Proteome Analysis Of Rat Hippocampal Plasma Membrane And The Application Of Two-dimensional Liquid-phase Chromatography In Toxinome

The hippocampus is an important brain region playing a central role in short-term memory formation. The adult rat hippocampal plasma membrane was purified by differential velocity centrifugation and continuous sucrose density-gradient centrifugation sequentially. Validation of the obtained plasma membrane was performed with Western blotting. The results showed that there were two plasma membrane bands within the gradient matrix, the major in the region of 32%~42% sucrose (density 1.13~1.18) and the minor in the region of 20%~25% sucrose (density 1.08~1.10); the region of about 39% sucrose (density 1.17) contained highest concentration and purity of plasma membrane. After analysis by SDS-PAGE and CapLC-MS/MS, totals of 135 proteins in rat hippocampal plasma membrane preparations were identified through database searching. Of the identified proteins, 70 were plasma membrane and membrane-associated proteins (accounting for 51.9%), including Na~+/K~+-ATPase, Glutamate/aspartate transporter, Lipophilin, GLAST la and so on. The present experimental results accumulated valuable data for understanding the function of hippocampus and constructing the protein database specific for rat hippocampus.The bird spider Selenocosmia huwena Wang [= Ornithoctonus huwena (Wang)] is one of the most venomous spiders in China. A number of studies have proved that its venom contains many biological proteins and peptides with low molecular weight (<10 kDa). In order to separate and identify these components, a novel two-dimensional liquid-phase separation method was developed. The method involves fractionation according to pI using isoelectric focusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous silica reversed-phase high-performance liquid chromatography in the second dimension (IEF-NPS RP HPLC). A peptide toxins pattern analogous to that of the two-dimensional ployacrylamide gel electrophoresis (2-D PAGE) image was created by using ProteoVue and DeltaVue software. Eluting fractions were collected by fraction collector (Gilson) and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Using IEF-NPS RP HPLC,～600 bands were resolved in a pI range from 3.5 to 9.0 and about 130 different peptides or proteins with different molecular masses ranging from 1 to 10 kD were analyzed and the sequence information on 26 toxins including 12 known ones was obtained.