Electronic supplementary information for:
OXIDATIVE MODIFICATIONS OF CEREBRAL TRANSTHYRETIN ARE ASSOCIATED
WITH MULTIPLE SCLEROSIS
Damiana
Pieragostino1,2(PhD),
Piero
Del
Boccio1,2(PhD),
Maria
Di
Ioia3(MD),
Luisa
Pieroni4,5(PhD), Viviana Greco4,5(PhD), Giovanna De Luca3(MD), Simona D’Aguanno4,5(PhD),
Claudia Rossi1,2(PhD), Diego Franciotta6(MD), Diego Centonze4(MD), Paolo Sacchetta1,2(PhD),
Carmine Di Ilio1,2(PhD), Alessandra Lugaresi3(MD) and Andrea Urbani4,5(PhD).
1
Research Centre on Aging (Ce.S.I), University “G. d’Annunzio” of Chieti-Pescara, Chieti. Italy;
2
Department of Experimental and Clinical Sciences, University “G. d’Annunzio” of Chieti-Pescara,
Chieti. Italy; 3Department of Neurosciences and Imaging, University “G. d’Annunzio” of ChietiPescara, Chieti. Italy; 4 IRCCS-Santa Lucia Foundation, Rome. Italy; 5 Department of Experimental
Medicine and Surgery, University of Tor Vergata, Rome. Italy; 6 Laboratory of Neuroimmunology
(IRCCS) National Neurological Institute C. Mondino, Pavia, Italy.
Keywords: CSF Transthyretin, Multiple Sclerosis, Post Translational Modifications
*Corresponding Author:
Dott.ssa Damiana Pieragostino, PhD
Department of Experimental and Clinical Sciences, University “G. d’Annunzio”, Chieti-Pescara,
Italy.
e-mail: dpieragostino@unich.it
Phone: +39 0871 541593
Fax: +39 0871 541598
Supplementary Figure captions
Figure S1: Identification of differential peaks highlighted
We identified the peaks 1-5 by fraction collection and MS/MS fragmentation analysis. Identified
peaks were isolated by the use of fraction collector after reverse phase chromatography (Fig. S1 A).
In Fig. S1 B was shown deconvoluted ESI+-Mass-Spectrum of the chromatographic peak eluted
between 42 and 43 minutes showing a low intense 13761 Da signal of the free monomer TTR; the
major molecular species at 13793 Da (differential peak 2 showed in Fig. 1) and 13841 Da
(differential peak 3, showed in Fig. 1). The fraction containing interesting peaks was analysed by
LC-nano-ESI-Q-TOF fragmentation analysis. All peaks were identified as TTR, a very abundant
protein in CSF. Fig. S1 C highlights tandem mass spectra and reconstructed sequences of TTR
peptides found in the chromatographic fraction analysed after digestion with Tryspin. Details of
identification experiments are shown in Table S2.
Figure S2: Serum TTR profiling
We analysed the serum taken at the same time of CSF to verify the presence of the differential
isoforms in peripheral compartment, for all MS patients included in the casuistry. As shown in Fig
S2 A average spectrum of CSF (upper profile) differs from the serum profiling (lower profile).
Peaks 1, 4 and 5 are visible in both biological fluids corresponding to canonical TTR isoforms.
Peaks 2 and 3 emerge only in CSF profile. This result is clear in Fig S2 B and C where intensity of
peak 2 and 3 (13793 Da and 13841 Da, respectively) in each acquired spectrum is shown. The
arrow indicates separation from CSF to serum samples.
Supplementary Tables captions
Table S1: Clinical data from patients affected by multiple sclerosis
Clinical information about the 43 patients affected by MS included in the present study with an
average age of 37.15 ± 9.36 and with a ratio female/male of 2.54.
Symbols:
POS: positive
NEG: negative
OCB = Oligoclonal Bands;
CIS = Clinically Isolated Syndrome evolving in MS at follow-up
RRMS = Relapsing-Remitting Multiple Sclerosis;
PPMS = Primary Progressive Multiple Sclerosis;
ND = Not Determined
Table S2: Tandem Mass Spectrometry experiments
Details of TTR identification by LC-MS/MS experiment.
Table S3: molecular weights of PTMs
Table of molecular weight of mixed disulfides associated to TTR (peaks 2 and 5). Peaks 2 and 3 are
atypical mixed disulfides with mass shift of + 32 Da and + 80 Da, in respect to free monomeric
specie (m/z= 13761 Da), and they are referable to S-sulfhydration (Cys-S-SH) and S-sulfonation
(Cys-S-SO3H) of the 10th cysteine of the protein.
Table S4: Correlation between TTR isoforms and anomalous TTR dimer
We compared the relative intensity in respect to free TTR of Cys-S-SH and Cys-S-SO3H TTR
isoforms obtained by MALDI-TOF analysis and the presence of anomalous dimer in the clinical
cohort investigated. Results demonstrates a proportional trend (p<0.05) between the % of
anomalous dimer and the relative intensity of sulfhydrated TTR isoform registered. This isoform
correlates inversely also with monomer volume (P< 0.05), but no correlation was registered with
the canonical dimer. Sulfonated TTR isoform slightly correlates with anomalous dimer in respect to
the sulfhydrated one. Moreover no correlation was found with monomer volumes.
Supplementary Figures
Figure S1
Figure S2
Supplementary Tables
Table S1
Code
DiseaseDuration
days
IgG index
<0.66
OCB
Blood-CSF bar r ier
<5.5
Disease's stage
Diagnosis
Sex
Concom itant m edications
MRI data
131
132
135
136
137
138
139
140
188
189
ND
60
870
720
1650
2160
540
20
360
1440
0.7
1.11
0.98
1.3
0.66
1.26
1.94
1.78
0.48
0.33
NEG
POS
POS
POS
POS
POS
POS
NEG
NEG
NEG
5.95
3.92
4.62
9.64
4.03
4.6
4.11
4.48
10.52
5.89
ND
active
active
active
active
stable
stable
active
active
stable
RRMS
RRMS
RRMS
RRMS
RRMS
PPMS
PPMS
RRMS
PPMS
RRMS+ VASCULAR LEOKOENCEPHALOPATY
F
F
F
M
F
F
F
F
M
F
ND
ACTIVE
ACTIVE
ACTIVE
ACTIVE
STABLE
STABLE
ACTIVE
STABLE
STABLE
190
191
193
200
203
204
205
207
208
213
214
216
219
221
222
223
248
225
235
236
237
245
246
209
226
242
64
67
71
72
85
93
84
30
90
60
90
390
510
ND
3960
45
1440
30
ND
360
120
90
810
ND
7
180
1080
ND
30
23
120
20
11
3240
ND
ND
3960
360
ND
360
0.65
0.31
0.4
0.74
0.66
0.9
0.48
0.51
0.69
0.61
2.83
0.58
0.56
1.41
0.77
2.21
0.41
0.8
0.43
0.45
0.44
0.5
0.63
0.65
0.56
1.49
0.77
1.08
0.72
0.54
0.45
0.47
1.85
POS
NEG
NEG
POS
POS
POS
POS
NEG
POS
NEG
POS
NEG
POS
POS
POS
POS
ND
POS
NEG
POS
ND
NEG
POS
POS
POS
POS
ND
POS
POS
POS
POS
NEG
POS
4.62
17.42
3.6
7.64
4.93
3.64
5.07
5.78
8
4.42
4.39
3.52
9.92
4.03
5.74
6.55
4.67
4.67
5.47
11.74
7.82
10.38
5.66
2.96
5.11
3.56
0.77
1.08
0.72
0.54
0.45
0.47
1.85
active
active
active
active
active
active
ND
active
stable
active
active
ND
active
active
active
active
ND
active
active
active
ND
active
active
active
active
stable
ND
active
active
active
stable
stable
active
RRMS. RADICULOPATHY L4-L5
RRMS, CORPUS CALLOSUM AGENESIS
CIS
RRMS
RRMS
RRMS
RRMS
PPMS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
CIS
CIS
RRMS
RRMS
RRMS
RRMS
RRMS
RRMS
CIS
RRMS
RRMS
CIS
RRMS
CIS
RRMS
RRMS
F
M
F
F
F
F
F
M
F
F
M
M
M
F
F
M
M
F
M
F
M
F
M
F
F
F
F
F
M
M
M
F
F
LEVOTHYROXINE SODIUM
NONE
NONE
NONE
NONE
ATENOL,
NONE
NONE
NONE
FOSINOPRIL, ETIZOLAM
ALENDRONIC ACID, GABAPENTINE, TRAMADOL ,
ALPRAZOLAM, METHYLPREDNISOLONE
NONE
PREGABALIN
NONE
EDRONAX, SERTRALINE
NONE
PROGESTOGENS
SERTRALINE
NONE
LEVOTHYROXINE SODIUM
NONE
NONE
NONE
NONE
NONE
NONE
NONE
PROGESTOGENS
NONE
DELAPRIDE LIVIAL
NONE
NONE
PREGABALIN
NONE
GABAPENTINE
NONE
NONE
NONE
NONE
TOPIRAMATE,FLUOXETINE
L-ACETYL CARNITINE
NONE
NONE
STABLE
ND
STABLE
ACTIVE
ACTIVE
ACTIVE
ND
ND
STABLE
ACTIVE
STABLE
ND
ND
STABLE
ACTIVE
ACTIVE
ND
STABLE
ATYPICAL
ACTIVE
ND
ACTIVE
ACTIVE
ACTIVE
ACTIVE
STABLE
STABLE
ND
STABLE
STABLE
ND
STABLE
STABLE
Table S2
Protein
name
Accession
number
Queries
Matched
Peptides sequenced
Transthyretin
BAA00059
6
GSPAINVAVHVFR
AADDTWEPFASGK
ALGISPMHEHAEVVFTANDSGPR
TSESGELHGLTTEEEFVEGIYKVEIDTK
Sequence Mascot
coverage
score
(%)
60
342
Table S3
m/z
Disulfide Bridge of Cys10
Peak 1
13761
-
Peak 2
13791
-SH
Peak 3
13841
-SO3H
peak 4
13880
-Cys
Peak 5
13937
-Cys-Gly
Table S4
Pvalue
t-test
TTR-SSH
TTR-SSH
X
TTR-SSO3H
Anomalous
Dimer
Normal
Dimer
Monomer
TTR-SSO3H
Anomalous Normal
Dimer
Dimer Monomer
0.0001
0.009
0.36
0.25
0.02
0.15
0.1
0.45
0.0001
-
X
-
-
X
-
-
-
X
0.0004
-
-
-
-
X