Reversed-phase chromatographic method development for peptide separations using the computer simulation program ProDigest-LC.
A computer program, ProDigest-LC, has been developed that assists scientists in devising methods of size-exclusion, cation-exchange and reversed-phase high-performance liquid chromatography for the analytical separation and purification of biologically active peptides and peptide fragments from enzymatic and chemical digests of proteins. ProDigest-LC accurately predicts the retention behaviour of peptides of known composition, containing 2-50 amino acid residues, and simulates the elution profiles in all three modes of chromatography. In addition, ProDigest-LC is a user-friendly program, designed as a teaching aid for both students and researchers in selecting the correct conditions for chromatography, that is, the mode of chromatography, column selection and mobile-phase selection, and has the ability to examine the effects of gradient-rate, flow-rate and sample size on the separation. The simulation capabilities of ProDigest-LC as they apply to the reversed-phase chromatography of peptides were examined. The development of the reversed-phase simulation features of the program is described, stressing the importance of peptide standards in the development, testing and practical use of ProDigest-LC. The ease of use of the program is clearly demonstrated by presenting a step-by-step procedure to produce several of the simulations illustrated in the paper. The predictive accuracy of the program was rigorously tested by its application to retention time prediction, at different gradient-rates and flow-rates, for a sample mixture containing peptides exhibiting a wide range of size (11-50 residues), charge (+1 to +8 net charge), hydrophobicity and conformation (random coil to considerable alpha-helical structure). The excellent accuracy of these peptide retention time predictions complemented the successful simulation (in terms of peptide retention times, peptide resolution, peak heights and peak widths) of the effects of gradient-rate and flow-rate on the elution profile of a mixture of closely related peptide analogues.
Mant
C T
CT
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Burke
T W
TW
Zhou
N E
NE
Parker
J M
JM
Hodges
R S
RS
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003198:Computer Simulation; D010455:Peptides / Q000032:analysis*; D012984:Software
D010455:Peptides