Proteomic Analysis Of Plasma Membrane From Rat Dorsal Root Ganglion Cells

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.