摘要
细胞质膜(plasma membrane,PM)是所有细胞的结构基础。质膜蛋白质执行细胞与外界环境以及细胞与细胞之间的物质交换、能量转换和信号传递等功能。在已经发现的药物靶标中,大约有2/3是质膜蛋白质。因此,细胞质膜蛋白质组学研究具有重要的理论意义和应用前景。然而,尽管近年来蛋白质化学与蛋白质组学研究技术取得了很大的发展,但膜蛋白尤其是整合膜蛋白的分析仍然是一个巨大的挑战,这是因为大多数膜蛋白质不仅丰度低,而且由于疏水性强而溶解性差和易于聚集沉淀,给蛋白质的提取、酶解和鉴定带来了很多困难,因此质膜蛋白质组学研究成为蛋白质组学研究中的难点。
背根神经节(dorsal root ganglion,DRG)细胞是一种初级感觉神经元,能传导触觉、痛觉、温觉等神经冲动。它们沟通脊髓与机体内外环境的联系,不仅传输和调节机体的各种感觉,而且还具有接受和传递机体各种伤害性感受的功能,因而在神经病理性疼痛的发生与维持中起着重要的作用。开展DRG细胞膜蛋白的研究具有重要意义,将为探讨神经系统疾病的发生机制、疾病的诊断和新药开发将提供重要的理论基础。由于大鼠DRG神经组织体积较小而质膜蛋白质又大多具有低丰度和强疏水性的特点,所以在样品的采集、溶解和蛋白质鉴定等方面给DRG细胞质膜蛋白质组学研究带来了更大的挑战。
为了从少量DRG细胞样品中综合性分析鉴定质膜蛋白质,本研究采用了差速离心结合双水相法富集大鼠DRG质膜的策略。Westernblotting结果显示经过这种富集策略处理的样品中质膜相对浓度是差速离心后的2.3倍,是粗匀浆样品的15倍。鉴于传统的二维凝胶电泳不适合膜蛋白质的分离,所以采用了SDS-PAGE来分离DRG质膜蛋白质样品。分离后的样品经过trypsin酶解,CapLC-MS/MS鉴定和生物信息学分析。通过IPI数据库的搜索,共得到了729个非冗余蛋白质,其中547个有GO注释信息,159(21.8%)个为质膜蛋白质。
为了更好地鉴定DRG细胞质膜上的蛋白质尤其是低丰度蛋质,随后的研究中,在DRG质膜的双水相富集后增加了高盐高pH洗涤的步骤和Shotgun消化策略。本次研究共鉴定了954个蛋白质,通过基于SDS-PAGE和Shotgun消化的策略分别鉴定了其中的620个和334个蛋白质。在954个鉴定的蛋白质中,有205个蛋白质明确定位在质膜。这些质膜蛋白质包括细胞结构蛋白质(22.8%),信号和受体蛋白质(25.7%),离子通道和转运蛋白质(24.3%),酶(8.3%),细胞黏附因子(12.1%)以及一些具有其它功能的蛋白质(6.8%)。更多的低丰度的质膜蛋白质(包括离子通道蛋白和受体蛋白)得到了鉴定,如chloride intracellμLar channel protein 4(IPI00208249),Sodium channel(IPI00326646),Isoform 2 of sigma 1-type opioidreceptor(IPI00202168)等。这些研究有助于我们加深对DRG细胞质膜蛋白质在细胞与外界环境进行物质和能量交换、信号传导等方面的理解。
大多数细胞的生物学功能都是由蛋白复合体而非单一蛋白质来完成的。因此,蛋白质复合物的研究已成为蛋白质组学中最重要研究内容之一。本研究用含温和去垢剂的缓冲液提取大鼠DRG细胞细胞浆和膜组分中的蛋白质复合物后,采用BN-PAGE结合SDS-PAGE的方法对复合物进行电泳分离并用CapLC-MS/MS进行分析。一共鉴定了460个非冗余蛋白质,包括属于若干细胞浆和膜组分的蛋白质复合物的亚基。
Plasma membrane (PM) is the structural base of all cells. Due to the presence of specific membrane proteins, PM plays important biological roles in exchange of substance, energy and information between cell and its environment as well as between cells themselves. Of the known drug targets, about two-thirds are PM proteins. Therefore, the proteomic analysis of PM has important theoretical significances and application prospects. However, although protein chemistry and proteomics have been greatly developed in recent years, the analysis of membrane proteins especially integral membrane proteins still presents an analytical challenge, because most membrane proteins not only are low-abundant, but also are not readily soluble in pure aqueous buffers due to their hydrophobicity and tendency of aggregation, thereby making the extraction, enzymolysis and identification of the proteins difficult. Therefore, the analysis of membrane proteome has become a difficult point in proteomics.
Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. DRG cells connect the spinal cord with internal and external environment of the organism. They not only transmit and accommodate sensations but also accept and convey nociception of the organism, so they play an important role in the origination and maintenance of neuropathologic pain. The proteomic analysis of DRG plasma membrane proteins is of outstanding significance and will lay a valuable theoretical base for investigating disease attack mechanism of neuron system, disease diagnosis and new drug development. DRG is a small-sized nerve tissue and most PM proteins are of hydrophobic and low abundant nature, thereby making the PM proteomic analysis of DRG more challenging in sample collection, dissolution and protein identification, etc.
To comprehensively identify proteins of plasma membrane from small amounts of DRG cells, this study utilized aqueous polymer two-phase partition in combination with differential velocity centrifugation to enrich PM. Western blot analysis showed that the concentration of PM in plasma membrane-enriched preapration was 2.3 times higher than that in crude plasma membrane, 15 times higher than that in whole tissue lysate. In view of that traditional two-dimensional gel electrophoresis is not suitable for the separation of membrane proteins, the present study adopted SDS-PAGE to separate the proteins in DRG PM sample, followed trypsin enzymolysis, CapLC-MS/MS and bioinformatics analysis. By searching against the rat IPI protein sequence database, a total of 729 non-redundant proteins were identified from the PM preparation, of which 547 had a gene ontology (GO) annotation for cellular component, and 159 (21.8%) were unambiguously identified as PM proteins.
To identify PM proteins especially low-abundance PM proteins from DRG cells even better, the subsequent study employed multiple analytical strategies including the addition of high salt and high pH solution washing after aqueous two-phase partition and the shotgun digestion. A total of 954 proteins were identified from DRG PM samples, of which 620 and 334 were identified with SDS-PAGE-based and shotgun-based strategies, respectively. Of the 954 identified proteins, 205 proteins were unambiguously identified as PM proteins, including 22.8% binding and structural proteins, 25.7% signal proteins and receptors, 24.3% ion channels and transporters, 8.3% catalytic proteins, 12.1% cell adhesion proteins. Other annotated proteins (6.8%) have activities such as protein folding and trafficking. More low-abundance PM proteins (including ion channel and receptors) were identified, such as chloride intracellular channel protein 4(IPI00208249), Sodium channel (IPI00326646), Isoform 2 of sigma 1-type opioid receptor (IPI00202168). The study would help to our understanding of the fundamental functions of DRG cellular PM proteins in the exchange of materials and energy between cells and its environment, and signal transduction, etc.
Most of the biological functions of cells are carried out by protein complexes rather than a single protein. Therefore, the study of protein complexes has become one of most important subjects in proteomics. In the study, protein complexes in cytoplasm and membranes were extracted with mild-detergent-contaning buffer, separated using BN-PAGE combined with SDS-PAGE, and analyzed by CapLC-MS/MS. A total of 460 non-redundant proteins were identified, including the protein subunits belonging to dozens of protein complexes from cytoplasm and membranes.
引文
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