The three major areas under investigation are: (1) development of novel separation methods of peptides/proteins; (2) multiple and simultaneous peptide purification by sample displacement chromatography (SDC); and (3) utility of reversed-phase chromatography (RPC) to monitor stability, folding and conformation of peptides. The ever-increasing demand for synthetic peptides has fueled a constant demand for efficient analytical and preparative purification techniques. Novel purification protocols, such as mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) with the development of volatile mobile phases, will rival RPC. Tailored RPC stationary phases will allow the advantageous manipulation of the elution patterns of peptide mixtures. A requirement for multiple peptide synthesis will be met by the development of a rapid, low pressure and simultaneous purification technique based on a combination of solid-phase extraction technology coupled with the novel SDC approach, allowing up to 100 peptides at a time to be purified in the absence of organic modifier. Combination of the sample displacement technique with microbore RPC to separate and concentrate synthetic impurities prior to direct elution into LC-MS will also allow at least a 100-fold increase in sensitivity over present methods to judge peptide homogeneity. RPC is a potent probe of polypeptide structural characteristics and it will be possible to correlate elution behaviour with amphipathicity and hydrophobicity of antimicrobial alpha-helical and cyclic beta-sheet peptides. Such work has major implications for the de novo design of more effective antimicrobial agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061855-02
Application #
6387244
Study Section
Special Emphasis Panel (ZRG1-BMT (01))
Program Officer
Edmonds, Charles G
Project Start
2000-07-01
Project End
2004-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$321,985
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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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