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1 Discovery and verification of panels of T-lymphocyte proteins as biomarkers of Parkinson's disease Tiziana Alberio a,b, Agnese C. Pippione a,b, Maurizio Zibetti c, Simone Olgiati a, Daniela Cecconi d, Cristoforo Comi e, Leonardo Lopiano c, Mauro Fasano a,b,* Supplemental material a Division of Biomedical Sciences, Department of Theoretical and Applied Sciences, University of Insubria, Busto Arsizio, Italy. b c d Center of Neuroscience, University of Insubria, Busto Arsizio, Italy. Department of Neuroscience, University of Torino, Torino, Italy. Proteomics and Mass Spectrometry Laboratory, Department of Biotechnology, University of Verona, Verona, Italy. e Division of Neurology, Department of Translational Medicine, University of Eastern Piedmont, Novara, Italy. * Corresponding author at: Division of Biomedical Sciences, Department of Theoretical and Applied Sciences, University of Insubria, via Alberto da Giussano 12, I Busto Arsizio, Italy. Tel: ; fax: address: mauro.fasano@uninsubria.it (M. Fasano). 1

2 Supplemental methods 2-DE gels were transferred to PVDF membranes as described in the Methods section. Membranes were saturated in 5% non-fat milk in TBS-T (0.1 M Tris-HCl ph 7.4, 1.5 M NaCl and 0.5% Tween-20) and incubated in the same buffer at 4 C overnight with goat anti-fibrinogen polyclonal antibody (Abnova), 1:10000 dilution, with mouse anti-αtubulin monoclonal antibody (Sigma), 1:10000 dilution, with goat anti-serpin B9 polyclonal antibody (Abcam), 1:2000 dilution, or with mouse anti-β-actin monoclonal antibody (GeneTex), 1:3000 dilution. Membranes were then washed with TBS-T and incubated with peroxidase-conjugated anti-goat-igg antibody (Millipore), 1:8000 in 5% milk-tbs-t, or anti-mouse-igg antibody (Millipore), 1:3000 in 5% milk-tbs-t, respectively, for chemiluminescence detection (Millipore). Image manipulation was performed with ImageJ ( For details on image registration see Natale M, Bonino D, Consoli P, Alberio T, Ravid RG, Fasano M, Bucci EM. A metaanalysis of two-dimensional electrophoresis pattern of the Parkinson's disease-related protein DJ-1. Bioinformatics 26, (2010). 2

3 Supplemental results In the LC-MS/MS identification of selected protein spots, a few double assignments occurred. In most cases, we refined them by matching with reference 2-DE maps available in the swiss-2dpage database ( 2dpage/). In particular, we referred to the LYMPHOCYTE_HUMAN map for disambiguation. In the case the ambiguous assignment was not resolved by matching, we checked the correct assignment by western blotting. Spot 382, for instance, was not confirmed as α-tubulin by Western Blot (Supplemental Fig. 2) and therefore we assigned it to filamin-a, the second LC- MS/MS identification with significant mascot score. Actually, alpha-tubulin is a highly abundant protein that localizes in the close proximity of spot 382, thus a contamination is expected to take place. In a similar case, we ambiguously assigned spot 598 to serpin B9 and moesin. Western blot validation displayed that serpin B9 has a different localization on the 2-DE map (Supplemental Fig. 3) and a repeated LC-MS/MS identification allowed us to assign its identity to moesin unambiguously. 3

4 Supplemental Table 1: LC-MS-MS identification of proteins Spot Protein name Uniprot ID Correlation Mascot score Seq Cov (%) PEPTIDES Mr. (Da) theor. pi theor. Mr. (Da) exp. pi exp. 86 Vinculin P18206 PD Vinculin P18206 PD Vimentin P08670 PD m/z charge start-end sequence AQQVSQGLDVLTAK QVATALQNLQTK VDQLTAQLADLAAR ALASQLQDSLK ELTPQVVSAAR MTGLVDEAIDTK Ox(M) LVQAAQMLQSDPYSVPAR Ox(M) ELLPVLISAMK Ox(M) ELLPVLISAMK Ox(M) MSAEINEIIR Ox(M) MLGQMTDQVADLR 2Ox(M) MLGQMTDQVADLR 2Ox(M) AQQVSQGLDVLTAK SLGEISALTSK QVATALQNLQTK VDQLTAQLADLAAR ALASQLQDSLK LLAVAATAPPDAPNREEVFDER ELTPQVVSAAR ELTPQVVSAAR MTGLVDEAIDTK Ox(M) SLLDASEEAIKK VAMANIQPQMLVAGATSIAR 2Ox(M) MFGGPGTASRPSSSR Ox(M) SYVTTSTR SLYASSPGGVYATR TNEKVELQELNDR VELQELNDR FANYIDK FANYIDK ILLAELEQLK ILLAELEQLKGQGK LGDLYEEEMR Ox(M) LGDLYEEEMR Ox(M) RQVDQLTNDK VEVERDNLAEDIMR Ox(M) VEVERDNLAEDIMR Ox(M) VEVERDNLAEDIMR Ox(M) EKLQEEMLQR Ox(M) EEAENTLQSFR LDLER KVESLQEEIAFLK QQYESVAAK FADLSEAANR QVQSLTCEVDALKGTNESLER Carbamidomethyl (C) Ion score

5 Talin-1 Q9Y490 PD Beta tubulin P07437 LOPD Fibrinogen beta chain P02675 PD Protein disulfide-isomerase A3 P30101 LOPD LQDEIQNMKEEMAR 2 Ox (M) LQDEIQNMKEEMAR 2 Ox(M) EYQDLLNVK MALDIEIATYR Ox(M) KLLEGEESR LLEGEESR LLEGEESR ETNLDSLPLVDTHSK ETNLDSLPLVDTHSK TLLIKTVETR KIFQAHK ILAQATSDLVNAIK ILAQATSDLVNAIK LAQAAQSSVATITR ALGDLISATK VGDDPAVWQLK VMVTNVTSLLK VMVTNVTSLLK Ox(M) ALEATTEHIR ALEATTEHIR SIAAATSALVK SIAAATSALVK QVAASTAQLLVACK Carbamidomethyl (C) ISVYYNEATGGK AILVDLEPGTMDSVR AILVDLEPGTMDSVR Ox(M) IMNTFSVVPSPK Ox(M) FPGQLNADLR FPGQLNADLR FPGQLNADLRK KLAVNMVPFPR Ox(M) LAVNMVPFPR Ox (M) LAVNMVPFPR Ox (M) LHFFMPGFAPLTSR Ox(M) LHFFMPGFAPLTSR Ox(M) ALTVPELTQQVFDAK ALTVPELTQQVFDAK YLTVAAVFR KGGETSEMYLIQPDSSVKPYR NYCGLPGEYWLGNDK Carbamidomethyl ( C) MGPTELLIEMEDWK MGPTELLIEMEDWK EDGGGWWYNR EDGGGWWYNR YYWGGQYTWDMAK YYWGGQYTWDMAK Ox(M) IRPFFPQQ SDVLELTDDNFESR SDVLELTDDNFESR LAPEYEAAATR LAPEYEAAATR QAGPASVPLR FVMQEEFSR Ox(M) FLQDYFDGNLK EATNPPVIQEEKPK

6 365 Fibrinogen beta chain P02675 PD EEAPSLRPAPPPISGGGYR SILENLR QGFGNVATNTDGK Fibrinogen beta chain P02675 PD NA a Fibrinogen beta chain P02675 PD Filamin-A P21333 PD Vimentin P08670 LOPD Lymphocyte-specific protein 1 P33241 PD Septin-6 Q14141 PD L-plastin variant Q59GX5 LOPD DNENVVNEYSSELEK IRPFFPQQ EAGAGGLAIAVEGPSK LTVSSLQESGLK TYSLGSALRPSTSR SLYASSPGGVYATR TNEKVELQELNDR VELQELNDR VELQELNDR FANYIDK ILLAELEQLK LGDLYEEEMR Ox(M) VEVERDNLAEDIMR Ox(M) VEVERDNLAEDIMR Ox(M) DNLAEDIMR Ox(M) EEAENTLQSFR KVESLQEEIAFLK FADLSEAANR FADLSEAANR EYQDLLNVK MALDIEIATYR Ox(M) MALDIEIATYR Ox(M) AEASSDPGAEER Acetyl (Protein N-term) EGPGPEDTVQDNLGAAGAEEEQEEHQK LIDRTESLNR SQPDLPISK QASIELPSMAVASTK Ox(M) STLMDTLFNTK Ox(M) STLMDTLFNTK Ox(M) SLDLVTMK Ox(M) VNIIPIIAK SLDDEVNAFK GSVSDEEMMELR 2Ox(M) EITENLMATGDLDQDGR Ox(M) MINLSVPDTIDER Ox(M) EGESLEDLMK Ox(M) LSPEELLLR Vimentin P08670 PD MALDIEIATYR Ox(M) Moesin P26038 PD KPDTIEVQQMK ALELEQER ALELEQER EALLQASR ISQLEMAR ESEAVEWQQK AQMVQEDLEK AQMVQEDLEK Ox(M) KTANDMIHAENMR Ox(M) TANDMIHAENMR

7 TANDMIHAENMR Ox(M) Gelsolin P06396 PD AGALNSNDAFVLK AGALNSNDAFVLK TPSAAYLWVGTGASEAEK TGAQELLR Transaldolase P37837 PD LLGELLQDNAK Transaldolase P37837 PD Purine nucleoside phosphorylase P00491 LOPD Twinfilin-2 Q6IBS0 PD Rho GDP-dissociation inhibitor 2 P52566 PD Glutathione S-transferase P P09211 LOPD Beta actin b P60709 PD protein epsilon P62258 PD Programmed cell death 6-interacting protein Q8WUM4 LOPD LSSTWEGIQAGK TIVMGASFR ALAGCDFLTISPK Carbamidomethyl (C) ALAGCDFLTISPK Carbamidomethyl (C) LLGELLQDNAK LLGELLQDNAK FEVGDIMLIR Ox(M) LGADAVGMSTVPEVIVAR Ox(M) TEISVESK ETIELVHTEPTDVAQLPSR YHFFLYK APEPHVEEDDDDELDSK ELQEMDKDDESLIK Ox(M) TLLGDGPVVTDPK TLLGDGPVVTDPK APNVVVTR ASCLYGQLPK Carbamidomethyl (C) FQDGDLTLYQSNTILR TLGLYGK ALPGQLKPFETLLSQNQGGK AGFAGDDAPR DLTDYLMK Ox(M) GYSFTTTAER SYELPDGQVITIGNER KEITALAPSTMK Ox(M) EITALAPSTMK Ox(M) YDEMVESMKK VAGMDVELTVEER VAGMDVELTVEER Ox(M) NLLSVAYK IISSIEQK HLIPAANTGESK YLAEFATGNDR YLAEFATGNDRK EAAENSLVAYK EAAENSLVAYK AASDIAMTELPPTHPIR EALQDVEDENQ LANQAADYFGDAFK LLDEEEATDNDLR TMQGSEVVNVLK Ox (M) DLQQSIAR a: identification by 2-DE map matching; b: fragment. 7

8 Supplemental Table 2: Coefficients for linear combinations of spots to build a function that tracks disease severity (H&Y score) and duration (Years from onset). Spot No. Protein H&Y Score Coefficients Years from onset 86 Vinculin Beta-fibrinogen Beta-fibrinogen Beta-fibrinogen Beta-fibrinogen Lymphocyte-specific Protein Vimentin Moesin epsilon

9 Supplemental Table 3: Linear discriminant analysis coefficients and weights Spot No. Protein 20-spot model 14-spot model 9-spot model Coefficient Weight Coefficient Weight Coefficient Weight 86 Vinculin Vinculin Vimentin Talin Beta-fibrinogen Beta-fibrinogen Beta-fibrinogen Beta-fibrinogen Filamin-A Lymphocyte-specific Protein Septin Vimentin Moesin Gelsolin Transaldolase Transaldolase Twinfilin Rho GDP dissociation inhibitor isoform beta actin fragment epsilon

10 Supplemental Table 4: Power analysis of the 20 spots showing the minimum number of subjects to be included in validation studies. Spot No. Number of subjects >

11 Supplemental Table 5: Linear discriminant analysis coefficients and weights (EO PD vs. LO PD patients). Spot Protein Coefficient Weight 351 Beta tubulin Protein disulfide isomerase A Vimentin Plastin Purine nucleoside phosphorylase Glutathione S-transferase P PDCD6 interacting protein

12 Supplemental Table 6: Demographic and clinical data of research participants. Code Gender Age Age at onset Daily L-DOPA dose Dopamine agonists Familiarity H&Y score (mg/day) CTF_NO021 F 63 n.a CTF_TO035 F 68 n.a CTF_TO027 F 63 n.a CTF_TO033 F 63 n.a APF_TO047 F APM_NO015 M CTM_TO059 M 44 n.a CTF_TO063 F 59 n.a CTF_TO065 F 55 n.a CTF_TO043 F 65 n.a CTM_NO022 M 51 n.a APF_TO042 F CTM_NO027 M 64 n.a CTM_TO058 M 43 n.a CTM_TO051 M 60 n.a CTF_TO060 F 47 n.a CTM_TO054 M 51 n.a EOM_NO017 M

13 EOF_TO048 F EOM_NO018 M EOM_TO049 M EOM_TO008 M EOM_TO022 M EOF_TO050 F EOM_NO028 M LOM_TO040 M LOF_TO021 F LOF_TO016 F LOM_TO006 M LOM_TO064 M LOF_NO023 F LOM_TO066 M Subjects are encoded as CT (control), EO (early-onset), LO (late-onset) or AP (atypical parkinsonism). H&Y, Hoehn and Yahr score. n.a., not applicable. 13

14 Captions to Supplemental figures. Supplemental Figure 1: Representative 2-DE maps for a patient with atypical parkinsonism and for a PD patient, in comparison with the reference map of a healthy control subject. Supplemental Figure 2: Verification of spot 382 as alpha-tubulin. Upper left: a detail of the original 2-DE map as shown in Figure 1. Lower left: antialpha-tubulin Western blot, with the region of interest in the red box. Upper right: registered Western blot after background subtraction and rescaling of the area boundaries using molecular weight markers and the anti-beta-fibrinogen Western blot as landmarks, with the corresponding region of interest in the red box. Lower right: overlay of the 2-bit Western blot (in red) with the original 2-DE map region. Supplemental Figure 3: Verification of spot 598 as serpin B9. Upper left: a detail of the original 2-DE map as shown in Figure 1. Lower left: anti betaactin (left) and anti-serpin B9 (small spot on the right) Western blot, with the region of interest in the orange box. Upper right: registered Western blot after background subtraction and rescaling of the area boundaries using molecular weight markers, the anti-beta-fibrinogen Western blot and the anti-beta-actin Western blot as landmarks, with the corresponding region of interest in the orange box. Lower right: overlay of the 2-bit Western blot (in orange) with the original 2-DE map region. 14

15 Supplemental Figure 4: Pearson correlation analysis of spots 86 (vinculin), 362 (vinculin), 365 (β-fibrinogen), 368 (β-fibrinogen), 369 (β-fibrinogen), 405 (lymphocyte-specific protein 1), 598 (moesin) and 1641 ( epsilon) with the disease stage, expressed as Hoehn and Yahr score. P values refer to slope different from zero. Correlation coefficients are calculated according to Eq. 1. Supplemental Figure 5: Pearson correlation analysis of spots 369 (β-fibrinogen), 591 (vimentin), 598 (moesin) and 1641 ( epsilon) with the disease duration, expressed as years from onset. P values refer to slope different from zero. Correlation coefficients are calculated according to Eq. 1. Supplemental Figure 6: Aggregate nesting based on linear Pearson correlation (Eq. 1) of relative volumes of spot pairs. Bars indicate clustering of the four β- fibrinogen spots. 15

16 Supplemental Figure 1 16

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20 Supplemental Figure 5 20

21 Supplemental Figure 6 21