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Salivary α-synuclein and DJ-1: Potential biomarkers for Parkinson’s disease
Ivana Devic1,3,†, HyeJin Hwang1,†, J. Scott Edgar2, Kenneth Izutsu3, Richard Presland3,4,
Catherine Pan1, David R Goodlett2, Yu Wang1,5, Jeff Armaly1, Vitor Tumas6, Cyrus Zabetian7, 8,
James B Leverenz7, 8, Min Shi1, and Jing Zhang1,*
Supplemental Documents
1) Supplemental Materials and Methods:
Saliva Sample Collection and Preparation
Whole saliva was collected from patients with Parkinson’s disease (PD) (n=24; M/F=17/7;
average age=63.5), as well as healthy controls (n=25; M/F=11/14; average age=58.0). All
patients met UK PD Society Brain Bank clinical diagnostic criteria for PD as determined by a
movement disorder specialist. Control subjects had no history of parkinsonism and were either
spouses of PD patients or healthy volunteers from the local community. The subjects refrained
from eating within 60 minutes prior to sample collection. Five minutes before collection, the
subjects rinsed their mouths with water for one (1) minute to remove any excess tissue and
debris present. Sample saliva was collected in a resting, unstimulated state (i.e., no food,
chewing gum, etc). The saliva was simply collected by drooling into a 50ml vial which were
placed on ice, with head tilted forward, allowing the saliva to accumulate in the mouth. After
collection, the whole sample was immediately placed on ice, and then precleared by a low spin
at 2,600  g for 15 minutes at 4°C, followed by a high spin at 15,000  g for 15 minutes at 4°C to
remove particles such as bacteria, mucosal cells, etc. The samples were then treated with
Protease Inhibitor Cocktail (100 µl/1 ml of whole saliva; Sigma Aldrich, St. Louis, MO, USA,
Cat# P2714) to minimize protein degradation. The treated samples were divided into 0.5ml
aliquots and stored at -80°C before use.
For Western blotting, proteins in a 1 ml pooled saliva sample were precipitated with 20%
trichloroacetic acid, washed with cold acetone and dissolved with a urea/loading buffer (8 M
urea, 2% sodium dodecyl sulphate, 10% glycerol, 62.5 mM Tris, pH 6.8). Protein concentrations
were measured using a BCA protein assay kit (Pierce, Rockford, IL). To measure the amount of
the target protein in saliva cellular component, the pellet was washed three times with PBS and
then re-dissolved in the urea/loading buffer.
Immunoprecipitation of α-synuclein and DJ-1:
Immunoprecipitation (IP) or immunodepletion of alpha-synuclein (α-Syn) and DJ-1 was
performed by using the ExactaCruz IP kits from Santa Cruz Biotechnology (Santa Cruz, CA,
USA) according to the manufacturer’s instructions. Briefly, 5 µg of purified mouse monoclonal
anti-α-Syn antibody (BD Transduction Laboratories, San Jose, CA, USA, Cat# 610787) or 30 µg
of rabbit anti-DJ-1 antibody (Novus Biologicals, LLC, Littleton, CO, Cat# NB100-483) were
coupled to 150 µl of IP matrix/beads in PBS (pH 7.2) overnight at 4˚C on a rotator. 500 µl of
saliva was precleared by incubating it with 50 µl of the pre-clearing matrix for 30 minutes at 4˚C
while rotating, and then incubated with the antibody-conjugated IP matrix at 4˚C on a rotator for
48 hrs. After incubation, the IP matrix was pelleted at 15,000  g for 30 seconds at 4˚C. The
supernatant was transferred into a new tube and the remaining proteins were precipitated with
20% trichloroacetic acid. The pelleted matrix was washed 4 times with PBS (500 µl) and the
bound proteins were finally eluted in a SDS-PAGE loading buffer.
Quantitative Western Blotting:
Western blotting was performed following a standard protocol (Hong et al., 2010; Hong et al.,
2009). Samples were loaded onto 10-20% sodium dodecyl sulphate polyacrylamide gels for
electrophoresis. Recombinant human DJ-1 (Covance, Berkeley, CA, USA) and α-Syn (rPeptide,
Athens, GA, USA) proteins were used as standards. For α-Syn, the membrane was incubated
with a rabbit anti-α-Syn antibody ASY-1 (Fjorback et al., 2007; Lindersson et al., 2004) (1:1000),
a gift from our collaborator Dr. Jensen in Denmark, followed by a HRP-conjugated goat antirabbit secondary antibody (Sigma Aldrich, St. Louis, MO, USA; 1:20,000). For DJ-1, the
membrane was incubated with a biotinylated anti-human DJ-1 antibody (R&D Systems,
Minneapolis, MN, USA; Cat# BAF3995; 1:500) followed by Streptavidin-peroxidase (Sigma
Aldrich, St. Louis, MO, USA; 0.02 µg/ml). Blots were scanned and band intensities were
quantified by densitometric analysis using a VersoDoc 3000 imaging system (Bio-Rad,
Hercules, CA, USA) and the Quantity One software (version 4.6.3, Bio-Rad).
2
In-gel Digestion and Mass Spectrometry:
In-gel digestion and mass spectrometry identification of α-Syn and DJ-1 in saliva were
performed as described previously (Hong et al., 2010) with minor modifications. For α-Syn, 10ml
of saliva was used for IP, and the enriched proteins were loaded and separated on a 10–20%
Tris–HCl gel. Lanes of protein matching the corresponding western blot were excised from the
gel and destained. In-gel digestion was carried out sequentially with trypsin and endoproteinase
Asp-N for 16h each. For DJ-1, proteins from saliva sample were precipitated with 20%
trichloroacetic acid, and 200 µg of proteins were separated on a 12.5% Tris-HCl gel, then
stained using Coomassie blue G-250. Excised proteins were digested by trypsin for 16h.
To validate the presence of α-Syn and DJ-1 in human saliva, both proteins were identified by a
4800 matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometer
(Applied Biosystems, Foster City, USA) as described by Hong and colleagues (Hong et al.,
2010) as well as an LTQ-Orbitrap hybrid mass spectrometer (Thermo Scientific Corp, San Jose,
CA, USA). For both DJ-1 and -Syn on LTQ, peptides were separated online with home-packed
columns (0.75µm i.d.×20cm for -Syn; 0.75µm i.d.×25cm for DJ-1; 100Å Magic C18AQ:
Michrom Bioresources, Auburn, CA, USA) with a linear gradient of 5-35% acetonitrile with 0.1%
formic acid over 60 minutes using a Nano-Acquity (Waters, Milford, MA, USA). Electrospray was
performed by applying an electric field to a liquid microtee junction (Yi EC, 2003). All ions were
measured in positive mode and Automatic Gain Control was used to maintain constant ion
populations. Spectral information was searched using SEQUEST (version 27) against the
International Protein Index (IPI) human protein database (version 3.68) and protein
identifications were filtered using PeptideProphet (Nesvizhskii et al., 2003).
Luminex detection of α-synuclein and DJ-1:
Salivary α-Syn and DJ-1 levels were measured using established Luminex assays as described
previously (Hong et al., 2010) with minor modifications. Briefly, saliva was treated with equal
volume of 2 radioimmunoprecipitation assay (RIPA) buffer for 1h on ice, for a 1:2 dilution. For
α-Syn detection, the samples were then loaded (100 µl/well) directly; for DJ-1 detection, the
samples were further diluted with assay diluent (0.1% bovine serum albumin/phosphate
buffered saline, pH 7.2) for a 1:16 dilution (1:32 final) and loaded (100 µl/well). After an
overnight incubation (~18 h) at 4°C on a plate shaker (600 rpm), the sample solution was
removed. The detecting antibodies in assay diluent [2 µg/ml biotinylated anti-human α-Syn
3
antibody (R&D systems) or 1 µg/ml biotinylated anti-human DJ-1 antibody (R&D systems)] were
added at 100 µl/well and incubated for 3 h at room temperature on a plate shaker (600 rpm).
After the detecting antibody solution was removed, diluted streptavidin-R-PE (0.33 ng/ml for αSyn and 1 ng/ml for DJ-1; Prozyme, San Leandro, CA, USA) in assay diluent was added (100
µl/well) and incubated at room temperature for 0.5 h on a plate shaker (600 rpm). The plate was
read on a LiquiChip Luminex 200TM Workstation (Qiagen). For each set of microspheres, 100
beads were analyzed and median values were reported. A series of recombinant, full-length
human α-Syn (rPeptide) or DJ-1 (Covance) standards diluted in assay diluent solution were run
in parallel. The assay diluent was used as a blank, and as internal standard, aliquots of one
previously prepared pooled saliva specimens with known target-protein concentrations were
included in each plate. The assays’ accuracy was measured by spiking human recombinant
proteins in saliva and the recovery rate for both proteins was close to 100%.
References:
Fjorback AW, Varming K, Jensen PH. Determination of alpha-synuclein concentration in human
plasma using ELISA. Scandinavian journal of clinical and laboratory investigation.
2007;67(4):431-5.
Hong Z, Shi M, Chung KA, Quinn JF, Peskind ER, Galasko D, et al. DJ-1 and alpha-synuclein in
human cerebrospinal fluid as biomarkers of Parkinson's disease. Brain. 2010 Mar;133(Pt
3):713-26.
Hong Z, Zhang QY, Liu J, Wang ZQ, Zhang Y, Xiao Q, et al. Phosphoproteome study reveals
Hsp27 as a novel signaling molecule involved in GDNF-induced neurite outgrowth. J Proteome
Res. 2009 Jun;8(6):2768-87.
Lindersson E, Beedholm R, Hojrup P, Moos T, Gai W, Hendil KB, et al. Proteasomal inhibition
by alpha-synuclein filaments and oligomers. The Journal of biological chemistry. 2004 Mar
26;279(13):12924-34.
Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by
4
tandem mass spectrometry. Anal Chem. 2003 Sep 1;75(17):4646-58.
Yi EC LH, Aebersold R, Goodlett DR. Rapid Communications in Mass Spectrometry
2003;17.:2093-98.
5
2) Supplemental Tables and Figures
Suppl. Table 1. Summary of demographics and salivary α-synuclein and DJ-1 values of
donors.
Control
Parkinson’s Disease
25
24
11/14
17/7
58.0 ± 10.4
63.5 ± 11.3
40 - 81
39 - 82
-
24
Mean ± SD
-
8.5 ± 6.4
Range
-
1 - 27
-
18/24
Mean ± SD
-
9.4 ± 6.9
Range
-
1 - 27
A: All cases
-
20:2:1:1
B: Cases with UPDRS Motor Scores
-
15:2:1:0
Mean ± SD
980.8 ± 479.5
1060.7 ± 587
Range
359.6 - 2009
204.7 - 3174
Mean ± SD
0.11 ± 0.13
0.07± 0.08
Range
0.003 - 0.4
0.004 - 0.3
Mean ± SD
128 ± 116.4
185.7 ± 339
Range
13.7 - 447.1
5.11 - 1675.1
Number of Cases
Sex (M/F)
Age (years)
Mean ± SD
Range
Duration of Disease (years)
A: All cases
B: Cases with UPDRS Motor Scores
Cases of Drug Treatmenta
Saliva total protein (µg/ml)
Saliva α-Syn (ng/ml; all cases)
Saliva DJ-1 (ng/ml; all cases)
6
Number of patients with Parkinson’s disease who were treated with carbidopa/levodopa alone or
together with other anti-parkinsonism drugs versus those treated with dopamine agonists but not
levodopa versus those treated with other anti-Parkinson’s disease medications (e.g. monoamine oxidase
B inhibitors and amantadine) only versus those not treated with any anti-parkinsonism drugs (no
Parkinson’s disease med) when the saliva samples were obtained.
a:
Suppl. Table 2. Identification of α-Syn and DJ-1 proteins in human saliva by mass
spectrometry.
IPI No. / Protein name
IPI00024107
Coverage (%)
confidence
34.0
1.00
Isoform 1 of α-synuclein
IPI00298547
Protein DJ-1
45.0
1.00
Identified peptide sequence
Probability
Ions
TVEGAGSIAAATGFVK
1.0000
25/30
EGVLYVGSK
0.9909
14/16
TKEQVTNVGGAVVTGVTAVAQK
0.8603
32/84
GAEEM147ETVIPVDVM147R
1.0000
23/28
EGPYDVVVLPGGNLGAQNLSESAAVK
1.0000
32/100
VTVAGLAGKDPVQC160SR
1.0000
29/60
DVVIC160PDASLEDAK
0.9981
16/26
VTVAGLAGK
0.9980
13/16
ALVILAK
0.9916
11/12
LPGGNLGAQNLSESAAVK
0.9456
19/34
VTVAGLAGKD
0.8221
15/18
APLVLKD
0.7581
9/12
7
Suppl. Fig. 1. Comparison of α-Syn and DJ-1 between human whole saliva’s cellular
component and supernatant. Cellular component (CC) and supernatant (SP) from a
pooled saliva was separated by centrifugation. Proteins from equal fraction (2.08% and
0.20% for α-synuclein and DJ-1, respectively) of both the cellular component and the
supernatant were loaded on SDS PAGE and immunoblotted with or without the primary
8
antibody against α-synuclein (A) or DJ-1 (B). Recombinant human DJ-1 and α-Syn
proteins were used as standards (STD).
Suppl. Fig. 2. Identified peptide sequence of α-synuclein and DJ-1 in human saliva. Both
α-synuclein (A) and DJ-1 (B) proteins were identified in human saliva by a 4800 MALDITOF/TOF (matrix-assisted laser desorption ionization-time of flight-time of flight) mass
spectrometer and an LTQ-Orbitrap hybrid mass spectrometer. The whole amino acid
sequences of the proteins are shown with the identified peptides shown in red. The
amino acid coverage of α-synuclein and DJ-1 was 34% and 45%, respectively.
9
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