Study On Protein Complexomics And Peptidomics Of Rat Nerve Cells

Dorsal root ganglion (DRG) cells 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. 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 a mild-detergent-containing 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.Peptidome includes all endogenous peptides of organ, tissue, cell and body fluid in an organism. Peptidomics is a course which studies structure, function, change regularity of the peptides and the relationship between them. Peptidomics and proteomics both are supplements of genomics and peptidomics fills the gap between metabonomics and proteomics. Normal research strategies of proteomics are not fit for researching peptidomics, because they can not effectively identify the peptides in the low molecular weight range. A major factor affecting the analysis of peptidome is that level of the peptides in brain and most other tissues is lower than that of peptides resulted from protein degradation. Besides, the cathepsins would rapidly degrade proteins shortly after the animal died. However, the results of some analyses indicate that a conventional microwave oven can be used to efficiently prevent postmortem protein breakdown. Hippocampus is one of the most important tissues of the brain limbic system. It is critical for learning and memory. In order to investigate the variety, structure and property of peptide in rat hippocampus, the peptidome of the tissue was studied in the study. At first, the head of a rat was placed in a domestic conventional microwave oven and the temperature of the brain tissue was raised to 80℃within 10 s to inactivate various enzymes. Endogenous peptides were extracted from hippocampus using 0.25% acetic and 8 M urea, respectively. The extracts were passed through a 10 kDa molecular weight cutoff (MWCO) filter. The obtained peptide mixtures were desalted by RP-HPLC and 10 mM dithiothreitol (DTT) was added just before TOF-Q MS analysis. The subsequent data analysis was carried out using the Mascot software platform. The searches were conducted with following parameters:enzyme:none; mixed modifications:none; variable modifications:acetyl (N-term), amidated (C-term), deamidated (NQ), oxidation (M) and phosphorylation (ST).Through this study, we identified in total 81 peptides in extracts of the rat hippocampus tissue heat-treated in a domestic microwave oven, compared to 13 peptides were identified in hippocampus tissue without heat treatment in the microwave oven. Of the identified 82 peptides,66 were identified in the acetic acid extract and 26 in the urea extract. Further analysis indicated that some of the identified peptides were known peptides and others were the neuropeptides newly discovered in the present study.