About Laboratory of Protein Chemistry & Proteomics
The Laboratory of Protein Chemistry & Proteomics at Hunan Normal University is mainly focusing on two research projects:
1. Structure, function and evolution of the peptide toxins from Chinese spiders.
There are about 4,000 spider species in China . Spider venoms are incredibly complex cocktails of peptide and protein toxins, which are of interest as tools for studying neurophysiology and as potential lead structures for insecticides and pharmaceuticals. Even very conservative estimates of the peptide diversity of spider venoms leads to an estimated total of 2-4 million different spider venom polypeptides. It is of great significance to explore the spider toxins at the molecular level to find some peptide toxins having scientific value and to discover the rules of the relationships between structure and bioactivity of these toxins. In this project, several Chinese representative spider species have been selected as studying objects and the investigation of the purification, primary structures and three-dimensional structure determination as well as bioactivitiy analysis of these toxins have been conducted. In our previous work, more than 60 peptide toxins with novel amino acid sequences and different biological activities have been isolated from the Chinese spiders Ornithoctonus huwena, Ornithoctonus hainana, Macrothele raveni, Chilobrachys jingzhao and Lycosa singoriensis . The three-dimensional (3D) structures of 15 spider toxins have been determined using NMR technique and found that spider toxins exhibit high similarity in 3D structures and remarkable diversity in bioactivites. We also established the cDNA library from the venom glands of the Chinese spider C. jingzhao and S. huwena . We also generated 468 and 886 ESTs from the cDNA libraries of S. huwena and C. jingzhao, respectively . Phylogenetics and sequence analyses of them reveal several different superfamilies. The results have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms and for better understanding the relationship of spider toxin evolution based on the diversification of cDNA sequences, primary structure of precursor peptides, 3D structure motifs and biological functions. In this project we have also developed several methods for investigating spider toxins, including the stablishment of a novel method for collecting pure spider venoms, a novel method for determining the disulfide bond patterns of spider toxins and two novel derivatization reagents for C-terminal peptide sequencing.
2. Functional proteomics of the plasma membrane of neuron s
Proteomics was developed to study protein expression profile related to cell function. The current intense interest in proteomics is largely driven by that the human genome and the genomes of other organisms have been fully sequenced, making their proteomes accessible. The plasma membrane(PM) is a selectively permeable barrier and communication interface of cells. The PM proteome plays crucial roles in the fundamental biological processes including cell-cell interaction and signal transport. In our previous work, several methods were developed and optimized for the neuronal PM proteome analysis of rat hippocampus and dorsal root ganglion. These methods include an optimized two aqueous partition methods for PM purification, the using of sodium deoxycholate(SDC) to enhance the membrane protein solubilizaton and a method for protein de novo sequencing by using SPITC with MALDI-TOF mass spectrometry. We performed comprehensively comparative proteomics between PM from tissue and primary neurons. A batch of proteins were detected only in the PM fraction from primary neurons and could be regarded as neuronal PM marker candidates. We also applied two-dimensional Blue Native-SDS Gel Electrophoresis in combined with the HCT ion-trap mass spectrometry to study the protein complexes in hippocampal synaptic PM. A prefractionation strategy by adjusting the pH of Triton X-100 extraction of isolated SPM was used in the synaptic membrane preparation . Our analysis found many potential synaptic proteins and protein complexes in the SPM. The ombination of BN-PAGE and MS will allow us to analyze transient protein-protein interactions at the protein complex level. Our findings shed light on understanding the physiology and pathology of the synaptic PM.
In recent years we have published over 80 research papers with international impacts in the peer journals and we have also got 10 patents.