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.

Public Health Relevance

Separation science is a very important technology for peptide and protein analysis and is particularly critical for biomedical science. Our laboratory is continuing to demonstrate the ability of high-performance separation methods (both HPLC and CE) to understand biological activities of peptides and proteins since a better understanding of separation mechanisms can lead to practical improvements in the separation and purification of bioactive peptides (e.g., peptide antimicrobial agents) and have a tremendous impact on the availability, cost and purity of polypeptide-based pharmaceuticals.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
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Edmonds, Charles G
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University of Colorado Denver
Schools of Medicine
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Abraham, Thomas; Prenner, Elmar J; Lewis, Ruthven N A H et al. (2014) Structure-activity relationships of the antimicrobial peptide gramicidin S and its analogs: aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes. Biochim Biophys Acta 1838:1420-9
Huang, Yibing; Pan, Ling; Zhao, Lianjing et al. (2014) Structure-guided RP-HPLC chromatography of diastereomeric ?-helical peptide analogs substituted with single amino acid stereoisomers. Biomed Chromatogr 28:511-7
Mant, Colin T; Jiang, Ziqing; Boyes, Barry E et al. (2013) An improved approach to hydrophilic interaction chromatography of peptides: salt gradients in the presence of high isocratic acetonitrile concentrations. J Chromatogr A 1277:15-25
Mant, Colin T; Hodges, Robert S (2012) Design of peptide standards with the same composition and minimal sequence variation to monitor performance/selectivity of reversed-phase matrices. J Chromatogr A 1230:30-40
Mant, Colin T; Cepeniene, Dziuleta; Hodges, Robert S (2010) Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns. J Sep Sci 33:3005-21
Kirwan, J Paul; Hodges, Robert S (2010) Critical interactions in the stability control region of tropomyosin. J Struct Biol 170:294-306
Hodges, Robert S; Mills, Janine; McReynolds, Susanna et al. (2009) Identification of a unique ""stability control region"" that controls protein stability of tropomyosin: A two-stranded alpha-helical coiled-coil. J Mol Biol 392:747-62
Mant, Colin T; Kovacs, James M; Kim, Hyun-Min et al. (2009) Intrinsic amino acid side-chain hydrophilicity/hydrophobicity coefficients determined by reversed-phase high-performance liquid chromatography of model peptides: comparison with other hydrophilicity/hydrophobicity scales. Biopolymers 92:573-95
Jiang, Ziqing; Vasil, Adriana I; Hale, John D et al. (2008) Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides. Biopolymers 90:369-83
Mant, Colin T; Hodges, Robert S (2008) Mixed-mode hydrophilic interaction/cation-exchange chromatography: separation of complex mixtures of peptides of varying charge and hydrophobicity. J Sep Sci 31:1573-84

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