关键词: 串联质量标记, 定量蛋白质组学, 帕金森病, 外泌体, 血浆标志物

Abstract:

The cardinal clinical features of Parkinson’s disease (PD), a common neurodegenerative disease, include the irreversible impairment of movement coordination, such as tremors, gait rigidity, bradykinesia, and hypokinesia. Although various factors are associated with the pathological changes in PD, such as oxidative stress, mitochondrial dysfunction, and neuroinflammation, the availability of treatments to retard PD progression is limited. Therefore, novel biomarkers for PD diagnosis and therapeutic targets are urgently needed. The diagnosis of PD mainly depends on its clinical manifestations and has an error rate of approximately 20%. Studies have shown that α-synuclein (α-syn) levels are significantly increased in the cerebrospinal fluid of patients with PD; however, the invasive nature of lumbar puncture restricts further studies on its clinical applications. Hence, the development of novel peripheral blood markers would be helpful for the early diagnosis of PD. Exosomes are extracellular vesicles (EVs) released by various cell types under physiological and pathophysiological conditions. Because exosomes carry a variety of bioactive molecules, they play a key role in biological processes such as intercellular communication and the immune response. Central nervous system (CNS)-derived exosomes can be detected in the cerebrospinal and peripheral body fluids of patients with PD, and their contents are altered during the disease process, rendering them an attractive biomarker resource. Therefore, a comprehensive and high-throughput investigation of the plasma and its exosomes may enhance our understanding of PD. In this study, we isolated exosomes from plasma using standard differential centrifugation and performed tandem mass tag (TMT)-labeled quantitative proteomic analysis of plasma and plasma exosome samples from healthy individuals and patients with PD using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 724 proteins were quantified in the plasma samples, and 611 proteins were screened from the exosome samples. Among these 611 proteins, 413 were found in the Exosomal Protein Database (Exocarta). Using |log₂FC|>0.26 and P-value (P)<0.05 as the cutoff, five upregulated and six downregulated proteins were identified in the plasma samples of the PD group compared with the healthy group. In the plasma exosome samples, compared with the healthy group, the PD group showed six upregulated and seven downregulated proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted based on gene set enrichment analysis (GSEA). GO-cellular component (CC) analysis revealed that plasma-enriched proteins were mainly located in the nucleus whereas plasma exosome-enriched proteins were mainly located in the cytoplasm. According to the GO-molecular function (MF) analysis, the MFs of differentially expressed proteins in the plasma were mainly enriched in RNA, DNA binding, and complement binding. By contrast, the molecular functions of differentially expressed proteins derived from plasma exosomes were enriched in antioxidant activity, oxidoreductase activity, and peroxide acceptor activity. We then analyzed the enriched KEGG pathways of differentially expressed proteins derived from the plasma and plasma exosome samples. The enrichment pathways of differentially expressed proteins in the plasma samples included the lysosome pathway, cellular senescence, and protein processing in the endoplasmic reticulum. By contrast, the enrichment pathways of differentially expressed proteins in the plasma exosome samples included chemokine signaling and cytokine receptor interactions. Finally, we assessed the functions of some exosomal proteins in PD to elucidate their potential for PD diagnosis and treatment. Significant differences were observed between the plasma and plasma exosome protein profiles, and the functions of differentially expressed proteins in plasma exosomes were strongly related to the pathology of PD. Our study provides a reference for identifying the potential biomarkers and therapeutic targets of PD.

Key words: tandem mass tag (TMT), quantitative proteomics, Parkinson’s disease (PD), exosomes, plasma biomarkers