II. Supplemental Figures and Table
Suppl. Fig. 1.
Characterization of DJ-1 in human CSF
( A ) To determine the presence and the amount of DJ-1 in CSF, the reference CSF sample was analyzed by immunoblotting using a mouse anti-human DJ-1 antibody.
Proteins in the CSF were denatured by heating and the addition of a strong detergent
(SDS), thereby disassociating potential oligomeric forms that might exist in native human CSF. Lane 1 from the left, human (Hu) brain (cerebral cortex) extract used as a positive control; lane 3, proteins from 500 μL reference CSF precipitated with trichloroacetic acid (TCA); lanes 5–10, serially diluted recombinant human DJ-1 protein at 0, 6.25, 12.5, 25, 50 and 100 ng/mL; lanes 2 and 4 were intentionally left blank. DJ-1 in CSF was detected as a single band of 25 kDa corresponding to the positive control of human brain extract. These immunoreactive bands were not detected when only anti-mouse IgG was used (data not shown). DJ-1 levels in CSF were estimated to be 90±21 ng/mL according to a photometric standard linear curve obtained using serially diluted samples of recombinant DJ-1. ( B ) To further confirm the 25 kDa band detected by Western was indeed DJ-1, the band was cut from gels (a total of 16 lanes [300 μg/lane] were pooled) run in parallel with the Western analysis, and the in-gel digests were analyzed by MALDI-TOF-TOF mass spectrometry (MS).
Shown is the fragmentation spectrum of MS/MS for ion 1158.61 of DJ-1. The

sequence shown is EILKEQENR, with y- and b-series ions interpreted. The results demonstrate that five fragments, corresponding to a total of 63 residues of the 189 amino acids, of full-length DJ-1 were identified (see also Suppl. Table 1 ).
( C ) To further determine the nature of CSF DJ-1 (monomers vs. oligomers or species conjugated with other macromolecules), reference CSF samples were eluted by size exclusion chromatography (SEC) to segregate proteins based on molecular weight
(MW). The elution pattern can be found in Suppl. Fig. 2 . Proteins from CSF fractions were precipitated and subjected to immunoblot analysis using an anti-human DJ-1 antibody. Most DJ-1 signals appeared to be from the SEC fractions corresponding to
MWs of 25-30 kDa. F1, >75 kDa; F2, ~71.5 kDa; F3, ~57 kDa; F4, ~43 kDa; F5, ~29 kDa; F6, ~14 kDa; F7-10, <14 kDa. 2 and 4
 g of human (Hu) brain extracts were also loaded as controls. ( D ) Fractions of 6 mL were collected, concentrated to 1 mL, and 100 μl were analyzed by Luminex to determine DJ-1 levels. A similar distribution pattern was observed as the Western results in ( C ). Data shown are representative of at least three independent experiments.

Suppl. Fig. 2. Elution of human CSF on a HiPrep 16/60 Sephacryl S-100 HR size-exclusion column
Five mL of CSF sample was loaded and eluted with 160 mM ammonium acetate (pH
7.4) at a flow rate of 0.25 ml/min. Absorbance of the eluted proteins was monitored at
280 nm. The elution pattern of the following protein size markers on the same column is also superimposed: Blue dextran (Bl), >2000 kDa; conalbumin (Co), 75 kDa; ovalbumin (O), 43 kDa; carbonic anhydrase (CA), 29 kDa; ribonuclease A (RA), 13.7 kDa; aprotinin (Apr), 6.5 kDa.

Suppl. Fig. 3.
Characterization of SNCA in human CSF
( A ) ~300 µg of proteins precipitated from 500 µL of the pooled reference CSF sample
(lane 3) along with serially diluted samples of recombinant SNCA (lanes 5 – 10, at 0,
0.1, 0.25, 0.5, 2.5 and 5 ng/mL) were analyzed by immunoblotting using a mouse anti-human SNCA antibody. Human (Hu) brain (cerebral cortex) extracts were used as a positive control (lane 1). Lane 2 and 4 were intentionally left as blank. SNCA in
CSF was detected as a single band of 19 kDa corresponding to the human brain control. No signal was detected when only anti-mouse IgG was used (data not shown).
SNCA levels in CSF were estimated to be 1.36±0.35 ng/mL according to a photometric standard linear curve obtained using serially diluted samples of recombinant SNCA. ( B ) Reference CSF samples were eluted by size exclusion chromatography to segregate the protein based on molecular weight (MW). Proteins from CSF fractions were precipitated and subjected to immunoblot analysis using an anti-human SNCA antibody. Most SNCA species present in native CSF corresponded to MWs of 45 kDa with some signals detected beyond 45 kDa. MW of Fractions:
F1, >75 kDa; F2, ~71.5 kDa; F3, ~57 kDa; F4, ~43 kDa; F5, ~29 kDa; F6, ~14 kDa;
F7-10, <14 kDa. ( C ) Fractions of 6 mL were collected, concentrated to 1 mL, and 100
μl were analyzed by Luminex to determine SNCA levels. A Similar distribution pattern was observed as the Western results shown in ( B ). Data shown are representative of at least three independent experiments.

Suppl. Fig. 4.
Establishment and characterization of the DJ-1 Luminex assay system
( A ) To assess CSF DJ-1, DJ-1 was captured by a mouse anti-DJ-1 monoclonal antibody (CV-DJ) or a rabbit anti-DJ-1 antibody (NV-DJ), followed by detection with a biotinylated anti-DJ-1 antibody (RD-B). Quantification was accomplished by incubating with streptavidin-conjugated fluorochrome-phycoerythrin. Among multiple antibodies tested, two pairs, i.e., CV-DJ/RD-B and NV-DJ/RD-B, gave the most consistent results. However, NV-DJ/RD-B appeared to be at least 10-fold more sensitive (data not shown). Hence NV-DJ/RD-B was used in future analyses. Shown is a calibration curve for the DJ-1 Luminex assay using the NV-DJ/RD-B antibody pair. The low end of the curve is also shown in the insert. ( B ) DJ-1 levels were determined by the Luminex assay in 0.5 μg cerebral cortex extracts from DJ-1-null
(KO) and wild-type (WT) mice (gifts from Dr. Amy Manning-Bog at The Parkinson's
Institute, CA), 0.5 μg human cerebral cortex extract (human brain), 100 μL un-concentrated pooled native human CSF (CSF) and 100 μL human CSF with DJ-1 depleted using a mouse anti-DJ-1 antibody (Covance) which is not used in the final
Luminex assay (cleared CSF). The depletion was done by coupling the antibody to
Santa Cruz ExactaCruz IP matrix E and then incubating the coupled antibody with

CSF over night at 4 o
C. The assay specificity of the output signals for standards and samples was also confirmed by a few other methods. First, no signal was detected by performing assays without the detecting antibody or using other recombinant protein(s) in place of the recombinant DJ-1 protein. Second, only background values were detected in the standards or samples (human and mouse brain tissue, human whole blood, or three different pooled reference CSF) when the detecting DJ-1 biotinylated antibody was replaced with an antibody that did not recognize DJ-1. Furthermore, no signal was observed in standards or any of the samples discussed above when the capturing DJ-1 antibody was substituted with another antibody that did not recognize
DJ-1 (data not shown for these three types of experiments). ( C ) Characterization and optimization of CSF DJ-1 Luminex assay. Identical aliquots of cell-free, native, previously unfrozen pooled CSF were processed in parallel under the indicated conditions. While the addition of NP40 detergent (0.5% final) to 100 μL of CSF had no observed effect on the signal from DJ-1 in the native/original CSF, equal volume of 2

RIPA buffer treatment increased fluorescent signal strength by up to 57%.
When CSF was diluted by the assay diluent 0.1%BSA/PBS (dilu), the signal intensity increased 100%. Unfrozen samples (empty bars) and thawed samples that were frozen at –80 o
C for 3 months (solid bars) were also compared; T/C twice, freeze and thaw twice. ( D ) DJ-1 concentrations were measured in un-spiked CSF samples (native and diluted with 0.1%BSA/PBS) and samples spiked with human recombinant DJ-1 proteins at indicated concentrations. In native CSF and diluted CSF, the recovery rate was 23% and 81%, respectively.

Suppl. Fig. 5.
Establishment and characterization of the SNCA Luminex assay system
( A ) To establish the SNCA Luminex assay, multiple antibody pairs were tested, including 211/RD-SB (a biotinylated anti-human SNCA antibody from R&D),
LB507/RD-SB, LB507/211 and ASY-1/RD-SB. The ASY-1/RD-SB pair was chosen for the remaining investigation as it demonstrated best sensitivity and recovery rate from CSF. Shown is a calibration curve for the SNCA Luminex assay using the
ASY-1/RD-SB antibody pair. The low end of the curve is also shown in the insert. ( B )
SNCA levels in 0.5 μg cerebral cortex extracts from SNCA-null (KO) and wild-type mice (Jackson Laboratory, Bar Harbor, ME), 0.5 μg human brain (cerebral cortex) extract and 100 μL un-concentrated pooled native human CSF were determined by the
Luminex assay. The assay specificity of the output signals for standards and samples was also confirmed by the same strategies used in the DJ-1 assay shown in Suppl. Fig.
4B . No signal was detected when any element of the assays was replaced or deleted
(data not shown).
(C ) Characterization and optimization of CSF SNCA Luminex assay. Identical aliquots of cell-free, native, previously unfrozen pooled CSF were processed in parallel under indicated conditions. The addition of NP40 detergent
(0.5% final) to 100 μL of CSF only slightly increased (~10%) the signal of SNCA in native/original CSF; in contrast, RIPA buffer treatment (25 μL of CSF treated with

25μL of 2

RIPA buffer, and then diluted by 50 μL of 0.1%BSA/PBS) consistently increased the signal intensity by up to 400%. Diluting CSF with 0.1%BSA/PBS yielded no positive effects on the signal (data not shown). Unfrozen samples (empty bars) and thawed samples that were frozen at –80 o
C for 3 months (solid bars) were also compared; T/C 2nd and 3rd, freeze and thaw two and three times. ( D ) SNCA concentrations were measured in un-spiked CSF samples (native and treated with
RIPA) and samples spiked with human recombinant SNCA proteins at indicated concentrations. In native CSF and RIPA-treated CSF, the recovery rate was 25% and
93%, respectively.

Suppl. Fig. 6. DJ-1 and SNCA in whole blood
Whole blood (WB; lanes 1 and 2, 1 μl and 0.5 μl, respectively) along with human cerebral cortex (Hu-brain) (2 μg) and human CSF (TCA precipitates from 500 μl CSF) were run on 8-16% SDS polyacrylamide gels and analyzed on immunoblots probed with a DJ-1 (1:1000, Novus) or a SNCA (1:1000, BD) specific antibody.

Suppl. Fig. 7. Distribution of ages for subjects in different groups included in this study
CTL, healthy control; AD, Alzheimer’s disease; PD, Parkinson’s disease.

Suppl. Fig. 8. Relationship between the duration of Parkinson’s disease and CSF
DJ-1 or SNCA levels
CSF DJ-1 ( A , B ) and SNCA ( C , D ) levels were measured by Luminex. Data shown are before ( A , C , n=115) or after ( B , D , n=88) eliminating subjects containing high hemoglobin levels (200 ng/mL was used as a cutoff). The correlation coefficient and
P value of linear regression for controls are shown in each panel.

Suppl. Table 1. Human DJ-1 peptides identified by mass spectrometry in CSF
Protein Accession
DJ-1
Peptide fragment sequence Confidence Precursor
MW
IPI00298547.3 EILKEQENR
GAEEMETVIPVDVMR
MMNGGHYTYSENR
VEKDGLILTSR
DKMMNGGHYTYSENR
CAGPTALLAHEIGFGSK
99%
99%
99%
99%
99%
99%
1157.596
1674.767
1558.611
1229.667
1833.671
1638.816
Theoretical
MW
1157.604
1674.796
1558.629
1229.698
1833.741
1638.873