Proteomic Analysis of Human Parotid Gland Exosomes by Multidimensional Protein Identification Technology (MudPIT)

Human ductal saliva contributes over a thousand unique proteins to whole oral fluids. The mechanism by which most of these proteins are secreted by salivary glands remains to be determined. The present study used a mass spectrometry-based, shotgun proteomics approach to explore the possibility that a subset of the proteins found in saliva are derived from exosomes, membrane-bound vesicles of endosomal origin within multivesicular endosomes. Using MudPIT (multidimensional protein identification technology) mass spectrometry, we catalogued 491 proteins in the exosome fraction of human parotid saliva. Many of these proteins were previously observed in ductal saliva from parotid glands (265 proteins). Furthermore, 72 of the proteins in parotid exosomes overlap with those previously identified as urinary exosome proteins, proteins which are also frequently associated with exosomes from other tissues and cell types. Gene Ontology (GO) and KEGG pathway analyses found that cytosolic proteins comprise the largest category of proteins in parotid exosomes (43%), involved in such processes as phosphatidylinositol signaling system, calcium signaling pathway, inositol metabolism, protein export, and signal transduction, among others; whereas the integral plasma membrane proteins and associated/peripheral plasma membrane proteins (26%) were associated with extracellular matrix-receptor interaction, epithelial cell signaling, T-cell and B-cell receptor signaling, cytokine receptor interaction, and antigen processing and presentation, among other biological functions. In addition, these putative saliva exosomal proteins were linked to specific diseases (e.g., neurodegenerative disorders, prion disease, cancers, type I and II diabetes). Consequently, parotid glands secrete exosomes that reflect the metabolic and functional status of the gland and may also carry informative protein markers useful in the diagnosis and treatment of systemic diseases.

人类导管唾液可为全口腔液贡献超过一千种独特蛋白质。目前，大多数此类蛋白质由唾液腺分泌的具体机制仍有待阐明。本研究采用基于质谱的鸟枪法蛋白质组学（shotgun proteomics）方法，探究唾液中部分蛋白质源自外泌体（exosomes）的可能性——外泌体是多囊泡内体（multivesicular endosomes）中起源于内体的膜结合囊泡。本研究借助多维蛋白质鉴定技术（multidimensional protein identification technology, MudPIT）质谱法，对人类腮腺唾液外泌体组分中的491种蛋白质进行了编目。其中265种蛋白质此前已在腮腺导管唾液中被检测到。此外，腮腺外泌体中的72种蛋白质与此前已鉴定的尿液外泌体蛋白质存在重叠，而这类蛋白质也常与其他组织及细胞类型的外泌体相关联。基因本体论（Gene Ontology，GO）与京都基因与基因组百科全书通路（KEGG pathway）分析结果显示，胞质蛋白（cytosolic proteins）占腮腺外泌体蛋白质的最大类别（43%），参与磷脂酰肌醇信号系统、钙信号通路、肌醇代谢、蛋白质输出以及信号转导等多种生物学过程；而整合质膜蛋白（integral plasma membrane proteins）及相关/外周质膜蛋白（占比26%）则与细胞外基质-受体相互作用、上皮细胞信号转导、T细胞与B细胞受体信号转导、细胞因子受体相互作用以及抗原加工呈递等多种生物学功能相关。此外，这些推定的唾液外泌体蛋白质与多种特定疾病相关，例如神经退行性疾病、朊病毒病、癌症以及I型和II型糖尿病。综上，腮腺分泌的外泌体可反映该腺体的代谢与功能状态，同时还可能携带可用于全身性疾病诊断与治疗的功能性蛋白质标志物。