Predictor discoveryin training set

6

Training setSJIA (24 F, 14 Q)POLY (15 F, 10 Q)

1

DIGEraw gel images

SJIA (13 F, 13 Q)POLY (5 F, 5 Q)

Spot findingspot alignment

feature extraction

889 discrete spot features

Classifier training

Ten-foldcross-validation

ClassifySJIA F vs QPOLY F vs Q

DIGE analysis

Prediction analysis of microarray (PAM)

Class predictionin testing set

7

Testing setSJIA (24 F, 14 Q)POLY (15 F, 11 Q)

Predictors of 4 ~ 12 features

PAMclass prediction algorithm

Construct estimates ofpredicted class probabilities

Analysis of goodness of class separation

2

Cluster analysis

7 feature biomarker panel

2d hierarchicalclustering

heatmap plotting

Normalizationmanual review

Manual review

MSMS ID97 spots

P < 10-5

3

Mann Whitney

10 proteincandidates

4

Literature review+ literature candidates- check antibodyavailability

5

12 ELISA assays

Figure 1

8

Prospective StudyN.D.

DiscriminateSJIA F

KDFI

F Q F Q

SJIA POLY

Figure 2

F Q

SJIA

Figure 3

A2MCFHR1ATIIIATIIIGSNA2MGSNTTRAPOA1APOA1APOA1APOA1APOA1APOA1SAPAPOIVHPCRPHPHPHPHPHPHPMRP8MRP8MRP14SAASAASAA

SAPAPOAIVHPCRPHPA2MCFHR1GSNGSNA2MATIIIATIIITTRAPOA1APOA1APOIVAPOA1APOA1APOA1MRP14HPHPHPHPHPMRP8MRP8SAASAASAA

F Q F Q

SJIA POLYFigure 4

POLY QPOLY F POLY QPOLY F

TRAINING TESTING

Figure 5A

SJIA QSJIA F SJIA QSJIA F

TRAINING TESTING

Figure 5B

Figure 6AG

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POLY Training POLY Testing

128754Feature# 128754

Figure 6BG

oo

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s s

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SJIA Training SJIA Testing

127654Feature# 8 9 127654 8 9

SJIA QSJIA F SJIA QSJIA F

TRAINING TESTING

Figure 7

Training setn = 38

24 14

Clinicaldiagnosis F Q

n =

PAM

18 5

6 9

Classified as F

Classified as Q

PercentAgreementwith clinicaldiagnosis

75% 64%+ -

71%

Overall

P < 0.05

Testing setn = 38

24 14

Clinicaldiagnosis F Q

n =

PAM

21 4

3 10

Classified as F

Classified as Q

PercentAgreementwith clinicaldiagnosis

88% 71%+ -

82%

Overall

P < 0.001

A B

Figure 8

C

Biomarker panelof 7 members

1. HP

2. APO AI

3. A2M

4. SAP

5. CRP

6. MRP8/MRP14

7. SAA

SJIA SJIA

1213 12

SJIAF KD FI

SAP

SAA

MRP8

MRP14

HP

CRP

APOA1

A2M

Data setn = 27

13

Clinicaldiagnosis

SJIAF

NOT-SJIAF

n =

Unsupervised clustering

10

3

1

23

Clustered as SJIA FClustered

as NOT-SJIA F

PercentAgreementwith clinicaldiagnosis

77% 96%+ -

85%

Overall

P < 10-5

24

Figure 9

Spot Cluster ID Protein ID T test P value Mann Whitney Test P value

6 A2M 1.05 X 10-4 2.31 X 10-6

7 CFHR1 2.31 X 10-4 1.92 X 10-7

3 ATIII 1.73 X 10-6 5.77 X 10-6

2 ATIII 1.17 X 10-6 1.35 X 10-6

1 GSN 7.68 X 10-6 2.31 X 10-6

19 A2M 1.79 X 10-6 5.77 X 10-6

4 GSN 4.23 X 10-6 1.92 X 10-7

21 TTR 2.16 X 10-6 2.31 X 10-6

26 APOA1 1.84 X 10-6 2.31 X 10-6

16 APOA1 3.27 X 10-7 2.31 X 10-6

25 APOA1 4.28 X 10-6 3.85 X 10-7

22 APOA1 1.32 X 10-6 3.85 X 10-7

11 APOA1 1.10 X 10-6 7.69 X 10-7

17 APOA1 3.50 X 10-7 7.69 X 10-7

24 SAP 4.16 X 10-6 3.65 X 10-6

5 APOAIV 1.30 X 10-8 3.65 X 10-6

12 HP 3.93 X 10-9 3.65 X 10-6

14 CRP 8.99 X 10-7 7.69 X 10-7

27 HP 4.03 X 10-7 1.35 X 10-6

20 HP 8.23 X 10-8 8.65 X 10-6

29 HP 1.22 X 10-9 1.35 X 10-6

13 HP 5.38 X 10-11 3.85 X 10-7

28 HP 9.54 X 10-8 1.35 X 10-6

15 HP 8.86 X 10-10 3.85 X 10-7

9 MRP8 2.32 X 10-10 3.85 X 10-7

8 MRP8 1.97 X 10-9 3.85 X 10-7

18 MRP14 2.15 X 10-10 3.85 X 10-7

30 SAA 1.26 X 10-8 3.65 X 10-6

23 SAA 3.03 X 10-9 3.65 X 10-6

10 SAA 1.22 X 10-9 1.35 X 10-6

Supplement Table 1