Development Of Proteomics Methodology And The Application In Proteome Analysis Of Rat Hippocampal Plasma Membrane

A core component of proteomics is the ability to systematically identify every protein expressed in a cell or tissue as well as to determine the properties of each protein. The technology for such analyses integrates separation science for the separation of proteins, analytical science for the identification and quantification of the analytes, and bioinformatics for data management and analysis. In the past two years, incremental advances in standard proteome technology have increased the speed of protein identification with higher levels of automation and sensitivity. High-sensitivity, high-accuracy and high-throughput technology for proteins analysis is still the duty of proteomics studies.In this paper, we describe a series of advances in MALDI-TOF-MS technology for proteome analysis, including peptide mass fingerprint (PMF) method, post-source decay (PSD) de novo sequencing method, C-terminal peptide ladder de novo sequencing method. There are often cases in which a peptide sequence is needed to confirm the identification in proteomics research. Various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomics samples to facilitate de novo sequencing of the peptides produced. We have recently developed a novel method for de novo sequencing based on charge derivatization by 4-sulfophenyl isothiocyanate (SPITC). The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The improved labeling with a sulfonic acid group enhanced PSD sequencing of tryptic peptides, and is of potential application for protein structural analysis. The technique has been successfully applied to peptide mixtures, and to the unambiguous identification of proteins isolated through one- and two-dimensional gel electrophoresis. Furthermore, Phosphorylated peptide has been detected and identified very sensitively by incorporationof IMAC affinity capture and chemically assisted de novo sequecing followed SPITC modification. Moreover, an optimized procedure for protein and peptide C-terminal de nove sequencing by coupling carboxypeptidase Y digestion with MALDI-TOF-MS has been development. The sequence of the first 20 amino acid residues from the C-terminus of the peptide can been determined without gap in a signle mass spectrum.The hippocampus is an important brain region playing a central role in short-term memory formation. Although proteomic technology has been widely used with regard to different aspects of hippocampus, however, up to date, there is no report about the proteome analysis of the plasma membrane of hippocampus. The plasma membrane constitutes the interface between eukaryotic cells and their external environment. The membrane protein which inlay in lipid bilayer are important biological and pharmacological targets involved in intercellular communication, cellular development, cell migration, and drug resistance. Contemporary genomic analyses indicate that 20-30% of all open reading frames (ORFs) encode for integral membrane proteins. It is essential to analyzing the proteome of plasma membrane proteins of hippocampus for biological and pharmacological research.The comparative proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially an approach for the identification of membrane-associated proteins. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. In the present paper, we describe a modified protocol of the affinity labeling technique, which combines cell surface biotinylation with affinity enrichment by immobilized streptavdin beads, for plasma membrane isolation. This method has been successfully combined with SDS-PAGE, mass spectrometry, and bioinformatics for evaluation proteomics strategy. Western bolting analysis show that almost all actin, a major contaminant...