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ADVANCES IN THE METHODOLOGY OF
ELECTROPHORETIC SEPARATION
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Andreas
C. Chrambach, PhD, Head, Section
on Macromolecular Analysis |
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Our work aims at increasing the effectiveness of electrophoretic separations of biological macromolecules and subcellular particle-sized species by application of separation theory through development of computer programs and by instrumental and procedural optimization at a level that is practical for the vast majority of biochemical laboratories. We continued to work on improving the method of "direct electroelution" of proteins from polyacrylamide gels and that of "static noncovalent" coating of capillaries to reduce to a negligible value electroosmotic flow in capillary electrophoresis (CE). Using rat synaptic vesicles as a model, we have been attempting to develop a novel approach to deciphering protein complexes based on their cross-linking and fractionation by capillary electrophoresis. The complexes are to be continuously blotted to membrane, identified as SNARE protein complexes, and then followed by a mass-spectrometric (MS) characterization of interacting proteins. We have also been attempting to improve the resolution at low gel concentration of fluorescently labeled saccharides by using acetate as the trailing constituent of a disc electrophoresis buffer system and to replace 2D-PAGE in proteomic analysis of a representative membrane protein by a three-stage procedure of SDS-PAGE, electroelution, IPG-IEF, and band transfer into MS. Discontinuous buffer systems operative at pH 2.5 to 11, 0°C and 25°C made available on the Internet Chrambach; in collaboration with Wheeler During the past year, our section made available on the Internet, for the first time, buffer systems across the entire pH scale capable of concentrating a sample and enhancing resolution (http://www.ncbi.nlm.nih.gov/Class/wheeler/jovin.html); previously, these systems could be accessed only by mainframe computer, on microfiche and magnetic tapes. to 11, 0 and 25°C, available on the Internet. Electrophoresis; in press. Hydrophobic protein transfer from IEF to MS Buzás, Chrambach 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 all 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 to isoelectric focusing (IEF), the first dimension of 2-D PAGE in conventional proteomic analysis. Accordingly, we are attempting to circumvent the insolubility of membrane proteins on IEF by conducting SDS-PAGE as a first dimension, directly electroeluting the SDS-proteins of interest as
described (Radko et al.,
2002), and applying the electroeluted proteins to IEF in the most
effectively solubilizing medium presently
available, a mixture of weak detergent and chaotrope. Given
that the electroeluate concentration of SDS is very low and that lipid
moieties are likely to be removed during SDS-PAGE, that three-step
procedure is likely to make membrane proteins amenable to proteomic
analysis. Furthermore, the procedure allows for simultaneous IEF analysis
at a number of narrow pI-range immobilized pH gradient (IPG) gels,
replacing the intolerably laborious and sample-consuming present practice
of multiple 2-D PAGE analysis with first-dimensional IPGs of several
narrow pI ranges.
Size dependence of microparticle transfer from CE to MS Chrambach; in collaboration with Radko Using rat synaptic vesicles in lieu of the previously applied scarce sea urchin egg cortical membrane vesicles, our third advance during the year concerns the continuing search for fusogenic proteins involved in addition to the SNARE proteins in vesicle-vesicle fusion. We are attempting to identify such fusogenic membrane proteins by immunodetection of their linked SNARE proteins after chemical cross-linking and then separating them by capillary zone electrophoresis and characterizing them mass spectrometrically after severance of the cross-links, SDS-PAGE, and electroelution. Sequential multiple protein transfer from PAGE to MS Antal, Buzás, Chrambach We designed, constructed, and successfully applied an apparatus for sequential multiple protein band elution and collection on soluble agarose blocks prior to MS (Figure 6.1). Novex gels into a mass spectrometer. Electrophoresis; in press. Electrophoretic separation of fluorescently labeled glucose oligomers Buzás, Chrambach; in collaboration with Berna, Cabanes-Macheteau, Taverna Replacement of the conventional catholyte Tris-glycine with the more rapidly migrating catholyte Tris-acetate allows us to separate glucose oligomers, which, under conventional conditions, are stacked into a single band because the highly charged oligomers are unstacked behind the trailing constituent. Cabanes-Macheteau M, Chrambach A, Taverna M, Buzás Z, Berna P. Resolution of ANTS labeled glucose oligomers in PAGE at low gel concentration. Electrophoresis; in press. A capillary electrophoresis procedure with improved sensitivity and speed Chrambach; in collaboration with Chang, Chiu, Huang, Lin We developed a CE procedure providing increased sensitivity and speed by applying SDS to the background electrolyte at one-fifth to one-third of the injection volume before sample (SYPRO-red-labeled protein) injection. To concentrate the sample, PEG added to the anolyte migrates by electroosmosis counter to the migration of the protein. Separation of five model proteins by the procedure required 11 minutes with detection limits of 30 pM. The procedure was validated by analysis of casein (0.45 mM) in milk. Chiu TC, Lin YW, Huang CC, Chrambach A, Chang HT. A simple rapid and sensitive method for analysis of SYPRO red labeled SDS protein complexes by capillary electrophoresis with laser induced fluorescence. Electrophoresis 2003;24:1730-1736.
COLLABORATORS Patricia Ashburn, Center for Information Technology, NIH, Bethesda MD Patrick Berna, PhD, Pzer Global R&D, Fresnes, France Paul S. Blank, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda MD Marion Cabanes-Macheteau, PhD, Pzer Global R&D, Fresnes, France Huan-Tsung Chang, PhD, National Taiwan University, Taipei, Taiwan Tai-Chia Chiu, PhD, National Taiwan University, Taipei, Taiwan Chih-Ching Huang, PhD, National Taiwan University, Taipei, Taiwan Alfred Yergey, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda MD Joshua Zimmerberg, PhD, Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda MD For further information, contact acc@helix.nih.gov |
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