Specific changes of serum proteins in Parkinson's disease patients - PubMed (original) (raw)
Specific changes of serum proteins in Parkinson's disease patients
Wenwen Lu et al. PLoS One. 2014.
Abstract
The aim of this study is to identify and validate protein change in the serum from PD patients. We used serum samples from 21 PD patients and 20 age-matched normal people as control to conduct a comparative proteomic study. We performed 2-DE and analyzed the differentially expressed protein spots by LC-MS/MS. In PD group 13 spots were shown to be differentially expressed compared to control group. They were identified as 6 proteins. Among these, 3 proteins were confirmed by Western blot analysis. It showed that the frequency of fibrinogen γ-chain (FGG) appeared 70% in PD, which could not be detected in control group. The protein of inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4) was found to exist two forms in serum. The full size (120 kDa) of the protein was increased and the fragmented ITI-H4 (35 kDa) was decreased in PD group. The ratio of full size ITI-H4 to fragmented ITI-H4 in PD patients was 3.85 ± 0.29-fold higher than in control group. Furthermore, fragmented Apo A-IV (∼ 26 kDa) was mainly detected in control group, while it was rare to be found in PD group. Above findings might be useful for diagnosis of PD. When the expressions of FGG and 120 kDa ITI-H4 are increase, as well as ∼ 26 kDa Apo A-IV disappear would provide strong evidence for PD.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Serum depletion.
It was clear from 2-DE of whole serum and depleted serum proteins that many more proteins were visible in the depleted sample (B) than were in the whole serum sample (A) when both gels were loaded with the same overall amount of serum proteins.
Figure 2. Representative silver-stained 2-DE gels of serum proteins from controls (A) and PD patients (B).
The most dominant proteins in human sera derived from controls and PD patients were depleted by an immunoaffinity column (MARS) and then separated by 2-DE. Molecular weight is indicated on the left side in kDa.
Figure 3. Distribution of differentially expressed protein spots.
The samples were separated using IPG gel (pH 3–10, 18 cm) in the first phase and 12.5% SDS-PAGE; 120 µg of protein was used in each gel. The spots showing significant differences between PD patients and controls (see Table 1) were labeled in a 2-DE gel from PD group.
Figure 4. Expanded images of protein spots differentially expressed between PD patients and controls.
The protein FGG (spot 390, 213, 214 and 209) and haptoglobin (spot 14) were increased in abundance in the serum from PD patients, relative to the levels in controls, whereas the levels of prothrombin (spot 429 and 277), inter-alpha-trypsin inhibitor heavy chain H4 (spot 422 and 393), apolipoprotein A-I (spot 278 and 41) and apolipoprotein A-IV (spot 268 and 266) were decreased in PD serum relative to the levels in controls.
Figure 5. Representative results of the identification of protein (FGG) by LC-MS/MS.
(A) Chromatogram of peptide mixture. (B and C) MS/MS spectra of two peptides.
Figure 6. Expression of FGG, Apo A-IV, and ITI-H4 in individual depleted serum samples from 4 PD patients and 4 control subjects.
(A) Representative panel of Western blots. It shows that full size (46 kDa) and ∼26 kDa fragment of Apo A-IV and full size (120 kDa) and 35 kDa fragment of ITI-H4 existed in PD and control serum. (B) Quantitative comparison of the Western blot shown in (A). FGG was only detected in PD serum samples. The level of full size Apo A-IV was increased, whereas fragmented Apo A-IV was decreased in PD patients compared with controls. The level of full size ITI-H4 was increased, whereas fragmented ITI-H4 was decreased in PD patients compared with controls. Total protein concentration in each sample was determined by Bradford assay. Protein loadings were approximately equal for all samples (20 µg/lane). Data represent mean ± S.E.M. for 4 individual subjects per group. *, p<0.05 compared with control, Student's _t_-test.
Figure 7. Validation of FGG in individual whole serum samples from 20 PD patients and 20 control subjects.
(A) Representative panel of Western blots. (B) The frequency (14/20) of FGG detected in the serum of PD patients. None was detected in controls. Each number in the panels corresponds to an individual normal control or PD patient. Total protein concentration in each sample was determined by Bradford assay. Protein loadings were approximately equal for all samples (20 µg/lane). The immunoglobulin (IgG) was used as a loading control.
Figure 8. Expression of ITI-H4 in individual whole serum samples from 16 PD patients and 16 control subjects.
(A) Representative panel of Western blots. Full size (120 kDa) and a fragment (35 kDa) of ITI-H4 were detected in the serum. (B) The ratio of full size to fragment ITI-H4 in PD serum was significantly higher than in control. (C and D) Quantitative analysis of the Western blot for full size and the fragmented ITI-H4, respectively, shown in (A). The level of full size ITI-H4 was increased, whereas fragmented ITI-H4 was decreased in PD patients compared with controls. Each number in the panels corresponds to an individual patient or control. Total protein concentration in each sample was determined by Bradford assay. Protein loadings were approximately equal for all samples (20 µg/lane). Data represent mean ± S.E.M. for 16 individual subjects per group. *, p<0.05 compared with control, Student's _t_-test.
Figure 9. Expression of Apo A-IV in individual whole serum samples from 16 PD patients and 16 control subjects.
(A and C) Representative panel of Western blots for full size (46 kDa) and the fragmented (∼26 kDa) Apo A-IV. (B and D) Quantitative analysis of the Western blot for full size and the fragmented Apo A-IV shown in A and C. The level of full size Apo A-IV was similar in control group and in PD patients. However, the fragmented Apo A-IV was mainly detected in control group. Each number in the panels corresponds to an individual patient or control. Total protein concentration in each sample was determined by Bradford assay. Protein loadings were approximately equal for all samples (20 µg/lane). Data represent mean ± S.E.M. for 16 individual subjects per group. *, p<0.05 compared with control, Student's _t_-test.
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