| BackgroundThe application of proteomics and bioinformatics methods help to screen and identify the differentially expressed proteins in serum or plasma,and find reliable biomarkers that can assist clinical diagnosis.Proteomics based on Mass Spectrometry(MS)technology is a high throughput and large range of proteomics methods.Therefore,We using high performance liquid chromatography(HPLC high-performance liquid chromatography,technique combined with The quadrupole Orbitrap mass spectrometer(Q-Exactive mass spectrometry),screened differentially expressed proteins between early-stage and advanced-stage Parkinson’s disease(PD)patients and healthy controls in plasma,and to explore the possible biological markers for early diagnosis.ObjectiveTo investigate the proteomics changes and diagnostic biomarkers in plasma of patients with Parkinson’s disease in the early stage and advanced stage by using Q Exactive mass spectrometry.Methods 1 The collection and treatment of clinical samplesThe PD patients that fulfilled standardized clinical diagnostic criteria for PD(MDS-PD Criteria,2015)were enrolled.The severity of Parkinson’s disease was rated according to the Modified 7-grade Hoehn-Yahr scale,and sort as early-stage and advancedstage.Control subjects were healthy individuals of similar age and sex to those with PD with no apparent neurological or known psychiatric symptoms.Randomly selected 6 patients with early stage,6 advanced stage and 6 controls,quantification of the proteins was performed following the manufacturer’s instructions for the 2D Quant Kit.100 μg of total protein in each sample was used for reduction and alkylation and then digested by 1.5μg trypsin.The digestion products were lyophilized and the enriched peptides were desalted by using C18 spin columns.2 Peptide analysis by LC-MS/MSSamples were loaded on a trap column and peptides were ionized and atomized into Q Exactive mass spectrometry by a Nanospray III source.Precursor ions and product ions were extracted by using Protein Pilot Software and protein searching was conducted by using Mascot.3 Bioinformatics analysisThe differentially expressed proteins were enriched by R package cluster Profiler with Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways and gene ontology terms(GO),based on the given protein’s molecular function,cellular component,or biological process.And analysis of proteins interaction.4 Validation of differential protein by ELISAIn order to verify the results of mass spectrometry analysis,we expanded the samples.Then we assayed the proteins’ concentration in the corresponding samples by enzyme linked immunosorbent assay(ELISA),including apolipoprotein E(APOE),peroxiredoxin-2(PRDX2),and inflammatory response protein complement component 7(C7).ResultsA total of 97 differentially expressed proteins were revealed between the control and early-stage PD patients,and 68 differentially expressed proteins were revealed between the control and advanced-stage PD patients.Bioinformatics analysis revealed that the functions of these proteins were mainly related to the oxidative stress,inflammatory response,complement activation,and neuron projection regeneration.Among the differentially expressed proteins,three oxidative stress-related proteins,APOE,PRDX2 and C7 were selected for validation.A trend of down-regulation of APOE(P<0.05)and significant down-regulation of PRDX2(P <0.01)were found in the plasma of PD patients compare to control.However,there was no statistically significance of the expressed C7(P >0.05).Conclusion1 Our study demonstrated that widespread alterations of protein expression in the plasma between patients with Parkinson’s disease and healthy subjects.2 Our results indicated that APOE and PRDX2 may be the biomarkers for the early diagnosis of Parkinson’s disease,the results still need to be expanded. |
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