2D-DIGE as a strategy to identify serum protein biomarkers to monitor pharmacological efficacy in dopamine-dictated states of Parkinson’s disease and schizophrenia

Figures & data

Figure 1 Illustration of the methodology followed in the discovery phase of the experiments. Florescent labeling of proteins isolated from clinical phenotypes of schizophrenia and Parkinson’s disease was done with Cyanine 3 (green) and Cyanine 5 (red). Internal standard was labeled with Cyanine 2 (blue). Combined image after DIGE is shown under gels. Relative quantitation and consistency of expression were analyzed by DeCyder software. Differentially expressed spots were picked from preparative gels. Spots were digested with trypsin. Mass spectrometry analysis was done for protein identification.

Abbreviation: DIGE, difference in gel electrophoresis.

Table 1 Clinical profile of patients used in DIGE experiment

Code	Sex	Age (years)	Phenotype
MM25	Female	23	Schizophrenia naïve
MK35	Male	30	Schizophrenia naïve
AS52	Female	40	Schizophrenia naïve
AT75	Male	27	Schizophrenia naïve
BP68	Male	28	Schizophrenia naïve
SU16	Male	61	Parkinson’s naïve
HR63	Male	36	Parkinson’s naïve
BK83	Male	38	Parkinson’s naïve
MI24	Male	42	Parkinson’s naïve
KP05	Male	50	Parkinson’s naïve

Abbreviation: DIGE, difference in gel electrophoresis.

Table 2 Demographic profile of patients recruited for ELISA

Clinical phenotype	Average age (years)	Gender		Total
		Female	Male
Schizophrenia naïve	29.6	3	2	5
Schizophrenia treated	31.4	17	19	36
Schizophrenia treated with Parkinson’s-type side effects	45.7	2	2	4
Parkinson’s disease naïve	46.1	6	0	6
Parkinson’s disease treated	56.4	33	11	44
Parkinson disease treated with schizophrenia’s-type side effects	60.4	3	2	5
Total number of patients recruited in this study				100

Table 3 Clinical profile of patients receiving pharmacological therapy and showing side effects

Patient	Age (years)	Gender	Clinical phenotype	Treatment (generic)	Side effects
01MA	48	Female	Schizophrenia	Olanzapine, risperidone	Tremor of hands, slow walking, no proper body balance for 4 years
02RA	34	Male	Schizophrenia	Olanzapine, pramipexole, haloperidol	Impaired voice, body stiffness, fatigue, dizziness, slow body movements for 1.5 years
03MA	45	Female	Schizophrenia	Olanzapine, risperidone	Unable to write, tremor of hands, dizziness, poor balance for 3 years
04SH	56	Male	Schizophrenia	Olanzapine, haloperidol	Fatigue, daytime sleepiness, difficulty in walking, jaw stiffness for 3 years
05AM	79	Male	Parkinson’s disease	Levodopa, carbidopa ropinirole, rasagiline	Depression and visual hallucinations for 2 years
06TA	56	Male	Parkinson’s disease	Levodopa, carbidopa, ropinirole	Auditory hallucinations for 2 years
07LA	58	Female	Parkinson’s disease	Levodopa, carbidopa, ropinirole	Claustrophobia, auditory hallucinations and delusions for 2.5 years
08SI	52	Female	Parkinson’s disease	Levodopa, carbidopa, ropinirole	Visual hallucinations for 1 year
09SA	57	Male	Parkinson’s disease	Levodopa, carbidopa, ropinirole	Delusion and hallucinations for 1 year

Figure 2 Differential gel electrophoretic gel images show differential protein expression between the serum of patients with Parkinson’s disease and schizophrenia. A total of five biological replicate gels were run using five sets of serum samples from patients with Parkinson’s disease and schizophrenia. Spots that equally expressed in both clinical phenotypes are indicated by white arrows. Spots that are differentially expressed are indicated by red or green arrows. Spots that are differentially expressed, with similar relative ratios and consistently present across all the five experiments, are indicated by a blue box.

Figure 3 Magnified view of the gels showing the varying expression of proteins in patients with Parkinson’s disease and schizophrenia. Images correspond to the two phenotypes from five biological replicate gels. The protein spots that were picked for identification are indicated by black arrows.

Figure 4 Relative abundance of the differentially expressed proteins that are marked. Top panel shows spot intensities in terms of peak volume ratios (±3.0) based on DeCyder software analysis. Graphical representation of protein spots differentially expressed in serum of patients with Parkinson’s disease (P) as compared to schizophrenia (S) (P<0.05). Spot data from same gels are connected by dotted lines.

Table 4 Profile of identified protein spots

Number	Spot	Protein	Accession	Mass/isoelectric point (experimental)	Mass/isoelectric point (theoretical)	Ratio	Appearance in gels	Mascot score	Number of peptides	Coverage
1	1,570	β-globin	gi/66473265	12/7.2	15.9/6	3.5	5/5	126	3	30%
2	1,527	α-globin	gi/28549	13/9	15.9/5.2	3.5	5/5	102	1	11%

Table 5 Pearson correlation between α-globin concentrations and disease scoring, gender, and age

Clinical phenotype	Disease score (P-value)	Gender (P-value)	Age (P-value)
Parkinson’s disease	0.41	0.58	0.37
Schizophrenia	0.43	1.00	0.36

Table 6 Pearson correlation between β-globin concentrations and disease scoring, gender, and age

Clinical phenotype	Disease score (P-value)	Gender (P-value)	Age (P-value)
Parkinson’s disease	0.94	0.93	0.35
Schizophrenia	0.48	0.52	0.35

Figure 5 Expression of α- and β-globins in the serum of patients with Parkinson’s disease and schizophrenia analyzed by ELISA. Clinical phenotypes comprise of Parkinson’s disease naïve, Parkinson’s treated, Parkinson’s treated with schizophrenic side effects, schizophrenia treated with Parkinson’s disease-like side effects, schizophrenia treated and schizophrenia naïve patients. Mean ± standard error of mean of the values is shown by horizontal lines. The bars represent the concentrations as the average of duplicate readings of each patient sample. Trend lines of α-globin (y =-1.12x+16.4; R² =0.86) and β-globins (y =-0.85x+14.2; R² =0.81) across the six clinical phenotypes are shown in blue dotted lines in (A and B), respectively. Diagrammatic representation of the dopamine concentration in cerebrospinal fluid (CSF) is shown along the x-axis.^47,48 *Statistical significance with P<0.05.

Figure 6 Correlation analysis of α- and β-globins expression with duration of pharmacotherapy. “r” represents the correlation coefficient.

Figure 7 Western blot analysis of bisphosphoglycerate mutase. P1–P4 show four representative samples from Parkinson’s disease group; S1–S4 show four representative samples from schizophrenia group. +, Positive control; M, marker.

Table 7 Pharmacotherapeutic monitoring value of α-globin and β-globin to differentiate Parkinson’s disease and schizophrenia

Biomarker	Cut-off values to differentiate Parkinson’s disease from schizophrenia	Parameters		Likelihood ratio
		Sensitivity (%)	Specificity (%)
α-Globin	>10.7 ng	70.9	71.1	2.5
β-Globin	>10.1 ng	70.9	80	3.5

Figure S1 SDS-PAGE showing removal of the high abundant proteins, albumin and immunoglobulin heavy chain, from serum samples of patients with Parkinson’s disease (P) and schizophrenia (S).

Figure S2 Snapshot of mass spectrometry data analysis of spot number 1,570 for protein identification using MASCOT software.

Figure S3 Snapshot of mass spectrometry data analysis of spot number 1,527 for protein identification using MASCOT software.

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