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Sex Age (years)
Disease duration
RF CCP Erosions (X-Rays)
Treatment
1 F 54 2 + + - Leflunomide 2 F 54 28 + + + Methotrexate 3 F 36 3 - + + Methotrexate 4 F 46 4 + + + Methotrexate 5 M 40 8 + + + Methotrexate 6 F 64 30 + + + Ciclosporine 7 M 48 7 + + + None 8 F 37 3 + + + Methotrexate 9 F 52 20 + + + Etanercept 10 F 50 9 - + + Infliximab
Methotrexate 11 M 51 24 + + + Leflunomide 12 F 51 + + Leflunomide 13 F 35 2 + + + Methotrexate 14 F 20 3 + + + Sulfasalazine 15 F 57 25 + + + Infliximab
Methotrexate 16 F 36 3 + + + Methotrexate 17 F 54 9 + + + Methotrexate
Sulfasalazine 18 F 20 3 - + - Methotrexate 19 M 24 1.5 - + - Sulfasalazine 20 F 62 0.8 - + - None 21 F 29 1 + + + None 22 F 58 20 + + + None 23 M 62 1 + + + None 24 F 43 6 + + - Anakinra
Methotrexate 25 F 55 19 - + + None 26 M 46 4 + + + Leflunomide 27 M 63 12 + + + Infliximab 28 M 55 18 + + + Methotrexate
Sulfasalazine 29 F 29 2 + + - Methotrexate 30 M 46 6 + + + Methotrexate 31 F 25 1 + + + None 32 M 33 2 + + + Methotrexate 33 F 46 8 + + + None 34 F 49 16 + + + Sulfasalazine 35 F 72 14 + + Adalimumab 36 M 29 4 + + + Anakinra
Leflunomide 37 M 23 3.5 + + + Leflunomide 38 M 69 7 + + + Hydroxychloroquine 39 F 54 4 + + + Methotrexate 40 F 52 21 + + + Etanercept
Methotrexate 41 F 72 7 - + + Anakinra
Methotrexate 42 M 74 3 + + + Leflunomide 43 F 60 22 + + + None 44 F 57 8 + + + Adalimumab
Table 1
14,4 kDa
18,8 kDa
25 kDa
35 kDa
45 kDa
116 kDa
Figure 1
Healthy control
sera
Seco
ndar
y A
b RA sera
Antigenic targets wrist RA SM
Antigenic targets RA SM
Wris
t
Finge
r
HipKnee
RA seru
m
Health
y seru
m
RA seru
m
Health
y seru
m
RA seru
m
Health
y seru
m
RA seru
m
Health
y seru
m
A B
Figure 2
Identification of major spots obtained with RA wrist FLS
RA FLS
RA sera
RA FLS P3 RA FLS P4 RA FLS P5
1 2 3 4 5RA sera RA sera RA sera
1 2 3 4 5 1 2 3 4 5
A B
P60709
P02545-2*
P18206-2
Q6P707*
P08670
P38646P11142
P09211
P04406
P06733
P18669
P30041
P32119
P30086
P62937
P07737
O15460P07237
P30101
P31949
Q9BRY9
P04179
P78371
P11021*
P55072
P67936
P23381
P04083*
P50995*
P07355
P08758*
P29474
P27797
P23528
O43175
P09382
P05413
P04792
P10809*
49
64
37
26
15
19
82
115
Mass
(kDa)
IEFS
DS
-PA
GE
pI 3 10
2-D map
Figure 3
Table 2: Results of MALDI mass spectrometry analysis of 2D-PAGE isolated FLS cellular proteins. The identities of 40 spots are listed.
Maldi-MSTheoretical Observed Theoretical Observed Peptides Matching
O15460Prolyl 4-hydroxylase alpha 2a
61,01 62 5,5 5,4 12/52 24 2,38 (1)
O43175D-3-phosphoglycerate
dehydrogenase 57,39 56 6,3 6,1 5/48 8 1,39 (1)
P02545-2 Lamin A/C 65,12 64 - 65,5 6,4 6,1 - 6,6 17/60 37 2,25 (1)
P04083 Annexin A1 38,92 36 6,6 5,75 - 6,1 14/56 44 2,29 (1)
P04179 Superoxide dismutase (Mn) 22,24 22,6 / 23 6,9 6,45 / 7 8/40 38 2,41 (1)
P04406Glyceraldehyde-3-phosphate
dehydrogenase 36 36,4 8,6 8,6 / 9 7/40 23 1,8.10-8 (3)
P04792 Heat Shock 27 kDa protein 22,82 25,2 6 5,4 2/10 9 287,6 (3)
P05413Heart fatty acid-binding
protein13,28 16,45 7 5,85 5/57 37 6,2.10-3 (1)
P06733 Alpha-enolase 47 49 7 6,5 / 7 9/20 30 1,5.10-16(3)
P07237 Protein disulfide-isomerase 55 60 4,7 4,8 18/40 41 54 (2)
P07355 Annexin A2 38,79 30,4 - 33,5 7,7 6,2 15/71 37 2,42 (1)
P07737 Profilin 1 15 17 8,5 8,65 5 48 60,2 (3)
P08670 Vimentin 53,75 46,9 - 58 5 4,45 - 5,2 18/83 35 2,41 (1)
P08758 Annexin A5 35,84 34 5 4,75 8/40 25 2,27 (1)
P09211 Glutathione S-transferase P 23 23,8 5,4 5,35 6/29 41 3,5.10-11 (3)
P09382 Galectin-1 14,91 16 5,3 4,8 8/68 62 2,39 (1)
P10809 60 kDa heat shock protein 61,21 61 5,7 5,15 14/73 30 2,34 (1)
P1102178 kDa Glucose-regulated
Protein 71,03 74,4 / 75,8 5,2 4,9 / 5 10/31 20 2,37 (1)
P11142 Heat shock 70 kDa protein 71,11 70 5,4 5,2 7/40 18 2,5.10-20 (3)
P18206-2 Vinculin 116,59 115 5,83 5,8 9/17 11 4,6.10-14 (3)
P18669Phosphoglycerate mutase 1
28,9 27,4 6,7 6,35 / 6,6 5/42 26 2,27 (1)
P23381Tryptophanyl-tRNA
synthetase 53,41 54 5,7 5,7 9/46 19 2,36 (1)
P23528 COFILIN-1 18 19,5 8,3 8,4 6/43 39P27797 Calreticulin 48,29 58 4,3 4,4 7/52 21 2,38 (1)
P29474Nitric-oxide synthase,
endothelial135,05 31 7 6,95 5/57 7 1,05 (1)
P30041 Peroxiredoxin 6 25,13 25,3 / 25,7 6 6 7/70 27 2,17 (1)
P30086Phosphatidylethanolamine
binding protein 1 21,15 22,2 7 7,8 7/82 42 2,23 (1)
P30101Protein disulfide-isomerase
ER6057,18 57 6 5,55 8/32 15 2,39 (1)
P31949 Protein S100-A11 11,84 14 6,6 5,6 4/35 30 1,2 (1)
P32119 Peroxiredoxin 2 16,03 22,5 6,1 5,35 10/53 50 2,36 (1)
P38646 Heat shock 75 kDa protein 74,05 71,5 6 5,3 10/22 18 2,3 (1)
P50995 Annexin A11 54,71 57,9 7,7 7,2 12/87 21 65 (2)
P55072Transitional endoplasmic
reticulum ATPase0 95,3 5,1 5,1
12/91 17 2,36 (1)
P60709 Beta actin 42 45,8 / 50,2 5,3 5,15 6/38 31 2,36 (1)
P62158 Calmodulin 17 18 4,1 4 6/20 59 173,5 (3)
P62937Peptidyl-prolyl cis-trans
isomerase A 18 18,55 7,8 7,5 / 8 5/20 34 2,5.10-11 (3)
P67936 Tropomyosin 4 28,5 31,3 4,7 4,45 14/64 33 1,4.10-9 (1)
P78371T-complex protein 1subunit
beta 57,87 55 6 5,9 15/101 34 89 (2)
Q6P707 CALDESMON 210 78,9 5,01 5,85 et 5,9 4/15 26 54(2)
Q9BRY9 SPSB1 12,03 18,2 6,1 5,45 2/33 31 0,91 (1)
PI Sequence Coverage %
ScoreNumber Protein nameMolecular weight
Figure 4
14,4 kDa
18,8 kDa
25 kDa
35 kDa
45 kDa
116 kDa
Antigenic targets: RA FLS
67 kDa
Figure 5
Sen
siti
vity
Cutoff = 0,55 Sensitivity = 89 % Specificity= 93%AUC : 0
0,5
1
1,5
2
2,5
0 1 2 3
cut-off
RA Healthy controls
0,0 0,2 0,4 0,6 0,8 1,0
1 - Specificity
0,0
0,2
0,4
0,6
0,8
1,0
ROC Curve
p19
p26p30
p33
p37
0.466
0.6050.629
0.495
0.618
Coe
ffici
ent
for X
axi
s
Wes
tern
blo
t str
ip
Ant
igen
Iden
tity
RA
FL
S
A B
Figure 6
P37
64
15
19
26
49
82
Mass (kDa)
P57
B
C
64
15
19
26
49
82P26
P30
Mass (kDa)
RA serum 1
A
IEF IEFS
DS
-PA
GE
SD
S-P
AG
E
RA serum 2pI 3
pI 310 10
Protein nameRA
n=44HS
n=16 Sensitivity Specificity
0 8 15 18,18 6,25
1 Lamin A/C 17 1 38,64 93,75
178 kDa Glucose-regulated Protein =
BiP 16 0 36,36 100
1 Annexin A11 16 0 36,36 100
2 Lamin A/C or Annexin A11 30 1 68,18 93,75
2 Lamin A/C or BiP 26 1 59,09 93,75
2 Annexin A11 or BiP 28 0 63,64 100
3 Lamin A/C or Annexin A11 or BiP 36 1 81,82 93,75
Table 3
