High-performance liquid chromatography of amino acids, peptides and proteins

Probing the Binding Behavior and Conformational States of Globular Proteins in Reversed-Phase High-Performance Liquid Chromatography

“…Over the past several years, analytical gradient elution RP-HPLC and high-performance hydrophobic interaction chromatography (HP-HIC) have been increasingly employed as experimental tools to probe polypeptide conformation. For example, RP-HPLC and HP-HIC methods have been used to study the induced stabilization of coiled-coil, − amphipathic α-helical peptides, − or insulin-related polypeptides − by nonpolar ligands. Compared to these studies with peptides, much less work has been reported on the conformational behavior of globular proteins in similar hydrophobic environments.…”

Predicting Retention Time Shifts Associated with Variation of the Gradient Slope in Peptide RP-HPLC

“…The widely accepted theoretical description of the retention behavior of peptidic compounds is based on LSS theory as detailed by Stadalius et al The retention time of a peptide under gradient elution conditions is given as t g = t G / ( S Δ ϕ ) log ( 2.3 k 0 t 0 ( S Δ ϕ / t G ) + 1 ) + t 0 + t D where t 0 is the column dead-time, t D is the dwell-time of the gradient system, t G is the gradient time for the gradient of Δϕ. While eq is important in the theoretical description of peptide behavior in RP-HPLC systems and determination of S and k 0 values from gradient data, ,− the practical application of it for calculating retention times of peptides is limited. It requires precise measurements of the parameters of the RP HPLC system ( t 0 , t D ), as well as knowing the coefficients S and k 0 for a particular peptide.…”

“…Conversely, this equation is often employed for a reverse task: to estimate the coefficients S or k 0 using experimental retention times measured at different chromatographic conditions in a gradient separation mode. This approach was used to determine S and k 0 values for a number of protein and peptides by Snyder’s and Hearn’s research groups, ,− and more recently by Shinoda et al in proteomic experiments . It should be noted, however, that analytical solution of this equation for several different LC conditions (gradients, flow-rates) is usually obtained by applying numerical multiparameter fitting algorithms and may result in significant errors.…”

“…Another similar trend is noticeable that the critical point in understanding parameters affecting S was clarified when extensive proteomics derived data were considered. Researchers in 1980s and 1990s were dealing with rather limited sets of measured S -values, which precluded the development of an accurate predictive model. ,− It should be noted that we deal with the problem of peptide selectivity variation based on the assumption that LSS theory can adequately describe the behavior of polymeric compounds. This might not be the case for the sufficiently large peptides, which could fold up and exhibit intramolecule interactions. , While the majority of tryptic species used in our experiment seems to behave like small molecules within the framework of LSS theory, some larger analytes might show unpredictable tendencies in the S variation.…”

“…Their original assumption postulated a relation between slope and molecular weight of the analyte: S = 0.48(MW) 0.44 for a set of polypeptides ranging from 600 to 14 000 Da in molecular weight . In later studies, Hearn and co-workers tested this rule for sets of peptides with a much narrower MW variation and attempted to establish the effect of peptide hydrophobicity on S -values. − They concluded that there is no such direct correlation between slope and molecular weight but rather the “magnitude of hydrophobic contact area and the number of interaction sites” determine S . Thus to date there has been no quantitative model developed for predicting S for peptidic compounds, so they are typically put in a category of “irregular compounds” from the point of view of LSS theory, analytes with significant “nonpredictable” variation of S and resulting separation selectivity.…”