DJ-1 isoforms in whole blood as biomarkers of Parkinson disease

Xiangmin Lin, Travis J. Cook, Cyrus P. Zabetian, James B. Leverenz, Elaine R. Peskind, Shu-Ching Hu, Kevin C. Cain, Catherine Pan, John Scott Eagar, David R. Goodlett, Brad A. Racette, Harvey Checkoway, Thomas J. Montine, Min Shi, Jing Zhang

Supplemental Documents

1) Supplemental Materials and Methods:

Confirmation of HNE antibody specificity

4-hydroxy-trans-2-nonenal (HNE) was prepared by 4-Hydroxy-non-2-enal diethylacetal (HNE-DEA) (Oxis, Beverly Hills, CA, USA) hydrolysis with 1 mM HCl for 1 h at room temperature 1. HNE was incubated with DJ-1 (Covnace-Research, Dedham, MA, USA) or bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) at a 800:1 molar ratio at 37°C for 4 h, and HNE-DJ-1 or HNE-BSA were reduced with NaBH4 (100 mM final concentration) for 1 h at room temperature. The proteins were desalted to remove excess 4-HNE and a BCA assay was performed to measure protein concentration.

Dot Blotting

A PVDF membrane was pre-wetted in 100% methanol for 2 min and then equilibrated 2 min in TBST buffer, washed with MilliQ water several times, and 50 ng BSA or HNE-treated BSA, or 60 ng DJ-1 or HNE-treated DJ-1- were spotted on the membrane. After blocking with Tris buffered saline (pH 8.0) containing 0.1% (v/v) Tween (TBS-T) and 5% (w/v) BSA at room temperature for 1 h, the membrane was incubated for 1 h at room temperature with rabbit anti-reduced HNE antibody 2 at a dilution of 1:1000. After washing with TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody at a dilution of 1:20,000 in TBS-T containing 3% (w/v) BSA at room temperature for 1 h. The immunostained proteins were detected using ECL Plus (GE Healthcare,Buckinghamshire,UK) and exposed for 2 min prior to development.

Human Whole Blood Incubation with Dopamine

Human whole blood was incubated with either HNE (10M) or dopamine hydrochloride (100M) at 37oC for 24 h. Following incubation, samples were used in dot blotting or 2DE experiments followed by western blotting for HNE and DJ-1 as described in the main text. Results are shown in Suppl. Fig. 4.

References:

1.Aldini, G., Dalle-Donne, I., Vistoli, G., Maffei Facino, R. & Carini, M. Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction. J. Mass Spectrom.40, 946-954 (2005).

2.Montine, K. S. et al. Distribution of reducible 4-hydroxynonenal adduct immunoreactivity in Alzheimer disease is associated with APOE genotype. J. Neuropathol. Exp. Neurol.57, 415-425 (1998).

2) SupplementalFigures

Suppl. Figure 1. 2-DE map of whole blood after differential solubilization (DS). (A) Proteins were enriched from 50 µL of whole blood using the DS method before 2-DE. Shown is a representative silver staining map with 7 isoforms of DJ-1 indicated by red circles; (B) Enlarged picture of HNE-modified DJ-1 isoforms revealed by western blotting using an anti-HNE antibody. (C) Enlarged picture of the same membrane re-probed with an anti-DJ-1 antibody after stripping. The indicated 7 spots were submitted for identification by mass spectrometry.

Suppl. Figure 2. Confirmation of HNE-modified and phosphorylated DJ-1 by immunoprecipitation and western blotting. Proteins were enriched from 500 µL (for HNE DJ-1) or 4 mL (for phosphorylated DJ-1) of whole blood by immunoprecipitation using an anti-DJ-1 antibody. (A) 2-DE and western blotting using an antibody against HNE were then performed to reveal HNE-positive spots; (B) these spots were confirmed to be DJ-1 after re-probing the same membrane with an anti-DJ-1 antibody. (C) Similarly, a phosphorylated spot was identified by 2-DE western blotting using an antibody against phospho-threonine; (D) and was confirmed to be DJ-1 isoform 4 after re-probing with anti-DJ-1 antibody; weak signals were detected for isoforms 5 and 6 following longer exposure periods (data not shown).

Suppl. Figure 3. Confirmation of the specificity of anti-reduced HNE antibody. (A) Dot blotting of 50 ng BSA and 60 ng DJ-1 with or without HNE treatment or reduction by NaBH4. Only HNE-BSA and HNE-DJ-1 reduced with NaBH4 produced a positive signal. (B) 2-DE western blot of 50L whole blood samples enriched by DS methods with or without reduction by NaBH4 and using HNE antibody or pre-immunity serum at a dilution of 1:1000. Only samples reduced by NaBH4 and detected by HNE antibody produced a positive signal.

Suppl. Figure 4. Effects of dopamine treatment on HNE-modified proteins in human whole blood. Human whole blood was incubated with either dopamine hydrochloride (100 M) or HNE (10 M) as a positive control. (A) Total HNE-modifications following whole treatment with dopamine (DA) or HNE were evaluated by using dot blotting with an anti-HNE antibody. BSA and HNE-modified BSA were used as negative and positive controls, respectively. (B) Effects of treatment with DA or HNE on HNE-modified DJ-1 isoforms were evaluated using 2DE followed by western blotting with an anti-HNE antibody and then an anti-DJ-1 antibody. Quantification of HNE-modified DJ-1 isoforms #4 and #6 from two independent experiments is shown.

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