and previously known chamber markers are indicated by *. (C) Heatmap of 134 atrial-enriched proteinsof the human fetal proteome, fetal microarray, and adult microarray. There are 46 high protein and transcript expression in the atria with highintensities between fetal proteome and microarray, 69 high protein but low transcript expression, and 19 subthreshold transcript level. The humanand mouse cardiac phenotypes of the three groups were also shown as either present or absent. Proteome is depicted by red to green andtranscriptome by yellow to green for high to low. (D) The same analysis and heatmap as that in panel C but for the 81 ventricular-enriched proteins(45 high protein and transcript expression, 24 high protein but low transcript expression, and 12 subthreshold detection in fetal microarray).

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attributed to di"erence in protein and transcript stability,modi!cation, and rates of transcription versus translation.24

The fetal data was further compared to the adult microarrayfrom the Gene Expression Omnibus database by gene symbols,

with 12 253 genes having a probe intensity 3-fold above theglobal median. In order to better visualize the data, we clusteredthe atrial- and ventricular-enriched proteins according to thefetal proteome, fetal microarray, and adult microarray. Heatmap

Figure 5. Ventricular-enriched protein expression in Human Protein Atlas. Immunohistochemical (IH) staining of ventricular proteins with highprotein and gene expressions in both cardiomyocyte and noncardiomyocyte expression groups with the proteins labeled. In total, 32 proteinsexhibited cardiomyocyte and one with noncardiomyocyte speci!c patterns in the heart. Human heart sections were obtained from adult ventricularbiopsies in online database Human Protein Atlas. IH staining is ordered based on fold-changes at the proteome and transcript levels.

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and clustering for the 134 atrial-enriched proteins with theprotein and transcript data are shown in Figure 4C, and 81ventricular-enriched proteins are shown in Figure 4D.In order to determine which proteins have previously been

implicated in disease, we searched our chamber-enrichedproteins against the Human Association Genomic Databaseand Mouse Genome Informatics Database. This identi!ed that33 of the 134 atrial-enriched proteins were previouslyassociated with a human cardiovascular phenotype and 22with a mouse cardiovascular phenotype. Similarly, 29 out of the81 ventricular-enriched proteins had a previously associatedhuman cardiovascular phenotype and 20 in mouse. Informationabout previous cardiac phenotypes can be found in Tables S5and S6, Supporting Information. Analysis of 100 randomlyselected proteins from the chamber-enriched proteins showedthat on average 47% of the proteins had previous cardiovascularphenotypes in either human or mouse. In contrast, randomlyselected proteins from the 3012 proteins identi!ed in this studyshowed that on average only 33% of the proteins had previouscardiovascular phenotypes, with a p-value of 0.048 compared tochamber-enriched proteins.Cell-Type Speci!c Identi!cations

Finally, we assessed cellular origin of the proteomic signals byexamining chamber-enriched proteins found in fetal ventricularproteome data set against stained human heart sections in theonline database, Human Protein Atlas. This imaging allowed usto visualize protein expression within speci!c cell types in theheart, namely, cardiomyocytes and noncardiomyocytes. Sincethis immunohistochemistry imaging database has ventricularbiopsy sections, only ventricular proteins were considered. Outof the 45 ventricular proteins examined, all proteins hadantibody staining in heart sections, and 32 proteins showedspeci!c staining for cardiomyocytes and included immunohis-tochemical staining for all proteins in Figure 5. Similarly, weidenti!ed one protein (TNS1) to have noncardiomyocytespeci!c expression. Five ventricular-enriched proteins(ADHFE1, ATP1A3, GPD1L, PYGL, and PYGM) showedno clear heart staining pattern, and 7 proteins (CD36, LDHD,MACROD1, PDLIM1, PFKP, STRN, and SYNPO2) showedstaining in both cardiomyocytes and noncardiomyocytes.

! DISCUSSIONSystem approaches by proteomics and transcriptomics generatea large amount of data in a relatively short period of timeproviding potential insights into molecular pathways anddi"erences in protein abundance between multiple biologicalsamples and tissues. The accuracy of these large-scale studieshas increased tremendously with advances in instrumentationand computational tools. This study is the !rst to report acomprehensive characterization of the human fetal atrial andventricular proteome and transcriptome to identify chamber-enriched and speci!c proteins. All protein samples were run onthe Q-Exactive tandem mass spectrometer and showed highreproducibility between all three biological repeats (Figures2A,B) giving us greater con!dence in the data. The datashowed high reproducibility between the biological triplicateswith a slightly greater degree of reproducibility in the atriacompared to the ventricles. This might be attributed to the factthat the ventricles have greater abundance of structural proteinsthat might mask less abundant proteins.The majority of the atrial and ventricular proteome data from

this study were also detected in other fetal heart proteomes, but

the relative protein expression between the chambers examinedin this study help to broaden the scope of the previouslyestablished data (Figure 2C). Identi!cation of several novelproteins in this study might be attributed to di"erences inorigin and handling of sample, sample preparation, and massspectrometer utilized. Adult data showed an enrichment forimmune and defense responses attributed most likely to acombination of innate body defense mechanisms and the factthat the source of tissues in previous experiments was frompatient autopsies; highlighting the di#culties of obtaininghealthy human adult cardiac samples for studying chamberdi"erences. Finally, we identi!ed slow skeletal troponin I(TNNI1) and fetal form of tropomyosin (TPM1) in the massspectrometry data at high levels, which serve as excellenthallmarks of fetal hearts.25,26

The data generated in this study allowed us to examinechamber-enriched proteins and proteins commonly sharedbetween the two chambers (Figure 3). This study reportedmany novel chamber-enriched proteins. In the atria, the mostsigni!cantly enriched novel protein by p-value is the WWdomain binding protein 11 (WBP11), a protein with a role innucleocytoplasmic shuttling and pre-mRNA splicing,25,27 whilethe highest fold-di"erence expression in the atria is G proteinalpha activating activity polypeptide O (GNAO1). GNAO1plays an important role in cancer progression28 and regulationof intracellular Ca2+.29 In the ventricle, the protein that has themost enriched by p-value is ribosomal protein L3-like (RPL3L),with only one large scale study showing its expression inskeletal and cardiac muscles.30 By fold-di"erence, the mostdi"erentially expressed novel ventricular protein is myosin lightchain 5 (MYL5), a protein that binds Ca2+ ions and is involvedin muscle contraction.31 For a complete list of di"erentiallyexpressed proteins in the atria and ventricles, please refer toTables S2 and S3, Supporting Information, respectively.By cross-referencing publicly available adult heart chamber

microarray data we assessed expression di"erences between thefetal and adult hearts and identi!ed di"erences that occur inexpression during development. Among the fetal atrial highprotein and transcript expression group, 27 out of 46 proteinsalso showed high enrichment in the adult with p-value # 0.05.Of interest, atrial natriuretic factor (NPPA) was the mostdi"erentially expressed in the adult atrial data. Bonemorphogenetic protein 10 (BMP10) and PAM also showedhigher expression in the adult atrial data where BMP10 hasbeen previously shown to be involved in cardiac growth andchamber maturation;32 and PAM also showed higherexpression during development, but its function is stillunknown. The adult ventricular data showed greaterdiscordance compared to the fetal data with only 12 out of45 proteins (with high protein and transcript expressions)enriched in the adult ventricles. The two proteins with thehighest expression in the adult ventricles were structuralproteins, MYL2 and MYL3, essential for mechanical forcegeneration and cross-bridge formation. The similarity ofchamber-enriched protein expressions between the adult andfetal atria suggested a smaller degree of modi!cation duringdevelopment compared to the ventricles.Proteins with cardiac phenotypes from both chambers

showed an enriched biological processes of cell adhesion.Atrial-enriched proteins with cell adhesion GO terms includeATPase slow twitch 2 (ATP2A2), protein tyrosin kinase 7(PTK7), collagen type II (COL2A1), collagen type XIV(COL14A1), !bronectin 1 (FN1), !bulin 5 (FBLN5), neural

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cell adhesion molecule 1 (NCAM1), and periostin andosteoblast speci!c factor (POSTN); and ventricular proteinsinclude CD36, angiotension (AGT), armadillo repeat genedeletes in velocardiofatal syndrome (ARVCF), cadherin type 2(CDH2), catenin alpha 3 (CTNNA3), collagen type XV(COL15A1), plakophilin 2 (PKP2), sorbin and SH3 domaincontaining 1 (SORBS1), and thrombospondin 4 (THBS4).These proteins and other chamber-enriched proteins provideimportant candidates for understanding chamber di"erencesduring cardiac diseases. Furthermore, seven of the atrial-enriched gene products (MYL7, MYH6, TBX20, NPPA, FN1,TAGLN, and BMP10) with known cardiac phenotypes havebeen linked to congenital heart diseases including defects incardiac looping, septation, and heart morphology.33$38

Hundreds of novel chamber-enriched proteins identi!ed inthis study provide new avenues for cardiac chamber and diseasespeci!c research. We compared the high protein and transcriptexpression group against known human and mouse cardiacphenotype databases. The results showed that a signi!cantproportion of the proteins have been previously linked tocardiac abnormal phenotypes, but the phenotype in a speci!cchamber is not well explained especially in the atria. Themajority of proteins do not have any known cardiac phenotypesassociated with it, and hence, we provide increased knowledgefor proteins with new areas of chamber speci!c research. Lastly,we checked expression of the 45 ventricular high protein andtranscript expression proteins in human heart sections stainedby immunohistochemistry for expression exclusively to thecardiomyocytes or noncardiomyocytes. There was signi!cantlymore cardiomyocyte speci!c proteins compared to non-cardiomyocytes, a result consistent with observations that themajority of the myocardium is composed of cardiomyocytes, atleast by volume.

! CONCLUSIONSUtilizing mass-spectrometry-based proteomics to examinehealthy human fetal atrial and ventricular tissues identi!ed134 statistically enriched atrial and 81 statistically enrichedventricular protein groups. By further incorporating genomicstudies, this article identi!ed 46 atrial and 45 ventricularproteins with high protein and transcript expression. Thesenovel chamber-enriched proteins establish a reference forunderstanding disease mechanisms and for identifying speci!cproteins that may be involved in cardiac diseases that target aparticular chamber. The analysis of comprehensive proteomicdata with clinical phenotype and advanced genomic informa-tion will prove invaluable in understanding cardiac develop-ment and disease. Future experiments should focus on verifyingthe novel proteins in vivo to gain a better insight of how theseproteins are contributing to chamber speci!city and fordi"erent chamber functions.

! ASSOCIATED CONTENT*S Supporting Information

Analysis and documentation of peptide and protein identi-!cations. Table S1. List of total protein groups identi!ed byMaxQuant. Table S2. List of atrial-enriched protein groups bysigni!cant fold-change and p-value. Table S3. List ofventricular-enriched protein groups by signi!cant fold-changeand p-value. Table S4. List of biological processes for atrial-enriched proteins, ventricular-enriched proteins, and commonlyexpressed proteins. Table S5. List of di"erent atrial-enriched

protein clusters for high protein and transcript expression, highprotein but low transcript expression, and high protein butsubthreshold transcript expression. Table S6. List of di"erentventricular-enriched proteins clusters for high protein andtranscript expression, high protein but low transcriptexpression, and high protein but subthreshold transcriptexpression. This material is available free of charge via theInternet at http://pubs.acs.org.

! AUTHOR INFORMATIONCorresponding Authors

*(A.O.G.) Phone: +1-416-634-8813. Fax: +1-416-978-4940. E-mail: anthony.gramolini@utoronto.ca.*(T.K.) Phone: +1-416-581-7627. Fax: +1-416-581-7629. E-mail: thomas.kislinger@utoronto.ca.Notes

The authors declare no competing !nancial interest.

! ACKNOWLEDGMENTSThe authors thank Dr. Peter Backx for valuable discussions,Drs. Bob Hamilton and Vinayakumar Siragam for expertsurgical assistance and valuable discussions, and VladimirIgnatchenko for expert assistance with the bioinformaticsanalyses. This project was funded by the Canadian Institutesof Health Research (MOP-106538; GPG-102166), Heart andStroke Foundation of Ontario (T-6281 and NS-6636), and theOntario Research Fund$Global Leadership Round in Ge-nomics and Life Sciences (GL2-01012) to A.O.G. and T.K.A.O.G holds the Canada Research Chair in CardiovascularProteomics and Molecular Therapeutic. T.K. holds the CanadaResearch Chair in Proteomics in Cancer Research. Z.Q.L.received an Ontario Graduate Scholarship in Science andTechnology. A.S. was supported by a Department of MedicalBiophysics Excellence Award and by a Kristi Piia CALLUMMemorial Fellowship.

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