The work of the Section on Macromolecular Analysis aims at a methodology of electrophoretic separations of biological macromolecules and particles which avoids both conventional manual and trial-and-error approaches and high-tech solutions available to only a small segment of the biochemical community. During the past year, implementation of that aim has been advanced by making available on the Internet (www.ncbi.nlm.nih.gov/Class/wheeler/jovin.html), for the first time, buffer systems across the entire pH scale capable of concentrating the sample and enhancing resolution which could previously be accessed only by mainframe computer, on microfiche and magnetic tapes. A second advance initiated during the past year concerns the frequent inapplicability of 2-dimensional electrophoresis in the proteomics of membrane proteins which constitute about one-half of the cellular proteins. That inapplicability derives from the insolubility in detergent solutions of hydrophobic proteins attached to lipids once they are separated from SDS. Such separation with replacement of SDS by neutral or amphoteric detergents is a prerequisite of isoelectric focusing (IEF), the first dimension of 2-D PAGE in conventional proteomic analysis. To circumvent that problem, an attempt is made to avoid the insolubility of membrane proteins on IEF by conducting SDS-PAGE as a first dimension, directly electroeluting the SDS-proteins of interest by the procedure described in last year?s report and applying the electroeluted proteins to IEF in the most effectively solubilizing weak detergent ? chaotrope media presently available. Since the electroeluate concentration of SDS is very low and lipid moieties are likely to be removed during SDS-PAGE, that 3-step procedure is likely to make membrane proteins susceptible to proteomic analysis. Furthermore, the 3-stage procedure allows for simultaneous IEF analysis at a number of narrow pI-range immobilized pH gradient (IPG) gels, to replace the intolerably laborious and sample-consuming present practice of multiple 2-D PAGE analysis with first-dimensional IPG?s of several narrow pI ranges.The third research project within the Section continues the search for fusogenic proteins involved in addition to the SNARE proteins in vesicle-vesicle fusion, using rat synaptic vesicles in lieu of the previously applied scarce sea urchin egg cortical membrane vesicles. An attempt is in progress to identify such fusogenic membrane proteins by immunodetection of their linked SNARE proteins after chemical crosslinking, separate them by capillary zone electrophoresis and characterize them mass spectrometrically after severance of the crosslinks, SDS-PAGE and electroelution.
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