Nian E. Zhou

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.

Structure, function and application of the coiled-coil protein folding motif

Recent X-ray analyses and synthetic model studies of the coiled-coil motif have clarified roles for hydrophobic core residues and ionic interactions in determining stability, selectivity, stoichiometry and orientation of alpha-helices in this structure. Although much remains to be learnt, current knowledge now enables this motif to be used in novel constructs and points the way to a more explicit understanding of native coiled-coil formation and protein folding in general.

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.

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.

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.

Chapter 13 Amino acids and peptides

Publisher Summary This chapter discusses amino acids and peptides, and the detection methods used to identify them. All primary amino acids form the same complex following reaction with ninhydrin, making this reagent unsuitable for pre-column derivatization. Most cation-exchange high-performance liquid chromatography (HPLC) columns utilized for ninhydrin-based post-column amino acid analysis still employ sulfonated polystyrene/divinylbenzene resins. Silica-based ion-exchange packings, popular for peptide separations, have poor resistance to the high pH values, ionic strength, and elevated temperature required to elute free amino acids. Modern automated amino acid analyzers based on post-column ninhydrin derivatization are capable of providing highly reliable and reproducible analyses. Phenylisothiocyanate (PITC), also known as Edmans reagent, has long been used for the sequencing of polypeptides and proteins, and was introduced for the analysis of amino acids in the early eighties. It is currently the most extensively used reagent for pre-column derivatization. Derivatization following cation-exchange chromatography of free amino acids has distinct advantages, provided that the instrumentation yields reproducible flow rates and reaction temperatures [lo]. Moreover, there is no need for derivatization to proceed to completion, and problems of derivative stability are not significant. The distinction among a peptide, polypeptide, and protein, in terms of the number of peptide residues they contain, is somewhat arbitrary. However, peptides are usually defined as containing 50 amino acid residues or less. Although molecules of more than 50 residues usually have a stable three-dimensional structure in solution, and are referred to as proteins, conformation can be an important factor in peptides as well as proteins.