HPLC and CE of Peptides and Proteins

University Of Colorado Denver, Aurora CO

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Abstract

The continuing development of efficient high-performance liquid chromatography (HPLC) protocols is critical for all areas of biomedical science and biotechnology. The major areas under investigation in our laboratory are: (1) development of novel and improved HPLC (both analytical and preparative) and capillary electrophoresis (CE) separation protocols for peptides and proteins;and (2) utility of reversed-phase HPLC (RP-HPLC) to monitor folding and stability of peptides and proteins. There is an ever-increasing demand for efficient analytical and preparative purification techniques for peptides and proteins, with HPLC being used routinely in our laboratory for purification of native proteins and protein fragments, recombinant proteins and synthetic peptides, the latter of which are a class of compounds with increasing therapeutic importance. Novel purification protocols, such as mixed-mode HILIC/CEX with the development of volatile mobile phases, will continue to rival RP-HPLC. The current shortage of acetonitrile also underlines the necessity of investigating alternative organic modifiers such as methanol, together with RP-HPLC columns with unique selectivities for peptides, for analytical and preparative protocols. Our novel Ion-Interaction-Capillary Zone Electrophoresis (IICZE) approach will serve as a convenient tool to study solution-based interactions of biomolecules in general, as well as being a powerful separation method in its own right. RP-HPLC represents a potent monitor of polypeptide structural characteristics and it should be possible to correlate the elution behavior of biologically active peptides with their amphipathicity and self-association, thus illustrating the advantages of HPLC in understanding the mechanism of action of biologically active peptides. The development of these methods will have a major impact on proteomics as well.