Analysis of synthetic peptides by high-performance liquid chromatography.
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Amino Acid Sequence↗
Biomedical subjects
C T Mant
Publications and source records attributed to C T Mant.
Analysis of peptides by high-performance liquid chromatography.
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Chromatography, High Pressure Liquid↗
Selectivity due to conformational differences between helical and non-helical peptides in reversed-phase chromatography.
The reversed-phase retention behaviour of two series of peptides, one non-helical and the other alpha-helical, was studied under various linear AB gradients in order to determine the effect of peptide conformation on selectivity of the separation. The non-helical series, designated X1, with the sequence Ac-XLGAKGAGVG-amide, exhibited negligible alpha-helical content in a hydrophobic medium; whereas, the amphipathic alpha-helical series, designated AX9, with the sequence Ac-EAEKAAKEXEKAAKEAEK-amide, exhibited high alpha-helical content in a hydrophobic medium. We have shown that plots of log k vs. phi (where k is the median capacity factor and phi is the median volume fraction of organic solvent) are very similar for any one peptide conformation, i.e., peptides from either the non-helical or amphipathic alpha-helical series exhibit similar S (solute parameter) values and the b (gradient steepness parameter) values are also similar for 17 different amino acid substitutions within each series of peptides. If mixtures of peptides from the two different series are separated using either increasing or decreasing gradient rates, large increases in resolution occur due to selectivity, which may be attributed to the difference in the log k vs. phi plots for each series of peptides. In addition, by using a polymer of an X1 peptide, which is 20 residues in length, it has been shown that the molecular mass difference between the X1 and the AX9 series of peptides is not sufficient to account for the selectivity difference. The S value of a non-amphipathic alpha-helical peptide further suggested that the difference in selectivity between the two series of peptides was due to differences in conformation. We believe that the peptide mixtures presented here provide a good model for studying selectivity effects due to conformational differences between peptides, an important concern when attempting to develop rational approaches to the prediction and optimization of peptide separation protocols from primary sequence information alone.
Amino Acid Sequence↗
Reversed-phase chromatography of synthetic amphipathic alpha-helical peptides as a model for ligand/receptor interactions. Effect of changing hydrophobic environment on the relative hydrophilicity/hydrophobicity of amino acid side-chains.
To mimic a hydrophobic protein binding domain, which is a region on the surface of a protein that has a preference or a specificity to interact with a complementary surface, we have designed amphipathic alpha-helical peptides where the non-polar face interacts with the non-polar surface of a reversed-phase stationary phase. Two series of potentially amphipathic alpha-helical peptides, a native Ala peptide (AA9) and a native Leu peptide (LL9), were designed where the native peptide contains 7 residues of either Ala or Leu, respectively, in its non-polar face. This design results in an overall hydrophobicity of the non-polar face of the Leu peptide that is greater than that of the non-polar face of the native Ala peptide. Mutants of the native Ala-face peptide, AX9, and the native Leu-face peptide, LX9, were designed by replacing one residue in the centre of the non-polar face in both series of peptides. Therefore, by changing the hydrophobicity of the environment surrounding the mutated amino acid side-chain, the effect on the hydrophilicity/hydrophobicity of each amino acid side-chain could be determined. Using the substitutions Ala, Leu, Lys and Glu, it was shown that the maximum hydrophilicity of these amino acid side-chains could be determined when the environment surrounding the mutation is maximally hydrophobic; whereas its maximum hydrophobicity can be determined when the environment surrounding the mutation is minimally hydrophobic. This procedure was further extended to the remaining amino acids commonly found in proteins and it was determined that this general principle applies to all 20 amino acids. These results have major implications to understanding the hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.
Alanine↗
Reversed-phase liquid chromatography as a useful probe of hydrophobic interactions involved in protein folding and protein stability.
We have evaluated the potential of reversed-phase liquid chromatography (RPLC) as a probe of hydrophobic interactions involved in protein folding and stability. Our approach was to apply RPLC to a de novo designed model protein system, namely a two-stranded alpha-helical coiled coil. It was shown that the reversed-phase retention behaviour of various synthetic analogues of monomeric alpha-helices and dimeric coiled-coil structures correlated well with their stability in solution, as monitored by circular dichroism during guanidine hydrochloride and temperature denaturation studies. In addition, an explanation is offered as to why amphipathic coiled coils, an important structural motif in many biological systems, are more stable at low pH compared to physiological pH values. The results of this study suggest that not only may RPLC prove to be a useful and rapid complementary technique for understanding protein interactions, but also the de novo designed coiled-coil model described here is an excellent model system for such studies.
Amino Acid Sequence↗
Effect of the alpha-amino group on peptide retention behaviour in reversed-phase chromatography. Determination of the pK(a) values of the alpha-amino group of 19 different N-terminal amino acid residues.
We have examined the contribution of the alpha-amino group to retention behaviour for peptides in reversed-phase chromatography using two series of peptide analogues, one containing an N alpha-acetylated terminal and the other containing an alpha-amino group (non-acetylated). The effect of the alpha-amino group, at pH 2, on the hydrophobicity of the side-chain of the N-terminal residue was obtained by referencing the retention time of the acetylated or non-acetylated peptide to the retention time of a glycine analogue. It was shown that the presence of an alpha-amino group could decrease or increase the hydrophobicity of the side-chain of the N-terminal residue with respect to the hydrophobicity of the side-chain in the absence of an alpha-amino group. The effect was also shown to be sequence dependent, with respect to the N-terminal residue. Increasing pH was shown to increase retention time dramatically for the non-acetylated analogues, through the deprotonation of the alpha-amino group. By separating pairs of acetylated/non-acetylated analogues over the pH range 2-9, it was possible to determine the pK(a) of the alpha-amino group, where it was shown that the pK(a) was dependent on two probable factors: (1) the inherent hydrophobicity of the stationary phase; and (2) the amino acid substituted in the N-terminal position. Interestingly, the pK(a) values determined were very similar to that found in proteins. It was also possible to determine the pK(a) values of some of the substituted amino acids containing ionizable side-chains. This study shows that, in order to understand fully the retention behaviour of peptides containing an alpha-amino group in reversed-phase chromatography, one must incorporate an alpha-amino group contribution and its effect on the hydrophobicity of the side-chain of the N-terminal residue.
Acetylation↗
Hydrophilic-interaction chromatography of peptides on hydrophilic and strong cation-exchange columns.
Hydrophilic-interaction chromatography (HILIC) was recently introduced as a potentially useful separation mode for the purification of peptides and other polar compounds. The elution order of peptides in HILIC, which separates solutes based on hydrophilic interactions, should be opposite to that obtained in reversed-phase chromatography, which separates solutes based on hydrophobic interactions. Three series of peptides, two of which consisted of positively charged peptides (independent of pH at pH less than 7) and one of which consisted of uncharged or negatively charged peptides (dependent on pH), and which varied in overall hydrophilicity/hydrophobicity, were utilized to examine the separation mechanism and efficiency of HILIC on hydrophilic and strong cation-exchange columns.
Amino Acid Sequence↗
Comparison of silica-based cyanopropyl and octyl reversed-phase packings for the separation of peptides and proteins.
The performance of a silica-based C8 packing was compared with that of a less hydrophobic, silica-based cyanopropyl (CN) packing during their application to reversed-phase high-performance liquid chromatography (linear trifluoroacetic acid-water to trifluoroacetic acid-acetonitrile gradients) of peptides and proteins. It was found that: (1) the CN column showed excellent selectivity for peptides which varied widely in hydrophobicity and peptide chain length; (2) peptides which could not be resolved easily on the C8 column were widely separated on the CN column; (3) certain mixtures of peptides and small organic molecules which could not be resolved on the C8 column were completely separated on the CN column; (4) impurities arising from solid-phase peptide synthesis were resolved by a wide margin on the CN column, unlike on the C8 column, where these compounds were eluted very close to the peptide product of interest: and (5) specific protein mixtures exhibited superior resolution and peak shape on the CN column compared with the C8 column. The results clearly demonstrate the effectiveness of employing stationary phases of different selectivities (as opposed to the more common optimization protocol of manipulating the mobile phase) for specific peptide and protein applications, an approach underestimated in the past.
Amino Acid Sequence↗
Multi-column preparative reversed-phase sample displacement chromatography of peptides.
Preparative reversed-phase sample displacement chromatography (SDC) of peptides was examined utilizing a multi-column approach. The effects of various SDC run parameters (flow-rate, run time and sample load) on the distribution of a single purified peptide and a mixture of three synthetic peptides was examined. The peptides in the mixture were closely related in hydrophobicity and mixed in a 1:4:1 ratio designed to mimic a typical preparative separation problem frequently encountered in crude synthetic peptide mixtures, that is, where there exist both hydrophobic and hydrophilic synthetic impurities close to the product of interest. Based on the results of these model systems, a SDC protocol was applied to the preparative purification of a crude synthetic peptide. The multi-column SDC approach provides rapid separations that are easy to employ because isocratic elution is utilized both in the separation process and in elution of the column segments. There is minimal fraction analysis, minimal use of organic solvents and increased utilization of the stationary phase such that the method involves considerably lower costs than traditional gradient-elution chromatography.
Binding, Competitive↗
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.
Chromatography, High Pressure Liquid↗
Correlation of protein retention times in reversed-phase chromatography with polypeptide chain length and hydrophobicity.
The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time behaviour on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption holds up well enough for small peptides (up to ca. 15 residues), it is now recognized that polypeptide chain length must be taken into account when attempting to equate retention time behaviour of larger peptides and proteins with their overall hydrophobicity. In the present study, we have examined the reversed-phase retention behaviour of 19 proteins of known sequence on stationary phases of varying hydrophobicity and ligand density. From the observed protein retention behaviour on C4, C8 and C18 stationary phases under gradient elution conditions, we have been able to correlate the observed retention times of proteins ranging in molecular weight from 3500 to 32,000 dalton and in chain length from 30 to 300 residues with their overall hydrophobicity (based on retention parameters derived from small peptides) and the number of residues in the polypeptide chain. The retention behaviour of the proteins on the C4, C8 and C18 columns was also compared to that obtained on supports containing lower ligand densities (phenyl ligands). The maintenance of native or partially folded protein conformation on the phenyl columns, resulting in lower retention times than would be expected for fully denatured proteins, underlined the importance of efficient protein denaturation for satisfactory correlation of protein retention times with protein hydrophobicity. In addition, the effectiveness of increasing temperature and/or ligand density of the stationary phase in denaturing proteins was also demonstrated.
Chromatography, High Pressure Liquid↗
Strong cation-exchange high-performance liquid chromatography of peptides. Effect of non-specific hydrophobic interactions and linearization of peptide retention behaviour.
Strong cation-exchange chromatography (strong CEX) is probably the most useful mode of high-performance ion-exchange chromatography (IEC) for peptide separations. Although the hydrophobic character of high-performance ion-exchange packings, often giving rise to mixed-mode contributions to solute separations, has long been recognized, a systematic approach to examining the effect and magnitude of the hydrophobicity of these packings during IEC of peptides has so far been lacking. In the present study, we report the synthesis of three series of positively charged peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5-50 amino acid residues): Ac-(Gly-Lys-Gly-Leu-Gly)n-amide, Ac-(Leu-Gly-Leu-Lys-Ala)n-amide and Ac-(Leu-Gly-Leu-Lys-Leu)n-amide (n = 1, 2, 4 6, 8, 10). We have examined non-specific hydrophobic interactions of these peptides with both silica-and polymer-based ion-exchange packings, demonstrating how these interactions are overcome by the addition of acetonitrile to the mobile phase. It was also shown that removal of non-specific hydrophobic interactions may be necessary just to elute peptides from the ion-exchange matrix. In addition, from the observed retention times of these three peptide polymer series and other peptides which vary substantially in charge density, net charge, polypeptide chain length and hydrophobicity, we have established a simple approach to linearization and, thus, prediction of peptide retention behaviour in CEX.
Chromatography, High Pressure Liquid↗
Computer simulation of high-performance liquid chromatographic separations of peptide and protein digests for development of size-exclusion, ion-exchange and reversed-phase chromatographic methods.
A computer program, called Pro Digest-LC, has been developed which 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. Pro Digest-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, Pro Digest-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, mobile-phase selection, and has the ability to examine the effects of flow-rate, gradient-rate, and sample size on the separation. We have designed a set of peptide standards for each mode of chromatography to aid the researcher in eliminating non-specific interactions, to standardize retention behaviour on the user's columns, to monitor column performance and to compare packing materials. In the development of each prediction mode, experimental peak heights, peak widths, and retention times from model synthetic peptide standards were incorporated directly into the program and can be used as default values. Pro Digest-LC is an interactive program, in that researchers can run peptide standards on their particular columns and enter the peak width at half-height, peak height, retention time and quantity injected to adjust the simulation to their particular column. The simulated experiments eliminate the time-consuming trial-and-error methods used to suitable separation or purification procedures. The researcher would perform the actual experiment only after predicting the optimized conditions, thereby saving valuable sample and research time. The general concepts of the program along with representative separations of protein digests are displayed.
Animals↗
Effect of peptide chain length on peptide retention behaviour in reversed-phase chromatography.
The use of amino acid retention or hydrophobicity coefficients for the prediction of peptide retention time and/or the elution order on hydrophobic stationary phases is based on the premise that amino acid composition is the major factor affecting peptide retention in reversed-phase chromatography. Although this assumption generally agrees well for small peptides (up to ca. 15 residues), the retention times of increasingly larger peptides are less than expected from a simple summation of retention coefficients. In the present study, we report the synthesis of four series of peptide polymers which vary significantly in overall hydrophobicity and polypeptide chain length (5-50 amino acid residues, Ac = acetyl): Ac-(G-L-G-A-K-G-A-G-V-G)n-amide (n = 1-5), Ac-(G-K-G-L-G)n-amide (n = 1, 2, 4, 6, 8, 10), Ac-(L-G-L-K-A)n-amide (n = 1, 2, 4, 6, 8, 10) and Ac-(L-G-L-K-L)n-amide (n = 1, 2, 4). From the retention behaviour of these peptide polymers on C4, C8 and C18 stationary phases under gradient elution conditions, we have clearly established the effect of polypeptide chain length and hydrophobicity on peptide retention. This, in turn, has enabled us to extend the utility of retention time prediction for peptides containing up to 50 residues by introducing a peptide chain-length correction.
Chromatography, High Pressure Liquid↗
Preparative purification of peptides by reversed-phase chromatography. Sample displacement mode versus gradient elution mode.
In this study, we describe a novel method of preparative liquid chromatography, applicable to analytical columns and instrumentation, where the reversed-phase column is operated in sample displacement mode (SDM). This technique takes advantage of the different relative hydrophobicities of components of a sample mixture, so that when a column is optimally loaded with an aqueous solution of the sample mixture, there is competition among the sample components for the adsorption sites on the hydrophobic stationary phase. The more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column. Thus, the major separation takes place in water. Subsequent treatment with an aqueous organic eluent is only required to wash retained components off the column and takes no part in the major separation process. A two-column (precolumn and main column, in series) SDM strategy was applied to the preparative purification of a peptide product from neighbouring hydrophilic and hydrophobic impurities. Hydrophobic impurities were isolated on a shorter precolumn trap while hydrophilic impurities were displaced from the main column in the aqueous mobile phase (0.05% aq. trifluoroacetic acid), leaving the main column filled with homogeneous product. Gradient elution was then used to remove the peptide product from the main column. The researcher can regulate the size of the precolumn trap depending on the amount of hydrophobic impurities in a particular sample, or the size of the main column depending on the amount of product desired. The simplicity and flexibility of the SDM approach to preparative-scale purification enabled rapid separation of a single peptide component from a complex multicomponent mixture and should prove to be valuable for researchers in the peptide/protein field.
Chemical Phenomena↗
High-performance liquid chromatography of amino acids, peptides and proteins. LXXXV. Evaluation of the use of hydrophobicity coefficients for the prediction of peptide elution profiles.
The gradient elution behaviour of five synthetic decapeptide analogues has been investigated using an octadecylsilica stationary phase and trifluoroacetic acid-water-acetonitrile mobile phases. The influence of gradient time and flow-rate on the relative retentions and bandwidths of these peptides was assessed using quantitative expressions derived from linear solvent strength theory and general plate height theory. Linear relationships between logarithmic median capacity factors, log k, and the mole fraction of organic solvent modifier, phi, were observed over the experimental range of conditions used. The slopes of these plots were different for all peptides, which indicates that divergences will occur in the prediction of peptide retention times due to conformation dependent changes in hydrophobic contact area occupancy at the stationary phase surface. However, the differences in S values (tangent to the curve obtained in a plot of log k versus phi) for these peptides were not substantial enough to seriously affect the prediction of peptide retention times at one gradient slope from those observed at another. In addition, significant differences existed between experimental and theoretical peak capacity data of these peptide analogues of similar molecular weight and overall polarity, particularly at lower flow-rates or longer residence times. These results once again demonstrate that additional diffusional and interactive processes occur during the reversed-phase separation of peptides and proteins which are not yet adequately formalized by current chromatographic theory.
Amino Acids↗
Size-exclusion high-performance liquid chromatography of peptides. Requirement for peptide standards to monitor column performance and non-ideal behaviour.
A series of five synthetic peptide polymers with the sequence Ac-(G-L-G-A-K-G-A-G-V-G)n-amide, where n = 1-5, was employed to assess the resolving power of high-performance size-exclusion columns in peptide separations. The peptide standards showed great versatility in monitoring both ideal (no interactions of solutes with the column material) and non-ideal (hydrophobic and/or ionic interactions of solutes with the column material) size-exclusion behaviour in volatile and non-volatile mobile phases. The effectiveness of adding salts or organic solvents to overcome non-specific interactions of solutes with the column materials was well illustrated by the standards. In addition, the advantageous use of non-ideal size-exclusion behaviour was highlighted. The ability to predict the position and/or elution order of peptides during size-exclusion chromatography (SEC) requires peptides to be separated by a pure size-exclusion process. Although the peptide standards demonstrated similar ideal size-exclusion profiles in non-denaturing medium on all the columns studied, this study suggested that, if the conformational character of a peptide-protein mixture in a particular mobile phase is uncertain and ideal size-exclusion behaviour is required, SEC should be carried out under highly denaturing conditions.
Acetonitriles↗
Effects of ion-pairing reagents on the prediction of peptide retention in reversed-phase high-performance liquid chromatography.
We have examined the resolution, on reversed-phase columns, of a series of model synthetic peptides and commercially available synthetic peptide standards under gradient elution conditions, using a water-acetonitrile mobile phase containing hydrophilic (phosphoric acid) or hydrophobic (trifluoroacetic acid, heptafluorobutyric acid) ion-pairing reagents. Increasing hydrophobicity or concentration of the ion-pairing reagents increased peptide retention times. It was clearly shown that these reagents effected changes in peptide retention time solely through interaction with the basic residues in the peptide. In general, each positive charge, whether originating from a lysine, arginine or histidine side-chain, or from an N-terminal alpha-amino group, exerts an equal effect on peptide retention. Different counterions have different effects on the change in peptide retention time per positively charged residue due to their differences in hydrophobicity. However, increasing concentrations of a specific counterion have an essentially equal effect per positively charged residue. These effects are also column dependent (n-alkyl chain length and ligand density). These results, demonstrating a simple relationship between peptide retention in different ion-pairing systems, enabled the determination of rules for prediction of peptide retention times in one ion-pairing system from observed or predicted retention times in another system. The small average deviation of predicted and observed retention times for a series of basic peptides was good evidence for the value of this predictive method. This study provides a clear understanding of the effect of changing counterion hydrophobicity or concentration on peptide retention, and thus can be extremely beneficial in the purification of peptides and for providing proof of peptide homogeneity.
Amino Acid Sequence↗