Development and Validation of a Protein-Based Risk Score for Cardiovascular Outcomes Among Patients with Stable Coronary Heart Disease

Home , Adapter molecule crk, Adenylate kinase 1, ALCAM, Angiostatin, ANGPT2, ANGPT4

Supplementary Online Content

Ganz P, Heidecker B, Hveem K, et al. Development and validation of a protein-based risk score for cardiovascular outcomes among patients with stable coronary heart disease. JAMA. doi: 10.1001/jama.2016.5951

eTable 1. List of 1130 Proteins Measured by Somalogic’s Modified Aptamer-Based Proteomic Assay eTable 2. Coefficients for Weibull Recalibration Model Applied to 9-Protein Model eFigure 1. Median Protein Levels in Derivation and Validation Cohort eTable 3. Coefficients for the Recalibration Model Applied to Refit Framingham eFigure 2. Calibration Plots for the Refit Framingham Model eTable 4. List of 200 Proteins Associated With the Risk of MI, Stroke, Heart Failure, and Death eFigure 3. Hazard Ratios of Lasso Selected Proteins for Primary End Point of MI, Stroke, Heart Failure, and Death eFigure 4. 9-Protein Prognostic Model Hazard Ratios Adjusted for Framingham Variables eFigure 5. 9-Protein Risk Scores by Event Type

This supplementary material has been provided by the authors to give readers additional information about their work.

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Supplemental Material

Table of Contents 1 Study Design and Data Processing ...... 3 2 Table of 1130 Proteins Measured ...... 4 3 Variable Selection and Statistical Modeling ...... 62 4 Recalibration for Validation...... 63 5 Table of Individual Proteins Associated with Cardiovascular Risk ...... 67 5.1 Biological Functions of 16 LASSO-selected Proteins ...... 74 5.2 9-Protein Model Specification ...... 77 6 Risk Score Distributions by Event Type ...... 79 7 References ...... 80

List of Figures and Tables

eTable 1: List of 1130 proteins measured by SomaLogic’s modified aptamer-based proteomic assay. ______4

eTable 2: Coefficients for Weibull recalibration model applied to 9-protein model. ______64

eFigure 1: Median protein levels in Derivation and Validation cohort. ______65

eTable 3: Coefficients for the recalibration model applied to refit Framingham. ______66

eFigure 2: Calibration plots for the refit Framingham model. ______666

eTable 4: List of 200 proteins associated with the risk of MI, stroke, heart failure and death. ______67

eFigure 3: Hazard ratios of LASSO selected proteins for primary end-point of MI, stroke, heart failure and death. 76

eFigure 4: 9-protein prognostic model hazard ratios adjusted for Framingham variables. ______78

eFigure 5: 9-protein risk scores by event type. ______79

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1 Study Design and Data Processing The flowchart shown in Figure 1 in the main article describes the key steps in the development and validation of the 9-protein model discussed in the main manuscript. Protein measurements in plasma samples were generated over a period of 3 weeks in 32 separate assay runs. Study samples were randomly assigned to assay runs along with a set of calibration and control samples. No identifying information was available to the laboratory technicians operating the assay.

Intra-run normalization and inter-run calibration were performed according to assay data quality control (QC) procedures defined in the good laboratory practice (GLP) quality system of SomaLogic, Inc. Inter- run calibration removes “batch effects” between the successive assay runs by matching the median signal over replicate observations of a pooled plasma calibrator sample included in each assay run to a fixed signal level reference. Typical calibration scale factors are close to unity and quality control (QC) acceptance criteria specify the acceptable range of scale factors as the median scale factor ± 40%. Intra- run normalization controls for “bulk” signal intensity biases that can result from either differential hybridization efficiency or differential sample dilution (or other collection protocol artifacts) that change the total protein concentration in the sample. The former effect is captured by a set of controls used to monitor the hybridization reaction for each sample and the latter uses the median of the ratio of median signal levels in each sample to the median signal level for the modified aptamers over all samples within the run. Typical normalization scale factors are close to unity and quality control (QC) acceptance criteria requires normalization scale factors for a sample to fall in the range [0.4, 2.5]. Seventy-six of the original 1130 proteins measured failed the inter-run calibration QC metrics in at least one of the 32 independent assay runs and were considered technical (analytical) failures unfit for analysis. Samples failing the intra- run normalization quality control (QC) metrics were technical failures unfit for analysis. Hemolyzed samples have a distinctive pattern of extreme hemoglobin and haptoglobin levels and extreme signal levels in more than 5% of proteins measured with the modified aptamer platform are indicative of additional sample degradation. Of the 2496 samples that passed the QC metrics, 73 (n=37 derivation, n=36 validation) were deemed unfit for analysis because they were hemolyzed (n=22 derivation, n=26 validation) or had at least 5% of the proteins measured (n=15 derivation, n=10 validation) exceed an outlier threshold defined as the median ± the maximum of 6 median absolute deviations and 5 times the median.

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2 Table of 1130 Proteins Measured

eTable 1 (below): List of 1130 Proteins measured by Somalogic’s modified aptamer-based proteomic assay, Version 3. The 76 proteins that failed quality control metrics were excluded from the analysis are indicated in the fourth column.

Excluded from Protein Analyte UniProt ID Gene Analysis sPLA2-Iia

14-3-3 protein family -- --

14-3-3σ | Stratifin P31947 SFN

15-hydroxyprostaglandin P15428 HPGD dehydrogenase | 15-PGDH | HPG-1

3-hydroxyacyl-CoA dehydrogenase Q99714 HSD17B10 type-2|ABAD | ERAB

3-hydroxyisobutyrate dehydrogenase P31937 HIBADH

4-1BB | CD137 Q07011 TNFRSF9

4-1BB ligand | CD137L P41273 TNFSF9

6Ckine | CCL21 O00585 CCL21

6-Phosphogluconate dehydrogenase P52209 PGD

Acid ceramidase-like protein | N- acylethanolamine-hydrolyzing acid Q02083 NAAA

amidase | ASAHL

Acid phosphatase 1, soluble | Adipocyte acid phosphatase | LMW- P24666 ACP1

PTP

Acidic fibroblast growth factor | β- P05230 FGF1 endothelial cell growth factor

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Excluded from Protein Analyte UniProt ID Gene Analysis

Acidic leucine-rich nuclear Q92688 ANP32B phosphoprotein 32 family member B

Activated leukocyte cell adhesion Q13740 ALCAM molecule | CD166

Activated protein C P04070 PROC

Activin A | Inhibin β-A homodimer P08476 INHBA

Activin AB | Inhibin β-A:β-B P08476, P09529 INHBA INHBB heterodimer

Activin receptor-like kinase 1 | ALK-1 P37023 ACVRL1

Activin serine-threonine-protein P36896 ACVR1B kinase receptor type-1B | ALK-4

ADAM metallopeptidase domain 12 O43184 ADAM12

ADAM metallopeptidase domain 9 Q13443 ADAM9

ADAM metallopeptidase with Q9UHI8 ADAMTS1 thrombospondin motifs 1

ADAM metallopeptidase with Q76LX8 ADAMTS13 thrombospondin motifs 13

ADAM metallopeptidase with Q8TE58 ADAMTS15 thrombospondin motifs 15

ADAM metallopeptidase with thrombospondin motifs 4 | O75173 ADAMTS4

Aggrecanase 1

ADAM metallopeptidase with thrombospondin motifs 5 | Q9UNA0 ADAMTS5

Aggrecanase 2

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Excluded from Protein Analyte UniProt ID Gene Analysis Adaptor protein Crk-I P46108 CRK

Adenylate kinase 1 | Myokinase P00568 AK1

Adenylosuccinate lyase P30566 ADSL

Adiponectin Q15848 ADIPOQ Y

Adrenocorticotropic hormone P01189 POMC Y

Afamin P43652 AFM

Aflatoxin B1 aldehyde reductase O43488 AKR7A2

Aggrecan core protein P16112 ACAN

Agouti-related protein O00253 AGRP

AITRL | Activation-induced TNFR member Ligand | GITRL | Q9UNG2 TNFSF18 Glucocorticoid-induced TNF receptor ligand

Albumin P02768 ALB

Alcohol dehydrogenase (NADP+) | Aldo-keto reductase family 1 member P14550 AKR1A1

Alkaline phosphatase, tissue- P05186 ALPL nonspecific isozyme

Alkaline sphingomyelinase Q6UWV6 ENPP7

Allograft inflammatory factor 1 P55008 AIF1

Aminoacylase-1 Q03154 ACY1

Amnionless Q9BXJ7 AMN Y

AMP kinase (α1β1γ1) Q13131, Q9Y478, P54619 PRKAA1 PRKAB1 PRKAG1

AMP kinase (α2β2γ1) P54646, O43741, P54619 PRKAA2 PRKAB2 PRKAG1

Amphiregulin P15514 AREG

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Excluded from Protein Analyte UniProt ID Gene Analysis Amyloid β A4 protein P05067 APP

Angiogenin P03950 ANG Y

Angiopoietin -related protein 4 Q9BY76 ANGPTL4

Angiopoietin-1 Q15389 ANGPT1

Angiopoietin-2 O15123 ANGPT2

Angiopoietin-4 Q9Y264 ANGPT4

Angiopoietin-like 3 Q9Y5C1 ANGPTL3

Angiostatin P00747 PLG

Angiotensin-converting enzyme 2 Q9BYF1 ACE2

Angiotensinogen P01019 AGT

Annexin A1 P04083 ANXA1

Annexin A2 | Annexin II P07355 ANXA2

Annexin A6 P08133 ANXA6

Anterior gradient protein 2 homolog O95994 AGR2

Anti-Mullerian hormone receptor, Q16671 AMHR2 type II

Antithrombin III P01008 SERPINC1

Apolipoprotein A-I P02647 APOA1 Y

Apolipoprotein B P04114 APOB

Apolipoprotein D P05090 APOD Y

Apolipoprotein E P02649 APOE Y

Apolipoprotein E (isoform E2) P02649 APOE Y

Apolipoprotein E (isoform E3) P02649 APOE Y

Apolipoprotein E (isoform E4) P02649 APOE Y

Apoptosis inhibitor 2 | C-IAP2 Q13489 BIRC3

Apoptosis regulator Bcl-2 P10415 BCL2

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Excluded from Protein Analyte UniProt ID Gene Analysis

Apoptosis regulator Bcl-X Q07817 BCL2L1

Arginase-1 P05089 ARG1

ARID domain-containing protein 3A Q99856 ARID3A

Artemin Q5T4W7 ARTN

Aryl hydrocarbon receptor-interacting O00170 AIP protein | XAP2

Arylsulfatase A P15289 ARSA

Arylsulfatase B P15848 ARSB

Asialoglycoprotein receptor 1 P07306 ASGR1

Aspartate aminotransferase | P17174 GOT1 Y Glutamic-oxaloacetic transaminase 1

ATP synthase β-subunit, mitochondrial P06576 ATP5B Y | ecto-ATPSβ

ATP-dependent DNA helicase II 70 kDa P12956 XRCC6 subunit | Ku70

Aurora kinase A O14965 AURKA

Aurora-related kinase 2 | Aurora B Q96GD4 AURKB

Azurocidin P20160 AZU1

B lymphocyte chemoattractant | BLC | O43927 CXCL13 BCA-1

B7 homolog 1 | CD274 Q9NZQ7 CD274

B7 homolog 2 | ICOS ligand O75144 ICOSLG

B7-2 | CD86 P42081 CD86

Bactericidal permeability-increasing P17213 BPI protein

Basal cell adhesion molecule P50895 BCAM Y

Basic fibroblast growth factor P09038 FGF2

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Excluded from Protein Analyte UniProt ID Gene Analysis

Basic fibroblast growth factor receptor P11362 FGFR1 1

BCAR3 breast cancer anti-estrogen O75815 BCAR3 resistance 3

B-cell maturation protein | BCMA Q02223 TNFRSF17

B-cell-activating factor | BAFF | BLyS | Q9Y275 TNFSF13B

B-cell-activating factor receptor | Q96RJ3 TNFRSF13C BAFF R

Bcl-2-related protein A1 Q16548 BCL2A1

Biglycan P21810 BGN

BMPER | BMP-binding endothelial Q8N8U9 BMPER regulator | Crossveinless-2

Bone marrow stromal cell antigen | Q10588 BST1 CD157

Bone morphogenetic protein receptor P36894 BMPR1A type IA | ALK-3

Bone morphogenetic protein type II Q13873 BMPR2 receptor

Bone morphogenetic protein-1 P13497 BMP1

Bone morphogenetic protein-10 O95393 BMP10 Y

Bone morphogenetic protein-14 | BMP-14 | Cartilage-Derived P43026 GDF5 Y Morphogenetic Protein-1 | CDMP-1

Bone morphogenetic protein-6 P22004 BMP6

Bone morphogenetic protein-7 P18075 BMP7

Bone sialoprotein 2 P21815 IBSP

Brain natriuretic peptide 32 | BNP-32 P16860 NPPB

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Excluded from Protein Analyte UniProt ID Gene Analysis

Brain-derived neurotrophic factor P23560 BDNF

Brain-specific serine protease 4 Q9GZN4 PRSS22

B-related factor 1 | Transcription Q92994 BRF1 factor IIIB 90 kDa subunit | TFIIIB90

Brevican Q96GW7 BCAN

C1-Esterase Inhibitor P05155 SERPING1

c-abl oncogene 1, non-receptor P00519 ABL1 tyrosine kinase

Cadherin-1 | E-cadherin (epithelial) P12830 CDH1

Cadherin-12 | BR-cadherin (brain) P55289 CDH12

Cadherin-15 | M-cadherin P55291 CDH15 (myotubule)

Cadherin-2 | N-cadherin (neuronal) P19022 CDH2

Cadherin-3 | P-cadherin (placental) P22223 CDH3

Cadherin-5 | VE-cadherin (vascular P33151 CDH5

Cadherin-6 | K-cadherin (kidney) P55285 CDH6

Calcineurin Q08209, P63098 PPP3CA PPP3R1

Calcineurin B α P63098 PPP3R1

Calcium-calmodulin-dependent Q14012 CAMK1 protein kinase I

Calcium-calmodulin-dependent Q8IU85 CAMK1D protein kinase ID

Calcium-calmodulin-dependent Q9UQM7 CAMK2A protein kinase II α

Calcium-calmodulin-dependent Q13554 CAMK2B protein kinase II β

Calcium-calmodulin-dependent Q13557 CAMK2D protein kinase II δ

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Excluded from Protein Analyte UniProt ID Gene Analysis

Calcium-calmodulin-dependent Q8N5S9 CAMKK1 protein kinase kinase 1 α

Calpain I P07384, P04632 CAPN1 CAPNS1

Calpastatin P20810 CAST

Calreticulin | Ro P27797 CALR

cAMP and cGMP phosphodiesterase Q9HCR9 PDE11A 11A | PDE11A

cAMP-dependent protein kinase P17612 PRKACA catalytic subunit α | PKA

cAMP-regulated phosphoprotein 19 P56211 ARPP19

cAMP-specific phosphodiesterase 4D | Q08499 PDE4D PDE4D

Carbohydrate sulfotransferase 15 Q7LFX5 CHST15

Carbohydrate sulfotransferase 2 Q9Y4C5 CHST2

Carbohydrate sulfotransferase 6 Q9GZX3 CHST6

Carbonic anhydrase I P00915 CA1

Carbonic anhydrase II P00918 CA2

Carbonic anhydrase III P07451 CA3

Carbonic anhydrase IV P22748 CA4 Y

Carbonic anhydrase IX Q16790 CA9

Carbonic anhydrase VI P23280 CA6

Carbonic anhydrase VII P43166 CA7

Carbonic anhydrase XIII Q8N1Q1 CA13

Carbonic anhydrase-related protein X Q9NS85 CA10

Carboxypeptidase E P16870 CPE

Cardiotrophin-1 Q16619 CTF1

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Excluded from Protein Analyte UniProt ID Gene Analysis

Carnosine dipeptidase 1 Q96KN2 CNDP1

Casein kinase II subunit α P68400 CSNK2A1 Y

Casein kinase II α1:β heterodimer P68400, P67870 CSNK2A1 CSNK2B

Casein kinase II α2:β heterodimer P19784, P67870 CSNK2A2 CSNK2B

Caspase-10 Q92851 CASP10

Caspase-2 P42575 CASP2

Caspase-3 P42574 CASP3

Catalase P04040 CAT

Cathepsin A P10619 CTSA

Cathepsin B P07858 CTSB

Cathepsin C P53634 CTSC

Cathepsin D P07339 CTSD

Cathepsin E P14091 CTSE

Cathepsin G P08311 CTSG

Cathepsin H P09668 CTSH

Cathepsin L2 | Cathepsin V O60911 CTSL2

Cathepsin S P25774 CTSS

Cathepsin Z/X/P Q9UBR2 CTSZ

CCL1 | CC chemokine 1 | I-309 P22362 CCL1

CCL18 | PARC |Macrophage P55774 CCL18 inflammatory protein 4

CCL28 Q9NRJ3 CCL28

CD109 Q6YHK3 CD109

CD200 | OX-2 membrane glycoprotein P41217 CD200

CD200 receptor 1 Q8TD46 CD200R1

CD22 | Siglec-2 P20273 CD22

CD23 | FCER2 P06734 FCER2

CD27 | TNFRSF7 P26842 CD27 Y

CD30 | TNFRSF8 | KI-1 P28908 TNFRSF8

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Excluded from Protein Analyte UniProt ID Gene Analysis

CD30 Ligand | TNFSF8 | CD153 P32971 TNFSF8

CD39 | Ecto-ATPDase 1 P49961 ENTPD1

CD40 ligand | TNFSF5 P29965 CD40LG

CD48 | SLAMF2 P09326 CD48

CD5 antigen-like O43866 CD5L

CD55 | Complement decay- P08174 CD55 accelerating factor | DAF

CD70 | TNFSF7 P32970 CD70

CD97 P48960 CD97

Cell adhesion oncogene-related CDO Q4KMG0 CDON

cGMP-binding cGMP-specific O76074 PDE5A phosphodiesterase | PDE5A

cGMP-inhibited cAMP Q14432 PDE3A phosphodiesterase 3A | PDE3A

Chemerin Q99969 RARRES2

Chitotriosidase-1 Q13231 CHIT1

Chordin-Like 1 Q9BU40 CHRDL1

Chromobox protein homolog 5 | HP1α P45973 CBX5

Chymase P23946 CMA1

Ciliary Neurotrophic Factor P26441 CNTF

Ciliary neurotrophic factor receptor α P26992 CNTFR

CKAP2 | Cytoskeleton-associated Q8WWK9 CKAP2 protein 2

CK-BB | Creatine kinase-BB P12277 CKB

CK-MB | Creatine kinase-MB P12277, P06732 CKB CKM

CK-MM | Creatine kinase-MM P06732 CKM

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Excluded from Protein Analyte UniProt ID Gene Analysis

Ck-β-8-1 | Macrophage inflammatory P55773 CCL23 protein 3 splice variant (aa 46-137)

Clusterin P10909 CLU Y CMRF-35A | CD300c Q08708 CD300C Y

Coagulation factor IX P00740 F9

Coagulation factor IXab P00740 F9

Coagulation factor V P12259 F5

Coagulation factor VII P08709 F7

Coagulation factor X P00742 F10 Y

Coagulation factor Xa P00742 F10

Coagulation factor XI P03951 F11 Y Cofilin-1 P23528 CFL1

Coiled-coil domain-containing protein Q76M96 CCDC80 80 | URB

Collagen α-1(VIII) chain P27658 COL8A1

Collagen α-1(XXIII) chain Q86Y22 COL23A1

Collectin Kidney 1 Q9BWP8 COLEC11

Collectin Placenta 1 Q5KU26 COLEC12

COMM domain containing 7 Q86VX2 COMMD7

Complement C1q P02745, P02746, P02747 C1QA C1QB C1QC

Complement C1q binding protein | Q07021 C1QBP Hyaluronan binding protein 1 | HABP1

Complement C1r P00736 C1R

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Excluded from Protein Analyte UniProt ID Gene Analysis Complement C1s P09871 C1S

Complement C2 P06681 C2

Complement C3 P01024 C3 Y

Complement C3a anaphylatoxin P01024 C3 Y

Complement C3a anaphylatoxin des P01024 C3 Arginine

Complement C3b P01024 C3 Y

Complement C3b, inactivated | iC3b P01024 C3 Y

Complement C3d P01024 C3

Complement C4 P0C0L4, P0C0L5 C4A C4B

Complement C4b P0C0L4, P0C0L5 C4A C4B

Complement C5 P01031 C5

Complement C5a P01031 C5 Y

Complement C5b,6 Complex P01031, P13671 C5 C6

Complement C6 P13671 C6

Complement C7 P10643 C7

Complement C8 P07357, P07358, P07360 C8A C8B C8G

Complement C9 P02748 C9

Complement factor B P00751 CFB

Complement factor D P00746 CFD

Complement factor H P08603 CFH

Complement factor H-related 5 Q9BXR6 CFHR5

Complement factor I P05156 CFI

Connective tissue growth factor | CCN2 | Hypertrophic chondrocyte- P29279 CTGF

specific protein 24 | HCS24

Connective-tissue activating peptide III P02775 PPBP | CTAP-III

Conserved dopamine neurotrophic Q49AH0 CDNF factor Contactin-1 Q12860 CNTN1

Contactin-2 Q02246 CNTN2

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Excluded from Protein Analyte UniProt ID Gene Analysis Contactin-4 Q8IWV2 CNTN4

Contactin-5 O94779 CNTN5

Copine-1 Q99829 CPNE1

Corticosteroid binding globulin P08185 SERPINA6

COX-2 | Prostaglandin H2 synthase 2 | P35354 PTGS2 Cyclooxygenase-2

C-reactive protein P02741 CRP

Cripto-1 P13385 TDGF1

CRTAM | cytotoxic and regulatory T O95727 CRTAM cell molecule

Cryptic protein P0CG37 CFC1

C-Src kinase | CSK P41240 CSK

C-type lectin domain family 1 member Q9P126 CLEC1B B | CLEC-2

Cutaneous T-cell-attracting chemokine Q9Y4X3 CCL27 | CTACK

Cyclin B1 P14635 CCNB1

Cyclin-dependent kinase 1:cyclin B P06493, P14635 CDC2 CCNB1 complex

Cyclin-dependent kinase 2:cyclin A P24941, P20248 CDK2 CCNA2 complex

Cyclin-dependent kinase 5:activator Q00535, Q15078 CDK5 CDK5R1 p35 complex

Cyclin-dependent kinase 8:cyclin C P49336, P24863 CDK8 CCNC complex

Cyclin-dependent kinase inhibitor p27 P46527 CDKN1B | p27kip1

Cyclophilin B | CypB P23284 PPIB Y Cystatin C P01034 CST3

Cystatin D P28325 CST5

Cystatin E/M Q15828 CST6

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Excluded from Protein Analyte UniProt ID Gene Analysis Cystatin F O76096 CST7

Cystatin S P01036 CST4

Cystatin SA P09228 CST2

Cystatin SN P01037 CST1

Cysteine-rich secretory protein 3 P54108 CRISP3

Cytochrome c P99999 CYCS

Cytochrome P450 3A4 P08684 CYP3A4

Cytokine-like factor 1:Cardiotrophin- O75462, Q9UBD9 CRLF1 CLCF1 like cytokine factor 1 Complex

Cytosolic non-specific dipeptidase 2 Q96KP4 CNDP2

Cytotoxic T-lymphocyte-4 P16410 CTLA4

dCTP pyrophosphatase 1 Q9H773 DCTPP1

D-dimer P02671, P02675, P02679 FGA FGB FGG

DEAD box RNA helicase 19B | DBP5 Q9UMR2 DDX19B

Death decoy receptor 3 | DcR3 O95407 TNFRSF6B

Death receptor 3 | DR3 Q93038 TNFRSF25

Death receptor 6 | DR6 O75509 TNFRSF21

Death-associated protein kinase 2 Q9UIK4 DAPK2

Decorin | Bone proteoglycan II P07585 DCN

Dectin-1 Q9BXN2 CLEC7A

Delta-like protein 1 O00548 DLL1

Dendritic cell-specific ICAM-3- Q9NNX6 CD209 grabbing nonintegrin 1 | DC-SIGN

Dendritic cell-specific ICAM-3- Q9H2X3 CLEC4M grabbing nonintegrin 2 | DC-SIGNR

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Excluded from Protein Analyte UniProt ID Gene Analysis

Dentin matrix protein 1 Q13316 DMP1

Dermatopontin Q07507 DPT Y

Desert hedgehog N-terminus O43323 DHH

Desmocollin-3 Q14574 DSC3

Desmoglein-1 Q02413 DSG1

Desmoglein-2 Q14126 DSG2

Dickkopf-related protein 1 O94907 DKK1

Dickkopf-related protein 3 Q9UBP4 DKK3

Dickkopf-related protein 4 Q9UBT3 DKK4

Dipeptidyl-peptidase II Q9UHL4 DPP7

Discoidin domain receptor 1 Q08345 DDR1

Discoidin domain receptor 2 Q16832 DDR2

DNA repair protein RAD51 homolog 1 Q06609 RAD51

DNA Topoisomerase I P11387 TOP1

DnaJ homolog subfamily C member 19 Q96DA6 DNAJC19

DNAX accessory molecule 1 | DNAM-1 Q15762 CD226

Dopa decarboxylase P20711 DDC

Dopamine responsive protein | DRG-1 Q9NP79 VTA1 | LIP5 | SBP1

Down-regulated in renal cell O95990 FAM107A carcinoma 1 | DRR1 | TU3A

Drebrin-like HIP-55 | Actin-binding Q9UJU6 DBNL protein 1 | mAbp1 | SH3P7

Drosophila delta homolog 4 Q9NR61 DLL4

Dual-specificity protein kinase 3 | O43781 DYRK3 Regulatory erythroid kinase | REDK

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Excluded from Protein Analyte UniProt ID Gene Analysis Dynactin subunit 2 Q13561 DCTN2

Dynein light chain 1 |DLC8 | LC8 P63167 DYNLL1

Dynein Roadblock light chain 1, RobI1 Q9NP97 DYNLRB1 | Km23

Ectodermal dysplasia receptor Q9UNE0 EDAR

Ectodysplasin-A, secreted form Q92838 EDA

Ectonucleoside triphosphate O75355 ENTPD3 diphosphohydrolase 3 | CD39L3

Ectonucleoside triphosphate O75356 ENTPD5 diphosphohydrolase 5 | CD39L4

EGF receptor | ErbB1 | HER1 P00533 EGFR

EGF-like module-containing mucin-like Q9UHX3 EMR2 receptor 2

EG-VEGF | PK1 | Endocrine-gland- derived vascular endothelial growth P58294 PROK1

factor

Elafin | Trappin-2 P19957 PI3

Elongation factor 1-β P24534 EEF1B2

Endocan Q9NQ30 ESM1

Endoglin | CD105 P17813 ENG

Endoplasmic reticulum Q9NZ08 ERAP1 aminopeptidase 1 | PILS | ARTS1

Endoplasmic reticulum resident P30040 ERP29 protein 29 | ERp29

Endostatin P39060 COL18A1

Endothelial cell-selective adhesion Q96AP7 ESAM molecule

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Excluded from Protein Analyte UniProt ID Gene Analysis

Endothelial-monocyte activating Q12904 AIMP1 polypeptide 2 | EMAP-2 | p43

Endothelin-converting enzyme 1 P42892 ECE1

Enterokinase | Enteropeptidase P98073 PRSS7

Eotaxin | CCL11 P51671 CCL11

Eotaxin-2 O00175 CCL24

EPH receptor A10 Q5JZY3 EPHA10

EPH receptor B2 P29323 EPHB2

EPH receptor B6 O15197 EPHB6

Ephrin type-A receptor 1 P21709 EPHA1

Ephrin type-A receptor 2 P29317 EPHA2

Ephrin type-A receptor 3 P29320 EPHA3

Ephrin type-A receptor 5 P54756 EPHA5

Ephrin type-B receptor 4 P54760 EPHB4 Y

Ephrin-A4 P52798 EFNA4

Ephrin-A5 P52803 EFNA5

Ephrin-B3 Q15768 EFNB3

Epidermal growth factor receptor Q9UBC2 EPS15L1 substrate 15-like 1 | Eps15R

Epiregulin O14944 EREG

Epithelial-derived neutrophil- P42830 CXCL5 activating protein 78 | ENA-78

ErbB2 | HER2 P04626 ERBB2

ErbB3 | HER3 P21860 ERBB3

ErbB3 binding protein Ebp1 Q9UQ80 PA2G4

ErbB4 | HER4 Q15303 ERBB4

Erythrocyte membrane protein 4.1 | P11171 EPB41 Y Protein 4.1R

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Excluded from Protein Analyte UniProt ID Gene Analysis Erythropoietin P01588 EPO

Erythropoietin receptor P19235 EPOR

E-Selectin | CD62E | ELAM-1 P16581 SELE

Esterase D P10768 ESD

Estrogen receptor 1 | ERα P03372 ESR1

Ethylmalonic encephalopathy 1 O95571 ETHE1

Eukaryotic translation initiation factor P38919 EIF4A3 4A-III | DEAD-box protein 48 | DDX48

Eukaryotic translation initiation factor Q13542 EIF4EBP2 4E-binding protein 2

Eukaryotic translation initiation factor P55010 EIF5 5

Eukaryotic translation initiation factor P63241 EIF5A 5A

Eukaryotic translation initiation factor P78344 EIF4G2 G2 | p97 | DAP5.

Extracellular matrix metalloproteinase P35613 BSG Y inducer | EMMPRIN | Basigin | CD147

Extracellular matrix protein-1 Q16610 ECM1

FACT complex subunit SSRP1 Q08945 SSRP1

Fas ligand, soluble fragment | CD95L P48023 FASLG

Fatty acid binding protein, epidermal- Q01469 FABP5 Y type | E-FABP | C-FABP

Fatty acid binding protein, heart-type P05413 FABP3

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Excluded from Protein Analyte UniProt ID Gene Analysis

Fc receptor-like protein 3 Q96P31 FCRL3

Ferritin P02794, P02792 FTH1 FTL

Fetuin B Q9UGM5 FETUB

Fibrinogen P02671, P02675, P02679 FGA FGB FGG

Fibrinogen γ chain dimer P02679 FGG

Fibroblast activation protein α Q12884 FAP

Fibroblast growth factor 10 | O15520 FGF10 Keratinocyte growth factor 2 | FGFA

Fibroblast growth factor 12 P61328 FGF12

Fibroblast growth factor 16 | FGFG O43320 FGF16

Fibroblast growth factor 17 | FGFH O60258 FGF17

Fibroblast growth factor 18 | zFGF5 | O76093 FGF18 FGFI

Fibroblast growth factor 19 | FGFJ O95750 FGF19

Fibroblast growth factor 20 | FGFK Q9NP95 FGF20

Fibroblast growth factor 23 Q9GZV9 FGF23

Fibroblast growth factor 4 P08620 FGF4

Fibroblast growth factor 5 P12034 FGF5

Fibroblast growth factor 6 | HST-2 P10767 FGF6

Fibroblast growth factor 7 | P21781 FGF7 Keratinocyte Growth Factor | KGF

Fibroblast growth factor 8 isoform A P55075 FGF8

Fibroblast growth factor 8 isoform B P55075 FGF8

Fibroblast growth factor 9 P31371 FGF9

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Excluded from Protein Analyte UniProt ID Gene Analysis

Fibroblast growth factor receptor 2 P21802 FGFR2

Fibroblast growth factor receptor 3 P22607 FGFR3

Fibroblast growth factor receptor 4 P22455 FGFR4

Fibronectin P02751 FN1

Fibronectin leucine rich Q9NZU1 FLRT1 transmembrane 1

Fibronectin-1 fragment 3 P02751 FN1

Fibronectin-1 fragment 4 P02751 FN1

Ficolin-1 O00602 FCN1

Ficolin-2 Q15485 FCN2

Ficolin-3 O75636 FCN3

Fms-related tyrosine kinase 3 ligand | P49771 FLT3LG Flt3-Ligand

Focal adhesion kinase 1 | FAK1 Q05397 PTK2

Follicle stimulating hormone P01215, P01225 CGA FSHB

Follistatin P19883 FST

Follistatin-like 3 O95633 FSTL3

Fortilin | Translationally-controlled tumor protein | TCTP | Histamine- P13693 TPT1

releasing factor

Fractalkine | CX3CL-1 P78423 CX3CL1 Y

Frizzled-related protein 1, secreted Q8N474 SFRP1 Y

Frizzled-related protein 3, secreted Q92765 FRZB

Fucosyltransferase 3 P21217 FUT3

Fucosyltransferase 5 Q11128 FUT5

G protein-coupled receptor associated Q96D09 GPRASP2 sorting protein 2 | GASP-2 | WFIKKN

Downloaded From: https://jamanetwork.com/ on 09/23/2021

Excluded from Protein Analyte UniProt ID Gene Analysis Galectin-2 P05162 LGALS2

Galectin-3 P17931 LGALS3 Y

Galectin-3 binding protein | MAC-2BP Q08380 LGALS3BP

Galectin-4 P56470 LGALS4

Galectin-8 O00214 LGALS8

GDF-11/8 | Growth-differentiation O95390 GDF11 factor 11/8

GDF-2 | Growth-differentiation factor Q9UK05 GDF2 2 | BMP-9

GDF-9 | Growth-differentiation factor O60383 GDF9 9

GDNF family receptor α1 P56159 GFRA1

GDNF family receptor α2 O00451 GFRA2

GDNF family receptor α3 O60609 GFRA3

Gelsolin P06396 GSN

GITR | TNFRSF18 Q9Y5U5 TNFRSF18

Glial fibrillary acidic protein P14136 GFAP

Glucagon P01275 GCG

Glucocorticoid receptor P04150 NR3C1 Y

Glucokinase (hexokinase 4) regulator | Q14397 GCKR GCKR

Glucose phosphate isomerase | Neuroleukin | Autocrine motility P06744 GPI

factor

Glutathione S-transferase A3 | GSTA3- Q16772 GSTA3 3

Glutathione S-transferase Pi 1 P09211 GSTP1

Glyceraldehyde-3-phosphate P04406 GAPDH dehydrogenase

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Excluded from Protein Analyte UniProt ID Gene Analysis

Glycogen synthase kinase-3 α/β P49840, P49841 GSK3A GSK3B

Glypican-2 Q8N158 GPC2

Glypican-3 P51654 GPC3

Glypican-5 P78333 GPC5

GP4 | CD36 | Platelet glycoprotein IV P16671 CD36

gp41 C34 peptide (HIV) Q70626 HIV env

G-protein coupled receptor 114 Q8IZF4 GPR114

GPT | Alanine aminotransaminase 1 | ALT | Gutamate pyruvate P24298 GPT

transaminanse

GPVI | Platelet glycoprotein VI Q9HCN6 GP6

Granulocyte chemotactic protein 2 | P80162 CXCL6 GCP-2 | CXCL6

Granulocyte colony-stimulating factor P09919 CSF3 Y | G-CSF

Granulocyte colony-stimulating factor Q99062 CSF3R receptor | CD114

Granulocyte-macrophage colony- P04141 CSF2 stimulating factor | GM-CSF

Granulysin P22749 GNLY

Granzyme A P12544 GZMA

Granzyme B P10144 GZMB

Granzyme H P20718 GZMH

GRAP2 | GRB2-related adaptor protein O75791 GRAP2 2 | Mona | Gads

Gremlin-1 O60565 GREM1

Growth arrest specific 1 P54826 GAS1

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Excluded from Protein Analyte UniProt ID Gene Analysis

Growth hormone receptor P10912 GHR

Gro-α | CXCL1 | KC P09341 CXCL1

Gro-β/γ | CXCL2/3 | MIP2-α/β P19876, P19875 CXCL3 CXCL2

GTP-binding nuclear protein Ran | P62826 RAN ARA24 Haptoglobin P00738 HP

Heat shock cognate 70 kDa protein 8 P11142 HSPA8

Heme oxygenase 2 P30519 HMOX2

Hemofiltrate CC chemokine 1 | HCC-1 Q16627 CCL14 | CCL14

Hemoglobin P69905, P68871 HBA1 HBB

Hemojuvelin | RGMC Q6ZVN8 HFE2

Hemopexin P02790 HPX

Hemopoietic cell kinase P08631 HCK

Heparan-sulfate 6-O-sulfotransferase O60243 HS6ST1 1 Heparin cofactor II P05546 SERPIND1

Heparin-binding EGF-like growth Q99075 HBEGF factor

Hepatocyte growth factor P14210 HGF

Hepatocyte growth factor activator Q04756 HGFAC

Hepatocyte growth factor receptor | P08581 MET c-Met

Hepatoma-derived growth factor- Q7Z4V5 HDGFRP2 related protein 2 | HRP-2

Heterogeneous nuclear P22626 HNRNPA2B1 ribonucleoprotein A2/B1

Heterogeneous nuclear Q99729 HNRNPAB ribonucleoprotein AB

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Excluded from Protein Analyte UniProt ID Gene Analysis

Heterogeneous nuclear P61978 HNRNPK ribonucleoprotein K | hnRNP K

Heterogeneous nuclear ribonucleoprotein Q | NSAP1 | GRY- O60506 SYNCRIP

RBP

High affinity cAMP-specific Q13946 PDE7A phosphodiesterase 7A | PDE7A

High affinity cAMP-specific O76083 PDE9A phosphodiesterase 9A | PDE9A

High temperature requirement serine O43464 HTRA2 peptidase A2 | OMI

High-mobility group box 1 | P09429 HMGB1 amphoterin

Histidine triad nucleotide binding P49773 HINT1 protein 1

Histidine-proline-rich glycoprotein | P04196 HRG Y HPRG

Histone acetyltransferase 1 O14929 HAT1

Histone acetyltransferase KAT6A | Q92794 KAT6A MOZ | MYST3

Histone deacetylase 8 Q9BY41 HDAC8

Histone H1.2 P16403 HIST1H1C

Histone H2A.z P0C0S5 H2AFZ

Histone H3-K9 methyltransferase 3 Q96KQ7 EHMT2

HMG-CoA reductase P04035 HMGCR

Homeobox transcription factor Nanog Q9H9S0 NANOG

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Excluded from Protein Analyte UniProt ID Gene Analysis

Homeodomain-interacting protein Q9H422 HIPK3 kinase 3 | YAK1

HSP 90α/β P07900, P08238 HSP90AA1 HSP90AB1

Hsp40 | HDJ1 P25685 DNAJB1

Hsp60 P10809 HSPD1

Hsp70 P08107 HSPA1A

Hsp90 co-chaperone Cdc37 Q16543 CDC37

Human chorionic gonadotropin P01215, P01233 CGA CGB

HVEM | TNFRSF14 Q92956 TNFRSF14

Hyaluronan and proteoglycan link P10915 HAPLN1 protein 1

Hydroxysteroid 17-β-dehydrogenase 1 P14061 HSD17B1

Iduronate 2-sulfatase P22304 IDS

IgA Fc receptor | CD89 P24071 FCAR

IgG Fc receptor I | CD64 P12314 FCGR1A

Immunoglobulin D P01880 IGHD IGK@ IGL@

Immunoglobulin E P01854 IGHE IGK@ IGL@

IGHG1 IGHG2 IGHG3 IGHG4 Immunoglobulin G P01857 Y IGK@ IGL@

Immunoglobulin G Fc region receptor O75015 FCGR3B III-B, low affinity | D16b

Immunoglobulin M P01871 IGHM IGJ IGK@ IGL@

IMP (inosine 5'-monophosphate) P20839 IMPDH1 dehydrogenase 1 | RP10

IMP (inosine 5'-monophosphate) P12268 IMPDH2 dehydrogenase 2

Importin β1 | Karyopherin β-1 | Q14974 KPNB1 Nuclear factor P97

Inducible T-cell co-stimulator Q9Y6W8 ICOS

Inferferon-α2 P01563 IFNA2

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Excluded from Protein Analyte UniProt ID Gene Analysis Inferferon-γ P01579 IFNG

Inferferon-γ receptor 1 P15260 IFNGR1

Inferferon-λ 1 Q8IU54 IL29

Inferferon-λ 2 Q8IZJ0 IL28A

Inhibitor of growth 1 Q9UK53 ING1 Y

Inorganic pyrophosphatase Q15181 PPA1 Y

Insulin P01308 INS

Insulin receptor | IR | CD220 P06213 INSR

Insulin-degrading enzyme | Insulysin P14735 IDE

Insulin-like growth factor I P05019 IGF1

Insulin-like growth factor I receptor P08069 IGF1R

Insulin-like growth factor II receptor P11717 IGF2R

Insulin-like growth factor-binding P08833 IGFBP1 protein 1

Insulin-like growth factor-binding P18065 IGFBP2 protein 2

Insulin-like growth factor-binding P17936 IGFBP3 Y protein 3

Insulin-like growth factor-binding P22692 IGFBP4 Y protein 4

Insulin-like growth factor-binding P24593 IGFBP5 protein 5

Insulin-like growth factor-binding P24592 IGFBP6 protein 6

Insulin-like growth factor-binding Q16270 IGFBP7 protein 7

Integrin α-I: β-1 complex | VLA-1 P56199, P05556 ITGA1 ITGB1

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Excluded from Protein Analyte UniProt ID Gene Analysis

Integrin α-IIb: β-3 complex | Platelet P08514, P05106 ITGA2B ITGB3 Membrane Glycoproteins gpIIbIIIa

Integrin α-V: β-5 complex | P06756, P18084 ITGAV ITGB5 Fibronectin Receptor

Intercellular adhesion molecule 1 | P05362 ICAM1 CD54

Intercellular adhesion molecule 2 | P13598 ICAM2 CD102

Intercellular adhesion molecule 3 | P32942 ICAM3 CD50

Intercellular adhesion molecule 5 Q9UMF0 ICAM5

Interferon-γ induced protein 10 | IP- P02778 CXCL10 10

Interferon-γ-inducible protein-9 | I- O14625 CXCL11 TAC

Interleukin-1 receptor 1 P14778 IL1R1

Interleukin-1 receptor accessory Q9NPH3 IL1RAP protein | IL-1 RAcP | IL1 R3

Interleukin-1 receptor accessory Q9NP60 IL1RAPL2 protein-like 2 | IL-1 sR9

Interleukin-1 receptor-like 1 | ST2 | Q01638 IL1RL1 Interleukin-1 receptor 4

Interleukin-1 receptor-like 2 | IL-1Rrp2 Q9HB29 IL1RL2

Interleukin-10 P22301 IL10

Interleukin-10 receptor β Q08334 IL10RB

Interleukin-11 P20809 IL11

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Excluded from Protein Analyte UniProt ID Gene Analysis

Interleukin-11 receptor α Q14626 IL11RA

Interleukin-12 P29459, P29460 IL12A IL12B

Interleukin-12 receptor β1 P42701 IL12RB1

Interleukin-12 receptor β2 Q99665 IL12RB2

Interleukin-13 P35225 IL13

Interleukin-13 receptor α1 P78552 IL13RA1

Interleukin-15 receptor α Q13261 IL15RA

Interleukin-16 Q14005 IL16

Interleukin-17 receptor A Q96F46 IL17RA

Interleukin-17 receptor B Q9NRM6 IL17RB

Interleukin-17 receptor C Q8NAC3 IL17RC

Interleukin-17 receptor D | SEF Q8NFM7 IL17RD

Interleukin-17A Q16552 IL17A

Interleukin-17B | IL20 | NIRF Q9UHF5 IL17B

Interleukin-17D | IL-27 Q8TAD2 IL17D

Interleukin-17E Q9H293 IL25

Interleukin-17F Q96PD4 IL17F

Interleukin-18 binding protein O95998 IL18BP

Interleukin-18 receptor α Q13478 IL18R1

Interleukin-18 receptor β O95256 IL18RAP

Interleukin-19 Q9UHD0 IL19

Interleukin-1α | IL-1F1 P01583 IL1A

Interleukin-1β | IL-1F2 P01584 IL1B Y

Interleukin-2 P60568 IL2

Interleukin-2 receptor α chain P01589 IL2RA

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Excluded from Protein Analyte UniProt ID Gene Analysis

Interleukin-2 receptor γ chain | P31785 IL2RG Cytokine receptor common chain

Interleukin-20 Q9NYY1 IL20

Interleukin-20 receptor subunit a Q9UHF4 IL20RA

Interleukin-22 Q9GZX6 IL22

Interleukin-22 receptor α-1 Q8N6P7 IL22RA1

Interleukin-22 receptor subunit α2 | Q969J5 IL22RA2 IL-22BP

Interleukin-23 P29460, Q9NPF7 IL12B IL23A

Interleukin-23 receptor Q5VWK5 IL23R

Interleukin-24 Q13007 IL24

Interleukin-27 Q8NEV9 IL27

Interleukin-27 receptor subunit α | Q6UWB1 IL27RA TCCR | WSX-1

Interleukin-3 P08700 IL3

Interleukin-3 receptor α P26951 IL3RA

Interleukin-34 Q6ZMJ4 IL34

Interleukin-37 Q9NZH6 IL1F7

Interleukin-4 P05112 IL4

Interleukin-4 receptor α chain P24394 IL4R

Interleukin-5 P05113 IL5 Y

Interleukin-5 receptor α Q01344 IL5RA

Interleukin-6 P05231 IL6

Interleukin-6 receptor subunit α P08887 IL6R

Interleukin-6 receptor subunit β | P40189 IL6ST gp130

Interleukin-7 P13232 IL7

Interleukin-7 receptor subunit α P16871 IL7R

Interleukin-8 | CXCL8 P10145 IL8

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Excluded from Protein Analyte UniProt ID Gene Analysis

Inter-α-trypsin inhibitor heavy chain Q14624 ITIH4 H4 | ITI-H4

Jagged-1 P78504 JAG1

Jagged-2 Q9Y219 JAG2

Janus kinase 2 O60674 JAK2

Junctional adhesion molecule B | JAM- P57087 JAM2 2 | VE-JAM

Junctional adhesion molecule C | JAM- Q9BX67 JAM3 3

Junctional adhesion molecule-like Q86YT9 AMICA1

Kallikrein 11 Q9UBX7 KLK11

Kallikrein 12 Q9UKR0 KLK12

Kallikrein 13 Q9UKR3 KLK13

Kallikrein 14 Q9P0G3 KLK14

Kallikrein 3 | PSA P07288 KLK3

Kallikrein 4 | Prostase Q9Y5K2 KLK4

Kallikrein 5 Q9Y337 KLK5

Kallikrein 6 | Neurosin Q92876 KLK6

Kallikrein 7 P49862 KLK7

Kallikrein 8 | Neuropsin O60259 KLK8

Kallistatin P29622 SERPINA4

Karyopherin α 2 | Importin α1 P52292 KPNA2

Keratin 18 P05783 KRT18

Killer cell immunoglobulin-like Q99706 KIR2DL4 receptor 2DL4

Killer cell immunoglobulin-like P43630 KIR3DL2 receptor 3DL2

Killer cell immunoglobulin-like Q14943 KIR3DS1 receptor 3DS1

Killer cell lectin-like receptor subfamily Q9NZS2 KLRF1 F, member 1 | NKp80

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Excluded from Protein Analyte UniProt ID Gene Analysis

Kinesin family member 23 | CHO1 | Q02241 KIF23 MKLP-1

Kininogen-1, HMW, Single chain P01042 KNG1

Kirrel3 | NEPH2 Q8IZU9 KIRREL3

KRAS P01116 KRAS

Kremen protein 2 Q8NCW0 KREMEN2

Kunitz-type protease inhibitor 1 | Hepatocyte growth factor activator O43278 SPINT1

inhibitor | HAI inhibitor type 1

Kunitz-type protease inhibitor 2A | O43291 SPINT2 HAI-2 Kynureninase Q16719 KYNU

Lactate dehydrogenase 1 (heart) P07195 LDHB

Lactoferrin P02788 LTF

Lactoperoxidase P22079 LPO

Lamin-B1 P20700 LMNB1

Laminin P25391, P07942, P11047 LAMA1 LAMB1 LAMC1 Y

Laminin receptor | p40 ribosomal P08865 RPSA protein SA

Langerin Q9UJ71 CD207

Layilin Q6UX15 LAYN

LD78-β | CCL3L1 P16619 CCL3L1

LEAP-1 | Hepcidin P81172 HAMP

Legumain | Asparaginyl Q99538 LGMN endopeptidase

Leptin | Obesity protein (OB) P41159 LEP

Leptin receptor P48357 LEPR

Leucine carboxyl methyltransferase 1 Q9UIC8 LCMT1

Leucine-rich repeat transmembrane Q86UE6 LRRTM1 neuronal protein 1

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Excluded from Protein Analyte UniProt ID Gene Analysis

Leucine-rich repeat transmembrane Q86VH5 LRRTM3 neuronal protein 3

Leucine-rich repeats and Ig-like Q6UXM1 LRIG3 domains protein 3

Leukemia inhibitory factor receptor P42702 LIFR extracellular domain

Leukocyte immunoglobulin-like receptor subfamily B member 1 | Q8NHL6 LILRB1

CD85j | ILT-2

Leukocyte immunoglobulin-like receptor subfamily B member 2 | Q8N423 LILRB2

CD85d | ILT-4

Leukotriene A-4 hydrolase P09960 LTA4H

LIGHT | TNFSF14 O43557 TNFSF14

Limbic system-associated membrane Q13449 LSAMP protein | LAMP

Lipocalin 2 | NGAL P80188 LCN2 Y

Lipopolysaccharide-binding protein P18428 LBP

Liver-expressed chemokine | HCC-4 O15467 CCL16

Livin | Baculoviral IAP repeat Q96CA5 BIRC7 containing 7 | ML-IAP

Low affinity immunoglobulin gamma P12318, P31994 FCGR2A FCGR2B Fc region receptor II-a/b

LRP8 | Apolipoprotein E receptor 2 Q14114 LRP8

L-Selectin | CD62L P14151 SELL

Luteinizing hormone P01215, P01229 CGA LHB

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Excluded from Protein Analyte UniProt ID Gene Analysis

Lymphatic vessel endothelial Q9Y5Y7 LYVE1 hyaluronic acid receptor 1

Lymphocyte activation gene-1 | LAG-1 Q8NHW4 CCL4L1 | CCL4L1

Lymphocyte antigen 86 | Myeloid O95711 LY86 differentiation 1 | MD-1

Lymphocyte-activation gene 3 | LAG-3 P18627 LAG3

Lymphocyte-specific protein tyrosine P06239 LCK kinase Lck

Lymphotactin P47992 XCL1

Lymphotoxin α1:β2 P01374, Q06643 LTA LTB

Lymphotoxin α2:β1 P01374, Q06643 LTA LTB

Lymphotoxin β receptor P36941 LTBR

Lyn kinase | Lyn A P07948 LYN

Lyn kinase | Lyn B P07948 LYN

Lysosome membrane protein 2 | Q14108 SCARB2 LIMPII | SCARB2

Lysozyme P61626 LYZ

M2-pyruvate kinase P14618 PKM2

Macrophage colony-stimulating factor P09603 CSF1 1 | M-CSF

Macrophage colony-stimulating factor P07333 CSF1R 1 receptor | M-CSF R

Macrophage inflammatory protein 1 α P10147 CCL3 | MIP-1a

Macrophage inflammatory protein 3 α P78556 CCL20 | MIP-3a

Macrophage inflammatory protein 3 β Q99731 CCL19 | MIP-3b

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Excluded from Protein Analyte UniProt ID Gene Analysis

Macrophage inflammatory protein 5 | Q16663 CCL15 Y MIP-1 delta | Leukotactin-1 | Lkn-1

Macrophage mannose receptor P22897 MRC1

Macrophage mannose receptor 2 | Q9UBG0 MRC2 uPARAP | Endo180

Macrophage scavenger receptor | SR- P21757 MSR1 A

Macrophage stimulatory protein | P26927 MST1 MSP

Macrophage stimulatory protein Q04912 MST1R receptor | RON

Macrophage-capping protein | Actin- P40121 CAPG capping protein CapG

Macrophage-derived chemokine | O00626 CCL22 MDC

Malate dehydrogenase, cytoplasmic P40925 MDH1

Mammaglobin B | Secretoglobin 2A1 O75556 SCGB2A1

Mannan-binding lectin serine P48740 MASP1 peptidase 1 | MASP3 catalytic domain

Mannose-binding protein C | MBL P11226 MBL2

MAPK 1 | MAP kinase 2 | ERK-2 |p42- P28482 MAPK1 MAPK

MAPK 11 | MAP kinase p38 β | SAPK2 Q15759 MAPK11

MAPK 12 | p38 γ P53778 MAPK12

MAPK 13 | p38 δ O15264 MAPK13

MAPK 14 | SAP KINASE 2A Q16539 MAPK14

MAPK 3 | ERK-1 | p44-MAPK P27361 MAPK3

MAPK 8 | JNK1 P45983 MAPK8

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Excluded from Protein Analyte UniProt ID Gene Analysis

MAPK kinase 1 | MEK1 Q02750 MAP2K1

MAPK kinase 2 | MEK2 P36507 MAP2K2

MAPK kinase 4 | SAPK-ERK kinase 1 | P45985 MAP2K4 JNKK

MAPK-activated protein kinase 2 P49137 MAPKAPK2

MAPK-activated protein kinase 3 Q16644 MAPKAPK3

MAPK-activated protein kinase 5 | Q8IW41 MAPKAPK5 PRAK

Marapsin | pancreasin Q9BQR3 PRSS27 Y

Matrilin-2 O00339 MATN2

Matrilin-3 O15232 MATN3

Matrix extracellular Q9NQ76 MEPE phosphoglycoprotein | OF45

MDM2 ubiquitin ligase Q00987 MDM2

Mediator complex subunit 1 | MED1 | Q15648 MED1 TRAP220

Megakaryocyte-associated tyrosine- P42679 MATK protein kinase | Hyl | CTK

Melanoma inhibitory activity | Cartilage-derived retinoic acid- Q16674 MIA

sensitive protein | CD-RAP

Membrane frizzled-related protein | Q9BY79 MFRP MFRP

Mesothelin Q13421 MSLN

Methionine aminopeptidase 1 P53582 METAP1

Methionine aminopeptidase 2 P50579 METAP2

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Excluded from Protein Analyte UniProt ID Gene Analysis

Methyl-CpG-binding domain protein 4 O95243 MBD4 | Base excision repair enzyme MED1

MHC class I polypeptide-related Q29983 MICA sequence A

MHC class I polypeptide-related Q29980 MICB sequence B

Microtubule-associated protein tau P10636 MAPT

Midkine P21741 MDK

MIF | macrophage migration P14174 MIF inhibitory factor

Milk fat globule-EGF factor 8 Q08431 MFGE8

Mitochondrial glutamate carrier 2 Q9H1K4 SLC25A18

Mitogen-activated protein kinase 9 | P45984 MAPK9 Y JNK2 α2

MMP-1 | Matrix metalloproteinase 1 | P03956 MMP1 Collagenase 1

MMP-10 | Matrix metalloproteinase P09238 MMP10 10 | Stromelysin 2

MMP-12 | Matrix metalloproteinase P39900 MMP12 12 | Macrophage metalloelastase

MMP-13 | Matrix metalloproteinase P45452 MMP13 13 | Collagenase 3

MMP-14 | Matrix metalloproteinase 14 | Membrane-type matrix P50281 MMP14

metalloproteinase 1

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Excluded from Protein Analyte UniProt ID Gene Analysis

MMP-16 | Matrix metalloproteinase 16 | Membrane-type matrix P51512 MMP16

metalloproteinase 3

MMP-17 | Matrix metalloproteinase 17 | Membrane-type matrix Q9ULZ9 MMP17

metalloproteinase 4

MMP-2 | Matrix metalloproteinase 2 | P08253 MMP2 Gelatinase A

MMP-3 | Matrix metalloproteinase 3 | P08254 MMP3 Stromelysin 1

MMP-7 | Matrix metalloproteinase 7 | P09237 MMP7 Matrilysin

MMP-8 | Matrix metalloproteinase 8 | P22894 MMP8 Neutrophil collagenase

MMP-9 | Matrix metalloproteinase 9 | P14780 MMP9 Gelatinase B

Moesin P26038 MSN

Monocyte chemoattractant protein 1 P13500 CCL2 | MCP-1

Monocyte chemoattractant protein 2 P80075 CCL8 | MCP-2

Monocyte chemoattractant protein 3 P80098 CCL7 | MCP-3

Monocyte chemoattractant protein 4 Q99616 CCL13 | MCP-4

MPIF-1 | Myeloid progenitor P55773 CCL23 inhibitory factor 1

Muellerian-inhibiting factor | MIS P03971 AMH

Myeloperoxidase P05164 MPO

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Excluded from Protein Analyte UniProt ID Gene Analysis Myoglobin P02144 MB

Myotonic dystrophy protein kinase- Q9Y5S2 CDC42BPB like β | MRCKB

N-acetyl-D-glucosamine kinase Q9UJ70 NAGK

N-acetylglucosamine-6-sulfatase P15586 GNS Y

NADPH-P450 Oxidoreductase P16435 POR

Nascent polypeptide-associated Q13765 NACA Y complex α subunit | αNAC

Natural cytotoxicity triggering O76036 NCR1 receptor 1 | NKp46

Natural cytotoxicity triggering O95944 NCR2 receptor 2 | NKp44

Natural cytotoxicity triggering O14931 NCR3 receptor 3 | NKp30

Natural killer group 2 member D | P26718 KLRK1 NKG2D

NCC27 | Chloride intracellular channel O00299 CLIC1 protein 1

NCK-α SH2-SH3 adaptor protein P16333 NCK1

Nectin-like protein 1 | IGSF4B | NECL1 Q8N126 CADM3 | TSLL1 | SynCAM3

Nectin-like protein 2 | IGSF4 | TSLC1 | Q9BY67 CADM1 SynCAM1

Nephroblastoma overexpressed gene P48745 NOV | NovH | CCN3 | IGFBP9

Neprilysin-2 Q495T6 MMEL1

N-ethylmaleimide-sensitive factor P54920 NAPA (NSF) attachment protein α | αSNAP

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Excluded from Protein Analyte UniProt ID Gene Analysis

Netrin receptor UNC5H3 O95185 UNC5C

Netrin receptor UNC5H4 Q6UXZ4 UNC5D

Netrin-4 Q9HB63 NTN4

Neural cell adhesion molecule 1 P13591 NCAM1 (CD56), 120 kDa isoform

Neural cell adhesion molecule L1 P32004 L1CAM

Neural cell adhesion molecule L1-like O00533 CHL1 protein | L1CAM-2

Neuregulin-1 | Heregulin Q02297 NRG1

Neurexin-1-β P58400 NRXN1

Neurexin-3-β Q9HDB5 NRXN3

Neurexophilin-1 P58417 NXPH1

Neuroblastoma suppressor of P41271 NBL1 tumorigenicity 1 | DAN

Neuroligin 4, X-linked Q8N0W4 NLGN4X

Neuropilin-1 O14786 NRP1

Neurotrophic tyrosine kinase receptor P04629 NTRK1 type 1 | TrkA

Neurotrophic tyrosine kinase receptor Q16620 NTRK2 type 2 | TrkB

Neurotrophic tyrosine kinase receptor Q16288 NTRK3 type 3 | TrkC

Neurotrophin-3 | NT-3 P20783 NTF3

Neurotrophin-4 | NT-4 | P34130 NTF4 Neurotrophin-5 | NT-5

Neutral ceramidase Q9NR71 ASAH2

Neutrophil elastase | Leukocyte P08246 ELANE elastase | Elastase 2 | PMN-elastase

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Excluded from Protein Analyte UniProt ID Gene Analysis

Neutrophil-activating peptide 2 | NAP- P02775 PPBP 2 | CXCL7

Nicotinamide phosphoribosyltransferase | Pre-B-cell P43490 NAMPT

colony-enhancing factor 1 | Visfatin

Nidogen | Entactin P14543 NID1

Nidogen-2 Q14112 NID2

N-myristoyltransferase 1 P30419 NMT1

Noggin Q13253 NOG Y

Nogo receptor | reticulon 4 receptor Q9BZR6 RTN4R

Non-pancreatic secretory P14555 PLA2G2A phospholipase A2 |

Notch 1 P46531 NOTCH1

Notch 2 Q04721 NOTCH2

Notch 3 Q9UM47 NOTCH3

NR1D1 | nuclear receptor subfamily 1, P20393 NR1D1 group D, member 1 | Rev-erbA-α

NRCAM | neuronal cell adhesion Q92823 NRCAM molecule

Nuclear oncogene protein SET | TAF-Iβ Q01105 SET Y | I-2PP2A

Nucleoside diphosphate kinase A | P15531 NME1 Y Nm23-H1

Nucleoside diphosphate kinase B | P22392 NME2 Nm23-H2

NudC domain-containing protein 3 | Q8IVD9 NUDCD3 NudCL

Olfactomedin-4 | hGC-1 | GW112 Q6UX06 OLFM4 Y

Oncostatin M P13725 OSM

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Excluded from Protein Analyte UniProt ID Gene Analysis

Opioid-binding cell adhesion molecule Q14982 OPCML | OBCAM

Osteoactivin | GPNMB Q14956 GPNMB

Osteomodulin | Osteoadherin Q99983 OMD

Osteonectin P09486 SPARC

Osteoprotegerin | OPG O00300 TNFRSF11B Y

Osteoprotegerin ligand | TRANCE | O14788 TNFSF11 sRANKL

Ovarian cancer immunoreactive antigen domain containing 1 | OCIA Q9NX40 OCIAD1

domain containing 1

OX40 Ligand | Tumor necrosis factor P23510 TNFSF4 ligand superfamily member 4

Oxidized low-density lipoprotein P78380 OLR1 receptor 1 | LOX-1

OXO | CD134 P43489 TNFRSF4

p21-activated kinase 3 O75914 PAK3 Y

p21-activated kinase 6 Q9NQU5 PAK6

p21-activated kinase 7 | KIAA1264 Q9P286 PAK7

p97 cofactor p47 Q9UNZ2 NSFL1C

PAFAH β subunit | Platelet-activating P68402 PAFAH1B2 factor acetylhydrolase IB subunit β

Pancreatic hormone P01298 PPY

Paraoxonase 1 P27169 PON1

Parathyroid hormone P01270 PTH

Parathyroid hormone-related protein P12272 PTHLH | PTHrP

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Excluded from Protein Analyte UniProt ID Gene Analysis

PARK7 | Parkinson protein 7 | DJ-1 Q99497 PARK7

PCSK9 | Proprotein convertase Q8NBP7 PCSK9 subtilisin kexin type 9

PDK-1 | 3-phosphoinositide- O15530 PDPK1 dependent protein kinase 1

Peptide YY P10082 PYY

Peptidoglycan recognition protein, O75594 PGLYRP1 short | PGRP-S

Peptidylprolyl isomerase A P62937 PPIA Y |Cyclophilin A

Peptidylprolyl isomerase D | Q08752 PPID Cyclophilin D | Cyclophilin 40

Peptidylprolyl isomerase E | Q9UNP9 PPIE Cyclophilin E

Peptidylprolyl isomerase F (Cyclophilin P30405 PPIF Y F)

Periostin | Osteoblast-specific factor 2 Q15063 POSTN | OSF-2

Peroxiredoxin-1 Q06830 PRDX1

Peroxiredoxin-5 P30044 PRDX5

Peroxiredoxin-6 | AOP2 | 1-cysPrx P30041 PRDX6

Persephin O60542 PSPN

Pescadillo O00541 PES1

Phosphatase and tensin homolog P60484 PTEN

Phosphatidylethanolamine-binding P30086 PEBP1 protein 1 | HCNP | RKIP

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Excluded from Protein Analyte UniProt ID Gene Analysis

Phosphatidylinositol-4,5-bisphosphate P48736 PIK3CG 3-kinase catalytic subunit γ | p110 γ

Phosphodiesterase 2A, cGMP- O00408 PDE2A stimulated

Phosphoglycerate kinase 1 P00558 PGK1

Phosphoglycerate mutase 1 P18669 PGAM1 Y

Phosphoinositide-3-kinase catalytic α polypeptide:regulatory subunit 1α P42336, P27986 PIK3CA PIK3R1

complex | p110 α:p85 α

Phospholipase A2, Group IB | type I P04054 PLA2G1B pancreatic sPLA2

Phospholipase A2, Group IIE Q9NZK7 PLA2G2E

Phospholipase A2, Group V P39877 PLA2G5

Phospholipase A2, Group X O15496 PLA2G10

Phospholipase C-II | PLCγ1 P19174 PLCG1

Pituitary adenylate cyclase-activating P18509 ADCYAP1 polypeptide 27 | PACAP-27

Pituitary adenylate cyclase-activating P18509 ADCYAP1 polypeptide 38 | PACAP-38

Placenta growth factor | PlGF P49763 PGF

Plasmin P00747 PLG

Plasminogen P00747 PLG

Plasminogen activator inhibitor 1 P05121 SERPINE1

Platelet endothelial cell adhesion P16284 PECAM1 molecule | CD31

Platelet factor 4 P02776 PF4 Y

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Excluded from Protein Analyte UniProt ID Gene Analysis

Platelet glycoprotein Ib α P07359 GP1BA

Platelet-activating factor Q13093 PLA2G7 acetylhydrolase | PAF-AH | Lp-PLA2

Platelet-derived growth factor A chain P04085 PDGFA homodimer

Platelet-derived growth factor B chain P01127 PDGFB homodimer

Platelet-derived growth factor C chain Q9NRA1 PDGFC homodimer

Platelet-derived growth factor P09619 PDGFRB receptor β-type

Pleiotrophin P21246 PTN Y Plexin C1 O60486 PLXNC1

Polymeric immunoglobulin receptor P01833 PIGR

Prefoldin subunit 5 | MM-1 Q99471 PFDN5

Pregnancy-associated plasma protein- Q13219 PAPPA A | Pappalysin-1 | DIPLA1

Prekallikrein P03952 KLKB1

Programmed cell death 1 ligand 2 | Q9BQ51 PDCD1LG2 PD-L2 | B7-DC

Progranulin P28799 GRN

Prolactin P01236 PRL

Prolactin receptor P16471 PRLR

Proliferating cell nuclear antigen | P12004 PCNA PCNA

Properdin | Factor P P27918 CFP

Proprotein convertase 7 Q16549 PCSK7

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Excluded from Protein Analyte UniProt ID Gene Analysis

Prostate-specific membrane antigen | Q04609 FOLH1 PSMA

Protease nexin 1 | PN1 P07093 SERPINE2

Proteasome activator complex subunit Q06323 PSME1 1 | PA28 α

Proteasome activator complex subunit P61289 PSME3 3 | REG-γ

Proteasome subunit p40 | MOV34 | P51665 PSMD7 Mov34 protein homolog

Proteasome subunit α type-1 P25786 PSMA1

Proteasome subunit α type-2 | α-type P25787 PSMA2 HC3 Proteasome subunit α type-6 | PROS- P60900 PSMA6 27 Protein C P04070 PROC

Protein C Inhibitor P05154 SERPINA5 Y

Protein disulfide isomerase A3 | P30101 PDIA3 ERp57 | GRp58 | ER-60

Protein disulfide-isomerase P07237 P4HB

Protein E7 (Human Papillomavirus P03129 HPV18 E7 Type 16)

Protein E7 (Human Papillomavirus P06788 HPV16 E7 Type 18)

Protein FAM107B Q9H098 FAM107B

Protein kinase B α/β/γ (RAC family) Family

Protein kinase C α P17252 PRKCA

Protein kinase C β type (splice variant P05771 PRKCB β-II)

Protein kinase C γ P05129 PRKCG Y Protein kinase C δ Q05655 PRKCD

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Excluded from Protein Analyte UniProt ID Gene Analysis

Protein kinase C ζ | PKCzeta Q05513 PRKCZ

Protein kinase C θ Q04759 PRKCQ

Protein kinase C ι P41743 PRKCI

Protein lin-7 homolog B | MALS-2 | Q9HAP6 LIN7B Veli-2

Protein Rev (HIV) P18093 HIV rev

Protein S P07225 PROS1

Proteinase-3 | PR3 P24158 PRTN3

Prothrombin | Coagulation factor II P00734 F2

Proto-oncogene serine/threonine- P11309 PIM1 protein kinase Pim-1

Proto-oncogene tyrosine-protein P09769 FGR kinase Fgr

Proto-oncogene tyrosine-protein P06241 FYN kinase Fyn | FYNT

Proto-oncogene tyrosine-protein P07949 RET kinase receptor Ret

Proto-oncogene tyrosine-protein P12931 SRC kinase Src | SRC N1

Proto-oncogene tyrosine-protein P07947 YES1 kinase Yes

P-Selectin | CD62P P16109 SELP

Pulmonary surfactant-associated P35247 SFTPD protein D | SP-D

Pyridoxal phosphate phosphatase Q96GD0 PDXP

Pyruvate dehydrogenase kinase, Q15118 PDK1 isozyme 1

Rab GDP dissociation inhibitor β P50395 GDI2

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Excluded from Protein Analyte UniProt ID Gene Analysis

RAGE, soluble | Advanced glycosylation end product-specific Q15109 AGER

receptor

RANTES | CCL5 P13501 CCL5

RAS p21 protein activator | p120GAP P20936 RASA1

Ras-related C3 botulinum toxin P63000 RAC1 substrate 1

Receptor activator of NF-KB | RANK Q9Y6Q6 TNFRSF11A

Receptor-type tyrosine-protein kinase P36888 FLT3 FLT3 | Flt-3 | Flk-2

RELT tumor necrosis factor receptor | Q969Z4 RELT TNFRSF19L

Renin P00797 REN

Repulsive guidance molecule A Q96B86 RGMA

Resistin | FIZZ3 | Adipose tissue- Q9HD89 RETN specific secretory factor | ADSF

Reticulon-4 | Nogo-A Q9NQC3 RTN4

Retinoblastoma 1 P06400 RB1

Retinol-binding protein 4 P02753 RBP4 Y

RGM domain family member B Q6NW40 RGMB

Ribosomal protein S3 P23396 RPS3

Ribosomal protein S6 kinase 5 | Mitogen- and stress-activated kinase 1 O75582 RPS6KA5

| MSK1 | RSK-like protein kinase

Ribosomal protein S6 kinase α-3 | P51812 RPS6KA3 RSK2 Ribosomal protein S7 P62081 RPS7

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Excluded from Protein Analyte UniProt ID Gene Analysis

Ribosome maturation protein SBDS Q9Y3A5 SBDS

RNA-binding motif protein 39 | Q14498 RBM39 CAPER-α Roof plate-specific spondin-2, isoform Q6UXX9 RSPO2 1

Roundabout axon guidance molecule Q9HCK4 ROBO2 2, ROBO2

Roundabout axon guidance molecule Q96MS0 ROBO3 3, ROBO3

S100A9 | calgranulin B | MRP-14 P06702 S100A9 Y

Scavenger receptor class F member 1 Q14162 SCARF1 | SREC-I

Scavenger receptor class F member 2 Q96GP6 SCARF2 | SREC-II

Scavenger receptor cysteine-rich type Q86VB7 CD163 1 protein M130 chain | Soluble CD163

Scavenger receptor for phosphatidylserine and oxidized low Q9H2A7 CXCL16

density lipoprotein | CXCL16

Secretin P09683 SCT

Secretory leukocyte protease inhibitor P03973 SLPI

Seizure 6-like protein 2 Q6UXD5 SEZ6L2

Semaphorin-3A Q14563 SEMA3A

Semaphorin-3E O15041 SEMA3E

Semaphorin-6A Q9H2E6 SEMA6A

Serine/threonine kinase 17b | DRAK2 O94768 STK17B

Serine-threonine-protein kinase 16 | O75716 STK16 PKL12

Serine-threonine-protein kinase Chk1 O14757 CHEK1

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Excluded from Protein Analyte UniProt ID Gene Analysis

Serine-threonine-protein kinase Chk2 O96017 CHEK2

Serine-threonine-protein kinase PLK1 P53350 PLK1

Serum amyloid A P0DJI8 SAA1

Serum amyloid P P02743 APCS

Sex hormone-binding globulin P04278 SHBG

SH2 domain-containing protein 1A | O60880 SH2D1A Y SLAM-associated protein (SAP)

SHC-transforming protein 1 P29353 SHC1

Shh receptor Boc Q9BWV1 BOC

Siglec-1 | Sialoadhesin Q9BZZ2 SIGLEC1

Siglec-14 Q08ET2 SIGLEC14

Siglec-3 P20138 CD33

Siglec-6 | CD327 O43699 SIGLEC6

Siglec-7 | CD328 Q9Y286 SIGLEC7

Siglec-9 Q9Y336 SIGLEC9

Signaling lymphocytic activation Q9UIB8 CD84 molecule 5 | SLAMF5

Sirtuin 2 Q8IXJ6 SIRT2

SLAM family member 6 | NTB-A Q96DU3 SLAMF6

SLAM family member 7 | CRACC | CS1 Q9NQ25 SLAMF7

SLIT and NTRK-like protein 5 O94991 SLITRK5

SMAC | DIABLO Q9NR28 DIABLO Y

Small glutamine-rich tetratricopeptide repeat-containing protein α | Vpu- O43765 SGTA

binding protein

Small nuclear ribonucleoprotein F P62306 SNRPF

Soggy-1 Q9UK85 DKKL1

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Excluded from Protein Analyte UniProt ID Gene Analysis Somatostatin-28 P61278 SST

Sonic hedgehog Q15465 SHH

Sortilin-related VPS10 domain Q96PQ0 SORCS2 containing receptor 2 | SorCS2

Sorting nexin 4 O95219 SNX4

SPARC-like 1 (hevin) Q14515 SPARCL1

S-phase kinase-associated protein 1 | P63208 SKP1 OCP2

Sphingomyelin phosphodiesterase, Q92484 SMPDL3A acid-like 3A | ASML3a

Sphingosine kinase 1 Q9NYA1 SPHK1

Sphingosine kinase 2 Q9NRA0 SPHK2

Spondin-1 | F-Spondin Q9HCB6 SPON1

Stabilin-2 Q8WWQ8 STAB2

Stanniocalcin-1 P52823 STC1

Stem cell factor receptor | CD117 | P10721 KIT SCF R | c-Kit

Stem cell growth factor-α Q9Y240 CLEC11A

Stem cell growth factor-β Q9Y240 CLEC11A

Stress-induced-phosphoprotein 1 P31948 STIP1

Stromal cell-derived factor 1 | SDF-1 P48061 CXCL12

SUMO-conjugating enzyme UBC9 | P63279 UBE2I UBE2I | Ubc9

Superoxide dismutase [Cu-Zn] P00441 SOD1

Superoxide dismutase [Mn] P04179 SOD2

Survivin O15392 BIRC5

Syntaxin 1A | HPC1 Q16623 STX1A

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Excluded from Protein Analyte UniProt ID Gene Analysis

T cell membrane protein TIM-3 | Hepatitis A virus cellular receptor 2 | Q8TDQ0 HAVCR2

TIM-3

TACI | Transmembrane activator and O14836 TNFRSF13B CAML interactor | TNFRSF13B

TAK1-TAB1 fusion O43318, Q15750 MAP3K7 TAB1

TANK-binding kinase 1 Q9UHD2 TBK1

Tartrate-resistant acid phosphatase P13686 ACP5 type 5 | TRACP | PAP

TATA-box-binding protein P20226 TBP

T-cell surface glycoprotein CD4 P01730 CD4

Tenascin-C P24821 TNC

Testican-1 | SPOCK1 Q08629 SPOCK1

Testican-2 | SPOCK2 Q92563 SPOCK2

TFF3 | Trefoil factor 3 | Intestinal Q07654 TFF3 trefoil factor | hITF

TGF-β receptor II P37173 TGFBR2

Thioredoxin domain-containing O95881 TXNDC12 protein 12 Thrombin P00734 F2

Thrombin-activatable fibrinolysis Q96IY4 CPB2 inhibitor

Thrombopoietin receptor P40238 MPL

Thrombospondin-1 P07996 THBS1 Y Thrombospondin-2 P35442 THBS2

Thrombospondin-4 P35443 THBS4

Thymic stromal lymphopoietin Q969D9 TSLP

Thymic stromal lymphopoietin protein Q9HC73 CRLF2 receptor | TSLP R

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Excluded from Protein Analyte UniProt ID Gene Analysis

Thymidine kinase 1, cytosolic P04183 TK1

Thymidylate synthase P04818 TYMS

Thymus and activation-regulated Q92583 CCL17 chemokine | TARC

Thymus expressed chemokine | TECK O15444 CCL25

Thyroglobulin P01266 TG

Thyroid peroxidase P07202 TPO

Thyroid stimulating hormone | TSH P01215, P01222 CGA TSHB

Thyroxine-binding globulin P05543 SERPINA7

TIMP1 | Tissue inhibitor of P01033 TIMP1 metalloproteinases 1

TIMP2 | Tissue inhibitor of P16035 TIMP2 metalloproteinases 2

TIMP3 | Tissue inhibitor of P35625 TIMP3 metalloproteinases 3

Tissue factor | Coagulation Factor III P13726 F3

Tissue factor pathway inhibitor P10646 TFPI

TLR4:MD-2 complex | Toll-like receptor 4:Myeloid differentiation O00206 Q9Y6Y9 TLR4 LY96

factor-2 complex

T-lymphocyte activation antigen CD80 P33681 CD80 | B7-1

T-lymphocyte surface antigen Ly-9 | Q9HBG7 LY9 CD229 | SLAMF3

TNF RI | Tumor necrosis factor P19438 TNFRSF1A receptor superfamily member 1A

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Excluded from Protein Analyte UniProt ID Gene Analysis

TNF RII | Tumor necrosis factor P20333 TNFRSF1B receptor superfamily member 1B

TNF-like ligand 1A | TL1A | TNFSF15 | O95150 TNFSF15 VEGF inhibitor | VEGI

TNF-α | Tumor necrosis factor ligand P01375 TNF superfamily member 2

TNF-β | Lymphotoxin-α | Tumor necrosis factor ligand superfamily P01374 LTA

member 1

Toll-like receptor 2 O60603 TLR2

tPA | Tissue-type plasminogen P00750 PLAT activator

TRAIL R1 | Tumor necrosis factor O00220 TNFRSF10A receptor superfamily member 10A

TRAIL R4 | Tumor necrosis factor Q9UBN6 TNFRSF10D receptor superfamily member 10D

Transcription factor MLR1 | Ligand- dependent nuclear receptor Q8N3X6 LCORL

corepressor-like protein

Transferrin P02787 TF

Transforming growth factor β induced Q15582 TGFBI protein ig-h3

Transforming growth factor β receptor Q03167 TGFBR3 type III

Transforming growth factor β-1 P01137 TGFB1

Transforming growth factor β-2 P61812 TGFB2

Transforming growth factor β-3 P10600 TGFB3

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Excluded from Protein Analyte UniProt ID Gene Analysis Transglutaminase 3 Q08188 TGM3

Transketolase P29401 TKT

Triosephosphate isomerase P60174 TPI1

TROP-2 | Tumor-associated calcium P09758 TACSTD2 signal transducer 2

Tropomyosin 1 P09493 TPM1

Tropomyosin β chain P07951 TPM2

Troponin I, cardiac P19429 TNNI3

TROY | TNFRSF19 Q9NS68 TNFRSF19

Trypsin P07477 PRSS1

Trypsin-2 P07478 PRSS2

Trypsin-3 P35030 PRSS3

Tryptase β-2 P20231 TPSB2

Tryptase γ Q9NRR2 TPSG1

TSG-6 | Tumor necrosis factor- P98066 TNFAIP6 inducible gene 6 protein

TWEAK |Tumor necrosis factor ligand O43508 TNFSF12 superfamily member 12

TWEAK receptor | TNFRSF12A Q9NP84 TNFRSF12A

Tyrosine kinase 2 P29597 TYK2 Y

Tyrosine kinase Bruton Q06187 BTK

Tyrosine kinase Etk | BMX non- P51813 BMX receptor tyrosine kinase

Tyrosine kinase Fer P16591 FER

Tyrosine phosphatase SHP-1 P29350 PTPN6

Tyrosine-protein kinase 6 | Brk Q13882 PTK6

Tyrosine-protein kinase receptor Tie-1, P35590 TIE1 soluble

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Excluded from Protein Analyte UniProt ID Gene Analysis

Tyrosine-protein kinase receptor Tie-2, Q02763 TEK soluble | Angiopoietin-1 receptor

Tyrosine-protein kinase receptor Q06418 TYRO3 TYRO3 | Dtk

Tyrosine-protein kinase Tec P42680 TEC

Tyrosine-protein kinase Q01973 ROR1 transmembrane receptor ROR1

Tyrosine-protein phosphatase non- P18031 PTPN1 receptor type 1 | PTP1B

Tyrosine-protein phosphatase non- Q06124 PTPN11 receptor type 11 | SHP-2

Tyrosine-protein phosphatase non- P17706 PTPN2 receptor type 2 | TC-PTP

Ubiquitin P62979 RPS27A Y

Ubiquitin C-terminal hydrolase-L1 | P09936 UCHL1 PGP9.5

Ubiquitin+1, truncated mutation for P62979 RPS27A Y UbB

Ubiquitin-conjugating enzyme E2 L3 P68036 UBE2L3

Ubiquitin-conjugating enzyme E2 N P61088 UBE2N

Ubiquitin-fold modifier 1 P61960 UFM1

Ubiquitin-fold modifier-conjugating Q9Y3C8 UFC1 enzyme 1

UL16-binding protein 1 | NKG2D Q9BZM6 ULBP1 ligand 1

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Excluded from Protein Analyte UniProt ID Gene Analysis UL16-binding protein 2 | NKG2D Q9BZM5 ULBP2 ligand 2

UL16-binding protein 3 Q9BZM4 ULBP3

UMP-CMP kinase P30085 CMPK1

uPA |Urokinase-type plasminogen P00749 PLAU activator

uPAR | Urokinase plasminogen Q03405 PLAUR activator surface receptor

v-abl Abelson murine leukemia viral P42684 ABL2 oncogene homolog 2 | ARG

Vaccinia virus VH1-related phosphatase | Dual specificity protein P51452 DUSP3

| VHR phosphatase 3

Vascular cell adhesion protein 1 | P19320 VCAM1 VCAM 1 | CD106

Vascular endothelial growth factor A | P15692 VEGFA VEGF 165

Vascular endothelial growth factor A, P15692 VEGFA secreted splice variant | VEGF 121

Vascular endothelial growth factor C P49767 VEGFC

Vascular endothelial growth factor P35968 KDR receptor 2

Vascular endothelial growth factor P35916 FLT4 receptor 3 | Flt-4

Vasoactive intestinal peptide P01282 VIP

von Willebrand factor P04275 VWF

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Excluded from Protein Analyte UniProt ID Gene Analysis

WAP, kazal, immunoglobulin, kunitz and NTR domain-containing protein 2 Q8TEU8 WFIKKN2 | GDF serum-associated protein-1 | GASP-1

Wingless-type MMTV integration site O00755 WNT7A family, member 7A | Wnt-7a

Wnt inhibitory factor 1 Q9Y5W5 WIF1

WNT1-inducible-signaling pathway O95388 WISP1 Y protein 1 | WISP-1 | CCN4

X-linked ectodysplasin-A2 receptor | Q9HAV5 EDA2R SF27 | XEDAR

X-Pro aminopeptidase 1 Q9NQW7 XPNPEP1

ZAP70 | 70 kDa zeta-associated P43403 ZAP70 protein kinase

α1-Antichymotrypsin P01011 SERPINA3

α1-antichymotrypsin complex | ACT P01011 SERPINA3 complex

α1-Antitrypsin P01009 SERPINA1

α2-Antiplasmin P08697 SERPINF2

α2-HS-Glycoprotein | Fetuin A P02765 AHSG

α2-Macroglobulin P01023 A2M Y

α2-Macroglobulin receptor-associated P30533 LRPAP1 protein

αII-Spectrin | α-fodrin Q13813 SPTAN1

α-L-iduronidase P35475 IDUA

α-S1-casein P47710 CSN1S1

β2-Microglobulin P61769 B2M

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Excluded from Protein Analyte UniProt ID Gene Analysis

β-Adrenergic receptor kinase 1 | GRK2 P25098 ADRBK1

β-Endorphin P01189 POMC Y β-Nerve growth factor P01138 NGF

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3 Variable Selection and Statistical Modeling The LASSO1 was used as a variable screening procedure to identify a subset of proteins jointly associated with increased cardiovascular risk. Cross-validation using coxnet2 in the R package glmnet3 was used to set the LASSO regularization parameter. The “one standard error” rule4 was used for setting the regularization level. To establish the sensitivity of the variable selection procedure to the cross- validation process the combination of LASSO + backward elimination was repeated 100 times with different cross-validation folds and a slight variant of the data pre-processing and outlier filtering steps. With this level of perturbation 7 of the 9 proteins were included in the final model at least 95% of the time. Two proteins appear in the final model less frequently under these perturbations – one approximately 60% of the time and another only 30% of time. For context, the other LASSO-selected proteins appear in the final model fewer than 2% of the time so the proteins that comprise the 9 protein model are generally what one would find using LASSO with regularization level set using the 1-standard error rule. To generate reproducible results for the ensuing analysis the random number seed was fixed at 1 prior to LASSO cross-validation. Initializing this value and setting the LASSO regularization parameter to the value 1 standard error above that which minimized the cross-validated partial likelihood deviance resulted in a LASSO model containing the 16 proteins discussed in the main manuscript. Ideally when using LASSO for variable selection the goal should be to identify a subset of variables that is relatively insensitive to the precise LASSO regularization level. In practice some of the variables selected are more sensitive to the specific regularization level than others so the concept of “stability5,6” was used to regularization level to guide the variable subset selection. Specifically, the sensitivity of individual variables to the LASSO regularization level was quantified by calculating the empirical selection probability for each of the 1054 proteins using an adaptation of stability selection6 for Cox proportional hazards models fitted using the R function glmnet. The method involves partitioning the data and sweeping the LASSO regularization parameter across its full range in uniform (logarithmic) steps. At each step the proteins that are included in the model at that specific regularization level were tabulated. Over many such random partitions this process generated a curve giving the empirical selection probability for each protein as a function of LASSO regularization level.

Using this method the median selection probability for the 9 proteins was 0.91 indicating that half were selected at least 91% of the time in models constructed using a wide range of LASSO regularization levels. Only three of the 9 proteins had selection probabilities below 0.75 and the least stable protein was still selected more than 60% of the time.

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That most of the proteins have selection probabilities at least 0.75 provided confidence that the proteins in the 9-protein model are not particularly sensitive to the specific choice of LASSO regularization parameter.

Though the variable selection procedure was stable as discussed above, it was nonetheless still possible to create alternative protein models from the set of LASSO-selected (or stability selection) proteins. Using a more stringent data standardization procedure an alternate 9 protein model was derived that differed in a single protein from the one discussed in the text. This alternate model had similar discrimination performance but owing to the improved data standardization it did not require recalibration before application to the validation cohort.

4 Recalibration for Validation The need for prognostic model recalibration is dictated by underlying differences in the baseline survivor function in the derivation and validation cohorts. For both the refit Framingham model and 9- protein model, the “validation by calibration” program outlined in van Houwelingen7 was followed, considering both Cox and Weibull AFT recalibration models for formal recalibration assessment in the validation cohort. Based on an empirical assessment of calibration performance, the 9-protein model was recalibrated with the Weibull accelerated failure time (AFT) recalibration model of the form,

log(퐻(푡|푃퐼)) = 훾0 + 훾1푃퐼 + 훾2푒 , where PI is the prognostic index, H(t|PI) is the cumulative hazard function with error term 푒 having an

PI extreme value distribution. Denoting the baseline cumulative hazard by H0(t) and using H(t|PI) = H0(t)e gives,

log(퐻0(푡)) = 훾0 + (훾1 − 1)푃퐼 + 훾2푒 . (1)

A formal calibration assessment tests the perfect calibration hypothesis, 퐻0: 훾0 = 0, 훾1 = 0, 훾2 = 1 . Fitting the model (1) using survreg from the R package survival8 gives the calibration coefficients listed in eTable 2.

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9 Protein Model Recalibration Model Coefficients Estimate 95% CI p-value 훾̂0 -0.230 -0.4189 -0.0412 0.02

훾̂1 − 1 -0.998 -1.1752 -0.8212 0.98 log (훾̂2) 0.149 0.0384 0.2598 0.008

eTable 2: Weibull recalibration model coefficients for 9-protein model.

The estimated value for the intercept term (훾̂0) is significant (p=0.02) and the resulting “recalibrated” prognostic index and extreme value scale parameter (shown below) was used in the validation cohort

PI=16.39 -1.54×ANGPT2 + 1.22×GDF11/8 - 2.11×C7 +2.64×SERPINF2 - 0.57×CCL18 - 1.02×ANGPTL4 - 1.43×KLK3.SERPINA3 - 0.72×MMP12 - 0.59×TNNI3 { s=0.98

It is not uncommon for differences in sample collection protocol to generate a systematic intensity differences for the signal levels for a large number of proteins. Blood samples in the validation cohort were collected using a more lenient collection protocol than those in the derivation cohort resulting in a systematic difference in “bulk” signal intensity between the cohorts. The data standardization procedures in use the time these samples were assayed removed this bulk difference between the cohorts. Post-hoc it was discovered that this “pre-processing” introduced an artifact in the signal levels for the 9-model proteins. eFigure 1 shows the median signal levels for the 9 proteins in the derivation and validation cohorts before (left) and after (right) pre-processing. While the pre-processing removes the bulk intensity differences in the vast majority of proteins measured in the validation cohort, it introduces a bias in these 9 proteins which are relatively insensitive to sample collection protocol differences.

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eFigure 1: Median protein level in relative fluorescent intensity units(RFU) in Derivation and Validation cohort for before (left) and after (right) data pre-processing with multiplicative scaling to remove bulk signal intensity differences in “most” proteins between the derivation and validation cohorts.

The intercept of the robust regression line in the right frame of eFigure 1 gives an estimate of the intensity bias introduced into protein signals. If we let ̂ denote this estimate then since the 9 protein model uses standardized covariates applying the model to an observation in the validation cohort resulted in the addition of the constant factor, 9 훽푗 ∆ ∑푗=1 (2), 휎푗

to what would otherwise be the contribution of the model intercept, 훽0. As a result, the signal intensity artifact introduced by the data pre-processing appears as a discrepancy in the time scale of the baseline

survivor function in the validation cohort, precisely the term associated with the parameter 훾0 in calibration model (1). Plugging the estimate ̂ = 0.058 from eFigure 1 into (2) and using the model

coefficients and population standard deviations introduced a constant factor that differed from (훾̂0) by 0.6% indicating that in fact the data processing artifact was largely responsible for the calibration result.

It was subsequently verified that applying more stringent data pre-processing procedures prior to the statistical analysis avoids the artifact discussed above resulting in a “null” recalibration model. The resulting prognostic model can be applied directly to the validation cohort without recalibration..

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To recalibrate the Framingham variable model for use in the validation cohort a single variable Cox proportional hazard recalibration7,9 model was used. This model gave superior calibration performance compared to the AFT-calibration model used for recalibrating the 9-protein model. Denoting the

baseline survivor function by S0(t), the calibrated 4-year risk score was

̂∗ 푒훽 × 푃퐼퐹푅 Prcal[푇 ≤ 푡] = 1 − 푆̂0(4) , ∗ where 훽̂ is the estimate from the calibration model and 푆̂0(t) is the Weibull model estimate of the survivor function. The table below (eTable 3) lists the resulting calibration model coefficient:

Refit Framingham model Recalibration model coefficients Estimated Cox Calibration 푆̂(4) Standard Error p-value 0 Coefficient (훽̂∗) Recalibration Coefficient 0.752 -0.5322 0.0702 <0.0001

eTable 3: Recalibration model coefficients for refit Framingham model.

Calibration plots for recalibrated proteomic model are shown in the main text and the recalibrated refit Framingham model below (eFigure 2):

eFigure 2: Calibration plots for derivation (left) and validation (right) cohorts for the refit Framingham model. Shown is the observed vs. predicted 4-year incidence of the primary outcome by deciles of predicted risk. Predicted 4-year incidence for each decile is the mean predicted risk score in each decile. Error bars indicate standard deviation. As explained above, the refit Framingham model was recalibrated for use in the validation cohort.

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5 Table of Individual Proteins Associated with Cardiovascular Risk In this study 1054 of the 1130 proteins measured by were deemed fit for analysis and 200 were significantly associated with cardiovascular risk. These 200 proteins are listed in eTable 4 Along with each protein we list the Universal Protein Resource (UniProt) (http://www.uniprot.org) identifier and the hazard ratio (both quintile and per standard deviation) along with the p-value for the latter.

eTable 4 (below): Proteins associated with risk of MI, stroke, heart failure and death at a 5% Bonferonni corrected level (correcting for 1054 comparisons) using single protein Cox Proportional hazard model. Quintile hazard ratio is the ratio of hazard for patients in the 5th (highest) quintile risk category compared to those in the 1st (lowest) quintile risk category.

Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

Angiopoietin-2 Angiopoietin-2 O15123 1.67 3.41 < 1.00E-16 MMP-12 Matrix metalloproteinase 12 P39900 1.65 3.71 < 1.00E-16 TIMD3 Hepatitis A virus cellular receptor 2 Q8TDQ0 1.61 3.44 < 1.00E-16 Insulin-like growth factor-binding IGFBP-2 P18065 1.58 3.35 < 1.00E-16 protein 2 Tumor necrosis factor receptor TNF sR-II P20333 1.56 3.72 < 1.00E-16 superfamily member 1B FSTL3 Follistatin-related protein 3 O95633 1.56 4.47 < 1.00E-16 HCC-1 C-C motif chemokine 14 Q16627 1.55 2.89 < 1.00E-16 b2-Microglobulin Beta-2-microglobulin P61769 1.54 4.39 < 1.00E-16 TSP2 Thrombospondin-2 P35442 1.54 3.80 < 1.00E-16 MMP-7 Matrix metalloproteinase 7 P09237 1.53 4.09 < 1.00E-16 Endostatin Endostatin P39060 1.52 2.92 < 1.00E-16 Cathepsin H Cathepsin H P09668 1.52 4.02 < 1.00E-16 EPHB2 Ephrin type-B receptor 2 P29323 1.50 2.53 < 1.00E-16 IL-18 BPa Interleukin-18-binding protein O95998 1.49 2.89 < 1.00E-16 CRDL1 Chordin-like protein 1 Q9BU40 1.49 2.80 < 1.00E-16 Cystatin C Cystatin-C P01034 1.49 3.65 < 1.00E-16 C9 Complement component C9 P02748 1.48 3.05 8.85E-14 PARC C-C motif chemokine 18 P55774 1.47 2.50 1.11E-16 C7 Complement component C7 P10643 1.47 3.84 < 1.00E-16 Tumor necrosis factor receptor RELT Q969Z4 1.47 3.72 < 1.00E-16 superfamily member 19L JAG1 Protein jagged-1 P78504 1.45 2.25 3.66E-15 UNC5H3 Netrin receptor UNC5C O95185 1.44 3.35 < 1.00E-16 Ephrin-A4 Ephrin-A4 P52798 1.44 3.70 < 1.00E-16

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Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

BSSP4 Brain-specific serine protease 4 Q9GZN4 1.44 2.92 < 1.00E-16 Neuroblastoma suppressor of DAN P41271 1.43 3.45 < 1.00E-16 tumorigenicity 1

Epithelial cell kinase Ephrin type-A receptor 2 P29317 1.43 3.28 < 1.00E-16

Spondin-1 Spondin-1 Q9HCB6 1.42 3.20 < 1.00E-16 Periostin Periostin Q15063 1.40 2.15 6.12E-12 VEGF Vascular endothelial growth factor A P15692 1.40 2.35 1.03E-12 SREC-I Scavenger receptor class F member 1 Q14162 1.39 3.45 < 1.00E-16 alpha-1- antichymotrypsin alpha-1-antichymotrypsin complex P01011 1.39 2.50 1.97E-14 complex CRK Adapter molecule crk P46108 1.39 3.06 < 1.00E-16 Ephrin-A5 Ephrin-A5 P52803 1.37 2.62 2.89E-15 Endothelial cell-selective adhesion ESAM Q96AP7 1.37 2.27 1.02E-14 molecule Glutathione S- Glutathione S-transferase P P09211 1.37 2.57 1.28E-12 transferase Pi Tumor necrosis factor receptor DR6 O75509 1.36 1.82 1.36E-10 superfamily member 21 CAPG Macrophage-capping protein P40121 1.36 3.24 < 1.00E-16 Coiled-coil domain-containing protein URB Q76M96 1.36 2.34 1.36E-10 80 LAG-3 Lymphocyte activation gene 3 protein P18627 1.35 2.11 2.48E-10 Ck-b-8-1 Ck-beta-8-1 P55773 1.35 2.14 1.26E-08 Elafin Elafin P19957 1.35 2.45 9.14E-12 TIMP-1 Metalloproteinase inhibitor 1 P01033 1.34 3.95 < 1.00E-16 HSP 70 Heat shock 70 kDa protein 1A/1B P08107 1.34 2.21 1.25E-10 Stanniocalcin-1 Stanniocalcin-1 P52823 1.34 2.61 2.65E-11 SLPI Antileukoproteinase P03973 1.34 2.36 7.96E-11 Low affinity immunoglobulin gamma Fc FCG3B O75015 1.34 2.13 2.53E-10 region receptor III-B Tumor necrosis factor receptor TRAIL R4 Q9UBN6 1.34 2.07 1.29E-09 superfamily member 10D MMP-3 Stromelysin-1 P08254 1.33 2.49 1.37E-09 PH Pancreatic hormone P01298 1.33 2.25 1.19E-10 ARMEL Cerebral dopamine neurotrophic factor Q49AH0 1.32 1.98 5.50E-10 CYTD Cystatin-D P28325 1.32 1.99 9.40E-10 GPVI Platelet glycoprotein VI Q9HCN6 1.32 1.85 3.10E-09 CATZ Cathepsin Z Q9UBR2 1.32 2.34 9.36E-10 DLL1 Delta-like protein 1 O00548 1.32 3.40 < 1.00E-16 MPIF-1 C-C motif chemokine 23 P55773 1.31 1.99 2.74E-09

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Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

Kallikrein 11 Kallikrein-11 Q9UBX7 1.31 2.03 1.22E-11 SLAF5 SLAM family member 5 Q9UIB8 1.31 2.60 9.99E-16 IL-1 R4 Interleukin-1 receptor-like 1 Q01638 1.31 2.08 1.92E-09 TFF3 Trefoil factor 3 Q07654 1.30 3.58 3.51E-13 Platelet-activating factor PAFAH beta subunit P68402 1.29 2.49 8.03E-14 acetylhydrolase IB subunit beta Insulin-like growth factor-binding IGFBP-1 P08833 1.29 1.90 1.12E-08 protein 1 CD48 CD48 antigen P09326 1.29 2.30 4.62E-10 Renin Renin P00797 1.29 1.77 1.68E-08 NLGNX Neuroligin-4, X-linked Q8N0W4 1.29 2.60 1.92E-12 BLC C-X-C motif chemokine 13 O43927 1.29 2.67 1.24E-11 Urokinase plasminogen activator suPAR Q03405 1.29 3.18 3.80E-13 surface receptor PAPP-A Pappalysin-1 Q13219 1.29 1.95 6.82E-08 resistin Resistin Q9HD89 1.28 1.87 2.27E-08 SDF-1 Stromal cell-derived factor 1 P48061 1.28 2.02 2.17E-07 FUT5 Alpha-(1,3)-fucosyltransferase 5 Q11128 1.28 2.04 8.33E-07 Tumor necrosis factor ligand TNFSF15 O95150 1.28 3.05 6.62E-13 superfamily member 15 Nidogen Nidogen-1 P14543 1.28 1.83 1.11E-07 HTRA2 Serine protease HTRA2, mitochondrial O43464 1.28 2.14 6.37E-09 Insulin-like growth factor-binding IGFBP-7 Q16270 1.28 1.82 1.55E-07 protein 7 IL-1 sRI Interleukin-1 receptor type 1 P14778 1.28 1.73 1.12E-06 Phospholipase A2, membrane NPS-PLA2 P14555 1.27 2.18 5.24E-09 associated ANGL4 Angiopoietin-related protein 4 Q9BY76 1.27 3.08 4.95E-11 FABP Fatty acid-binding protein, heart P05413 1.27 2.64 1.30E-10 LBP Lipopolysaccharide-binding protein P18428 1.27 2.04 1.81E-06 IGF-I sR Insulin-like growth factor 1 receptor P08069 1.27 1.84 9.91E-07 Tenascin Tenascin P24821 1.27 1.84 3.15E-07 Tumor necrosis factor receptor XEDAR Q9HAV5 1.27 3.99 < 1.00E-16 superfamily member 27 Troponin I Troponin I, cardiac muscle P19429 1.27 3.18 1.02E-12 BSP Bone sialoprotein 2 P21815 1.27 1.80 7.52E-08 Insulin-like growth factor-binding IGFBP-6 P24592 1.26 2.42 3.06E-11 protein 6 MATN2 Matrilin-2 O00339 1.26 2.12 4.44E-07 LY9 T-lymphocyte surface antigen Ly-9 Q9HBG7 1.26 1.75 1.79E-06 Layilin Layilin Q6UX15 1.26 2.84 1.43E-09

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Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

XTP3A dCTP pyrophosphatase 1 Q9H773 1.26 1.69 7.38E-06 Fibrinogen g-chain Fibrinogen gamma chain P02679 1.25 1.88 3.30E-06 dimer EphB6 Ephrin type-B receptor 6 O15197 1.25 1.85 3.27E-09 Oxidized low-density lipoprotein OLR1 P78380 1.25 1.85 2.38E-07 receptor 1 Carbonic anhydrase Carbonic anhydrase 3 P07451 1.25 1.93 5.34E-07 III CYTT Cystatin-SA P09228 1.25 1.80 2.83E-06 FGF7 Fibroblast growth factor 7 P21781 1.25 2.78 2.90E-12 AK1A1 Alcohol dehydrogenase [NADP(+)] P14550 1.25 1.91 1.86E-06 Tyrosine-protein kinase transmembrane ROR1 Q01973 1.24 1.95 1.40E-08 receptor ROR1 Midkine Midkine P21741 1.24 2.20 2.96E-09 Low affinity immunoglobulin gamma Fc P12318 FCG2A/B 1.24 1.83 7.23E-06 region receptor II-a/b P31994 IP-10 C-X-C motif chemokine 10 P02778 1.24 2.26 1.08E-06 MBL Mannose-binding protein C P11226 1.24 1.89 1.44E-05 NAGK N-acetyl-D-glucosamine kinase Q9UJ70 1.23 1.54 4.84E-06 MCP-3 C-C motif chemokine 7 P80098 1.23 1.98 3.56E-07 SHC1 SHC-transforming protein 1 P29353 1.23 1.96 6.47E-08 GPNMB Transmembrane glycoprotein NMB Q14956 1.23 1.64 2.29E-06 Macrophage Macrophage mannose receptor 1 P22897 1.23 1.83 1.30E-05 mannose receptor Dual specificity mitogen-activated MP2K2 P36507 1.23 1.81 1.01E-05 protein kinase kinase 2 Tumor necrosis factor receptor TNF sR-I P19438 1.23 3.71 < 1.00E-16 superfamily member 1A Cathepsin B Cathepsin B P07858 1.23 1.76 5.70E-06 vWF von Willebrand factor P04275 1.23 1.74 1.49E-05 TF Tissue Factor P13726 1.23 2.78 8.34E-13 Tumor necrosis factor ligand CD30 Ligand P32971 1.22 1.46 6.40E-07 superfamily member 8 IL-17 RC Interleukin-17 receptor C Q8NAC3 1.22 2.56 3.50E-09

Nectin-like protein 2 Cell adhesion molecule 1 Q9BY67 1.22 1.80 1.10E-05

CCL28 C-C motif chemokine 28 Q9NRJ3 1.22 1.64 1.94E-05 IL-18 Ra Interleukin-18 receptor 1 Q13478 1.22 1.58 2.90E-05 NRP1 Neuropilin-1 O14786 1.22 1.94 4.17E-08 LAG-1 C-C motif chemokine 4-like Q8NHW4 1.22 1.51 4.69E-06 Retinoic acid receptor responder TIG2 Q99969 1.22 1.80 1.58E-05 protein 2 sICAM-5 Intercellular adhesion molecule 5 Q9UMF0 1.21 1.91 1.79E-06

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Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

CD5L CD5 antigen-like O43866 1.21 1.62 1.06E-05 BNP-32 Brain natriuretic peptide 32 P16860 1.21 2.77 1.34E-09 EGF-like module-containing mucin-like EMR2 Q9UHX3 1.21 1.82 3.46E-05 hormone receptor-like 2 Neurogenic locus notch homolog Notch-3 Q9UM47 1.21 1.88 9.13E-07 protein 3 C34 gp41 HIV gp41 C34 peptide, HIV Q70626 1.21 1.72 2.56E-06 Fragment CYTN Cystatin-SN P01037 1.20 1.52 3.97E-05 B7-H1 Programmed cell death 1 ligand 1 Q9NZQ7 1.20 2.12 3.37E-06 YES Tyrosine-protein kinase Yes P07947 1.20 2.11 9.06E-08 IL-15 Ra Interleukin-15 receptor subunit alpha Q13261 1.19 3.21 4.51E-10 P31946, P62258, P61981, 14-3-3 14-3-3 protein family Q04917, 1.19 1.75 4.45E-05 P27348, P63104, P31947 OX2G OX-2 membrane glycoprotein P41217 1.19 1.44 2.85E-05 IL-8 Interleukin-8 P10145 1.19 2.10 1.60E-07 JAM-B Junctional adhesion molecule B P57087 1.19 1.73 1.95E-06 LIF sR Leukemia inhibitory factor receptor P42702 1.19 1.72 4.55E-05 FGF23 Fibroblast growth factor 23 Q9GZV9 1.18 2.41 7.74E-08 Tumor necrosis factor receptor 4-1BB Q07011 1.18 2.36 2.83E-08 superfamily member 9 MIP-1a C-C motif chemokine 3 P10147 1.18 2.42 1.53E-06 Factor D Complement factor D P00746 1.18 2.72 3.93E-07 Tumor necrosis factor receptor TAJ Q9NS68 1.18 2.16 1.99E-07 superfamily member 19 LD78-beta C-C motif chemokine 3-like 1 P16619 1.18 1.55 3.25E-05 SPINT2 Kunitz-type protease inhibitor 2 O43291 1.17 2.18 4.28E-06 PTHrP Parathyroid hormone-related protein P12272 1.17 1.51 3.45E-05 Ubiquitin-fold modifier-conjugating UFC1 Q9Y3C8 1.17 1.41 4.54E-05 enzyme 1 VEGF-C Vascular endothelial growth factor C P49767 1.16 2.49 7.62E-06 SLAF6 SLAM family member 6 Q96DU3 1.15 1.88 3.84E-05 MIS Muellerian-inhibiting factor P03971 0.84 0.50 6.70E-06 Antithrombin III Antithrombin-III P01008 0.84 0.55 2.07E-05 FCN2 Ficolin-2 Q15485 0.83 0.59 8.21E-06 Dual specificity mitogen-activated MP2K4 P45985 0.82 0.53 1.11E-05 protein kinase kinase 4 ASAH2 Neutral ceramidase Q9NR71 0.82 0.54 4.48E-05

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Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

Endothelin- converting enzyme Endothelin-converting enzyme 1 P42892 0.82 0.60 4.05E-05 1 Neurogenic locus notch homolog Notch 1 P46531 0.82 0.60 1.71E-05 protein 1 Heparin cofactor II Heparin cofactor 2 P05546 0.82 0.57 9.33E-06 Carbonic anhydrase Carbonic anhydrase 6 P23280 0.82 0.58 1.13E-05 6 Testican-1 Testican-1 Q08629 0.81 0.59 2.80E-05 PAI-1 Plasminogen activator inhibitor 1 P05121 0.81 0.60 1.27E-05

6-Phosphogluconate 6-phosphogluconate dehydrogenase, P52209 0.81 0.59 8.60E-06 dehydrogenase decarboxylating

Afamin Afamin P43652 0.81 0.44 7.50E-09 DRAK2 Serine/threonine-protein kinase 17B O94768 0.80 0.57 2.65E-05 Ectonucleoside triphosphate ENTP5 O75356 0.80 0.57 2.51E-06 diphosphohydrolase 5 Coagulation Factor Coagulation factor Xa P00742 0.80 0.48 3.43E-08 Xa Plasminogen Plasminogen P00747 0.80 0.58 7.86E-06 MDHC Malate dehydrogenase, cytoplasmic P40925 0.80 0.50 2.23E-06 Prothrombin Prothrombin P00734 0.80 0.44 4.72E-08 Opioid-binding protein/cell adhesion OBCAM Q14982 0.80 0.56 1.13E-05 molecule Aminoacylase-1 Aminoacylase-1 Q03154 0.80 0.52 8.51E-06 cGMP-stimulated cGMP-dependent 3',5'-cyclic O00408 0.79 0.57 3.51E-06 PDE phosphodiesterase Cytochrome P450 Cytochrome P450 3A4 P08684 0.79 0.48 1.97E-06 3A4 Ficolin-3 Ficolin-3 O75636 0.78 0.51 2.13E-08 Superoxide dismutase [Mn], Mn SOD P04179 0.78 0.52 7.13E-07 mitochondrial a2-HS-Glycoprotein Alpha-2-HS-glycoprotein P02765 0.78 0.48 1.16E-07 TrkB BDNF/NT-3 growth factors receptor Q16620 0.78 0.59 2.78E-06 C-type lectin domain family 4 member DC-SIGNR Q9H2X3 0.77 0.53 2.20E-08 M A disintegrin and metalloproteinase ATS13 Q76LX8 0.77 0.42 4.14E-09 with thrombospondin motifs 13 Kallistatin Kallistatin P29622 0.77 0.51 1.15E-08 Tyrosine-protein phosphatase non- PTP-1B P18031 0.77 0.52 6.46E-06 receptor type 1 FGF-20 Fibroblast growth factor 20 Q9NP95 0.76 0.50 1.13E-06 Cathepsin V Cathepsin L2 O60911 0.76 0.48 1.80E-08 Prekallikrein Plasma kallikrein P03952 0.76 0.49 1.99E-10

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Hazard Quintile Target Full Name UniProt Ratio Hazard P value per SD Ratio

NXPH1 Neurexophilin-1 P58417 0.75 0.57 1.11E-08 Soggy-1 Dickkopf-like protein 1 Q9UK85 0.75 0.48 2.58E-08 15-hydroxyprostaglandin HPG- P15428 0.75 0.44 1.93E-08 dehydrogenase [NAD(+)] Protein C Vitamin K-dependent protein C P04070 0.74 0.36 1.30E-13 SEPR Seprase Q12884 0.74 0.46 1.47E-06 Tumor necrosis factor ligand TWEAK O43508 0.74 0.44 4.07E-06 superfamily member 12 Vascular endothelial growth factor VEGF sR2 P35968 0.74 0.41 2.22E-10 receptor 2 Angiostatin Angiostatin P00747 0.73 0.41 5.92E-11

ERBB3 Receptor tyrosine-protein kinase erbB-3 P21860 0.72 0.38 2.04E-10

GDF-11/8 Growth/differentiation factor 11/8 O95390 0.72 0.41 8.79E-09 BMP-1 Bone morphogenetic protein 1 P13497 0.71 0.37 2.54E-13 Cell adhesion molecule-related/down- CDO Q4KMG0 0.70 0.37 9.14E-14 regulated by oncogenes CK-MM Creatine kinase M-type P06732 0.70 0.43 3.61E-11 CNDP1 Beta-Ala-His dipeptidase Q96KN2 0.69 0.33 < 1.00E-16 Dual 3',5'-cyclic-AMP and -GMP PDE11 Q9HCR9 0.69 0.36 5.19E-12 phosphodiesterase 11A Creatine kinase M-type:Creatine kinase P12277 CK-MB 0.69 0.36 5.08E-13 B-type heterodimer P06732 P-Cadherin Cadherin-3 P22223 0.67 0.39 1.11E-16 Proto-oncogene tyrosine-protein kinase RET P07949 0.66 0.38 4.93E-13 receptor Ret a2-Antiplasmin Alpha-2-antiplasmin P08697 0.64 0.34 < 1.00E-16 Growth hormone Growth hormone receptor P10912 0.63 0.27 < 1.00E-16 receptor ERBB1 Epidermal growth factor receptor P00533 0.60 0.27 < 1.00E-16

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5.1 Biological Functions of 16 LASSO-Selected Proteins The protein biomarkers identified in this analysis not only serve to derive a cardiovascular risk prediction model, but also inform understanding of the biology of cardiovascular disease (CVD) and identify potential drug targets and treatment options. Below, a brief description is provided of the known function(s) of the 16 proteins selected by LASSO.

GROWTH AND REMODELING Growth Differentiation Factor 11/8 (GDF11/8) is an example of biological discovery using unbiased proteomics assay tool with findings of potential clinical significance. Using modified aptamers, Lee and colleagues pinpointed age-related decline in circulating GDF-11 as the cause of age-related cardiac hypertrophy in mice10. GDF-11 is currently under active investigation for its role in suppressing cardiac hypertrophy and diastolic heart failure in humans. Interestingly, while GFD-11 concentrations are reduced with increasing cardiovascular event risk in this study, an inhibitor of GDF11, Follistatin-like 3 is positively associated with increasing cardiovascular risk (eTable 4). As the modified aptamer used in the present study does not distinguish GDF11 from its close homologue GDF8 as two the proteins are 90% identical in their active mature regions and have identical functions in vitro, this protein is designated as GDF11/811. Epidermal Growth Factor (EGF) receptor (EGFR) is expressed on monocytes and macrophages in atherosclerotic lesions. Activation by ligand binding stimulates cellular proliferation and chemotaxis12. Evidence from animal studies shows that EGF receptor protects against cardiac hypertrophy and supports appropriate vascular wall architecture and vessel reactivity13. Soluble forms of the Growth hormone receptor (GHR) and the epidermal growth factors can serve as both reservoirs and inhibitors of the circulating factors involved in mitogenesis, cell function, and have well-known roles in cancer. Interestingly, growth hormone receptor signaling, via stimulation of its anabolic mediator Insulin-like growth factor I, has previously been shown to have a negative correlation with risk of developing coronary artery disease14. Angiopoietin-2 (ANGPT2), which antagonizes Angiopoietin-1 activity on the Tyrosine-protein kinase receptor Tie-2 receptor and acts in concert with Angiopoietin-1 during angiogenesis, promotes relaxation of cell-matrix contacts and may induce endothelial cell apoptosis and vessel disruption during angiogenesis15. A member of the same gene family, Angiopoietin-related Protein 4 (ANGPTL4) is induced by hypoxia and not only affects vascular function and matrix-endothelial cell interaction, but also lipid metabolism as a potent inhibitor of lipoprotein lipase16.

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Controlled interactions of the extracellular matrix and cells are vital for normal organ physiology, during normal development, in response to vascular and myocardial injury, and during cancer metastasis. Matrix metalloproteinases and their inhibitors have several targets in the vascular extracellular matrix and have been associated with atherosclerotic plaque stability, aneurysm formation and other cardiovascular diseases17. Matrix metalloproteinase (MMP)-7 and MMP-12 are represented in the LASSO selected 16 proteins, while the metalloproteinase inhibitor TIMP1 also has significant association with cardiovascular risk (eTable 4). Thrombospondin-2 (THBS2) mediates vascular and cardiac cell-cell and cell-matrix interactions and has been implicated in the regulation of angiogenesis, thrombosis, and inflammation. Increased serum Thrombospondin-2 concentration is associated with the risk of cardiac mortality in older men18. Cell adhesion oncogene-related CDO (CDON) is a cell surface protein member of the Ig/fibronectin superfamily involved in myogenesis and muscle cell adhesion19. Its role in cell-cell interaction has been noted in tumor invasiveness but little is known about its relationship to the cardiovascular system.

INFLAMMATION Representing the complex roles of inflammation and immunity in cardiovascular disease, the 9-protein model incorporates the inflammatory chemokine Chemokine (C-C motif) ligand 18, previously known as Pulmonary and activation-regulated chemokine CCL18/PARC , which is a monocyte/macrophage- elaborated chemokine that appears to be involved in the recruitment of T cells20. Plasma levels of CCL18/PARC are elevated during episodes of unstable angina and have also been found to predict CV events in patients with stable angina21. The T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is involved in macrophage activation and other immune system activities22. Complement C7 (C7) is one of the 5 components that form the bioactive terminal complement complex (TCC). TCC deposited on endothelial cells results in cell proliferation, release of growth factors and inflammatory cytokines, and increased expression of tissue factor. TCC also stimulates proliferation of smooth muscle cells in atherosclerotic plaques23. In patients with symptomatic heart failure elevated serum soluble TCC predicts adverse outcome (death, urgent heart transplantation, or hospitalization with worsening heart failure)24. Complement C9, another member of TCC, is also elevated in our study.

PROTEASES

α1-antichymotrypsin complex (SERPINA3) complex represents the bound form of the protease inhibiter α1-Antichymotrypsin which has several biological substrates. It can modulate multiple acute

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and chronic disease processes including blood pressure25. α2-Antiplasmin (SERPINF2) is a serine protease inhibitor (SERPIN) that inactivates plasmin and thus reduces fibrinolysis26,27. Cathepsin H (CTSH) is a lysosomal cysteine proteinase important in the degradation of lysosomal proteins28. However, its relationship to CV disease until the present study has been uncertain29.

MYOCARDIAL NECROSIS MARKER Unlike many of the aforementioned proteins that are potentially involved in causal pathways of cardiovascular diseases, Troponin I is a well-established marker of end-organ damage (cardiomyocyte necrosis) and of cardiovascular risk30-33.

Univariate hazard ratios for the 16 LASSO selected proteins were largely consistent across the cohorts. The univariate hazard ratios per standard deviation for each of the 16 LASSO selected proteins are shown in eFigure 3 for the derivation (black) and validation (red) cohorts. Proteins included in the 9- protein model are denoted with (*).

eFigure 3: Hazard ratios (per standard deviation) for primary end-point of MI, stroke, heart failure and death with LASSO selected proteins in derivation (black) (n=938) and validation (red) cohort (n=971). Proteins marked with asterisks are included in the final 9 protein model after step-wise backwards elimination of the least important 7 proteins. Legend: MMP-7 = matrix metalloproteinase 7; MMP-12 = matrix metalloproteinase 12; TIM3 = T-cell immunoglobulin and mucin domain-containing protein 3; CCL18 = Chemokine (C-C motif) ligand 18, previously known as PARC = Pulmonary and activation-

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regulated chemokine; GDF11/8 = Growth differentiation factors 11 and 8; CDO = Cell adhesion associated oncogene regulated; EGF Receptor = epidermal growth factor receptor.

5.2 9-Protein Model Specification LASSO was used for variable selection only, as the fully parametric (Weibull) survival model was preferred as a final prognostic model. The latter has a simple representation and a mathematical form amenable7,34 to recalibration for use in external validation studies. Stepwise backward elimination starting from the full set of 16 LASSO-selected proteins was used to remove proteins from the Weibull model that were not significant predictors in the absence of the constraint imposed by the LASSO penalty. When using the Bayesian information criterion (BIC) stopping criteria to balance model performance and complexity, backward elimination discarded 7 proteins: Cathepsin H, EGF receptor, Growth hormone receptor, T cell membrane protein TIM-3, MMP-7, Cell adhesion oncogene-related CDO and Thrombospondin-2 resulting in the final 9-protein model.

For an accelerated failure time model, the probability of an event occurring at time T in the interval [0,t] is given by

퐿표푔(푡)−푃퐼 ( ) Pr[푇 ≤ 푡] = 1 − 푒−푒 푠 , where PI is the prognostic index (or linear predictor) and s is the associated extreme value distribution scale parameter. When fitting the model standardized variables were used. Absorbing the population mean and standard deviations for the proteins into the intercept term, the prognostic index (PI) and scale factor for the model applied to the derivation cohort are,

PI = 16.61-1.55×ANGPT2 + 1.22×GDF11/8 - 2.12×C7 + 2.64×SERPINF2 - 0.57×CCL18 - 1.02×ANGPTL4 - 1.43×SERPINA3 - 0.72×MMP12 - 0.59×TNNI3, s = 0.85 , where signal levels associated with the specified proteins are in taken to be in log10 relative fluorescence units(RFU). Legend: ANGPT2=Angiopoietin-2; GDF11/8=growth differentiation factor 11/8; C7=complement 7; SERPINF2= α2-antiplasmin; CCL18= chemokine (C-C motif) ligand 18; ANGPTL4= angiopoietin-related protein 4; SERPINA3= α1-antichymotrypsin complex; MMP12= matrix metalloproteinase-12; and TNNI3= troponin I.

While adjusting this model for clinical variables improved the fit slightly, these adjustments failed to produce a meaningful improvement in either the discrimination or calibration performance over that

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achieved with the “proteins only” model in the derivation cohort. Anticipating that this model might be refined based on the outcome of the validation results presented here (and additional studies in progress), various adjusted models in the main text are not presented at this time. The adjusted hazard ratio of the 9-protein model (listed above), with Prognostic index (PI) computed in the total (derivation + validation) cohort is shown in eFigure 4. The top dashed line separates the addition of Framingham variables from proteins alone and bottom dashed line separates proteins + Framingham variables + CRP, where adjustment was made for CRP for illustrative purposes as measurement of this protein is used in clinical practice.

eFigure 4: Hazard ratios (95% CI) associated with 9-protein model Prognostic Index (PI) for the primary end-point of MI, stroke, heart failure and death computed for the total (derivation + validation) cohort, adjusted for each of the Framingham variables (between dashed lines) and finally for CRP, in addition to the Framingham variables.

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6 Risk Score Distributions by Event Type The eFigure 5 below shows the distributions of 9-protein risk scores for participants with the specific cardiovascular endpoints: heart failure (CHF), myocardial infarction (MI), stroke, and death compared to participants without events (No Event) and participants that experienced any one of the events.

eFigure 5: Distributions of 9-protein risk score for participants with specific cardiovascular events in derivation (left) and validation (right). Legend: heart failure (CHF), myocardial infarction (MI).

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