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High-Speed Chromatographic Separation of Proteins and Peptides for High Sensitivity Microsequence Analysis

  • Robert L. Moritz
  • James Eddes
  • Richard J. Simpson

Abstract

Reversed-phase high-performance liquid chromatography (RP-HPLC) and polyacrylamide gel electrophoresis (PAGE) are two of the most widely-used high-resolution techniques for isolating proteins and peptides for structural analysis (Simpson et al., 1988, 1989; Matsudaira, 1993; Patterson, 1994). In recent years, the importance of these two technologies has been further enhanced by using them in tandem. For example, proteins from complex mixtures such as total cell lysates can be resolved by two-dimensional gel electrophoresis (2-DE) and, following proteolytic digestion, the generated peptides separated by microbore column RP-HPLC. Proteolytic digestion of 2-DE gel spots can be accomplished either in the gel matrix (Ward et al., 1990, Eckerskorn et al., 1990, Rosenfeld et al, 1992, Ji et al., 1994, and Hellman, U. personal communication) or on immobilizing membranes such as polyvinylidine difluoride (Fernandez et al., 1992) and nitrocellulose (Aebersold et al., 1987), following electrotransfer from the gel. Protein identification can be achieved by microsequence analysis of the isolated peptides using either automated Edman degradation or tandem mass spectrometry (Hunt et al., 1986; Burlingame et al., 1994). More recently, alternative means of protein identification such as peptide mass fingerprinting (Pappin et al., 1993; Mann et al., 1993; Henzel et al., 1993, James et al., 1994) and amino acid compositional analysis (Sibbald et al., 1991, Shaw, 1993) have emerged. Since these latter techniques have the potential for generating large quantities of data rapidly, there is now an increasing need for rapid protein and peptide isolation procedures.

Keywords

Colorectal Cancer Cell Line Mass Transfer Kinetic Linear Flow Velocity Internal Amino Acid Sequence Chromatographic Efficiency 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Robert L. Moritz
    • 1
  • James Eddes
    • 1
  • Richard J. Simpson
    • 1
  1. 1.Joint Protein Structure LaboratoryLudwig Institute for Cancer Research (Melbourne Branch) and The Walter and Eliza Hall Institute for Medical ResearchAustralia

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