Rational design of bifunctional chimeric peptides that combine antimicrobial and titanium binding activity

Bacterial biofilm formation remains a serious problem for clinical materials and often leads to implant failure. To counteract bacterial adhesion, which initiates biofilm formation, the development of antibiotic surface coating strategies is of high demand and warrants further investigations. In this study, we have created bifunctional chimeric peptides by fusing the recently developed antimicrobial peptide MGD2 (GLRKRLRKFFNKIKF) with different titanium-binding sequences. The novel peptides were investigated regarding their antibacterial potential against a set of different bacterial strains including drug-resistant Staphylococcus aureus. All peptides showed high antimicrobial activities both when in solution and when immobilized on titanium surfaces. Owing to the ease of synthesis and handling, the herein described peptides might be a true alternative to prevent bacterial biofilm formation.     

SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 mug of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides.     

Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters

Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.     

Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets

Synthetic peptides corresponding to the carboxyl terminus of the fibrinogen gamma chain inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to platelets, yet the active decapeptide sequence has only been found in fibrinogen to date. In contrast, all three proteins contain Arg-Gly-Asp sequences, and peptides containing Arg-Gly-Asp are potent inhibitors of their binding to activated platelets. We have analyzed the relationship between these peptide sets by direct binding assays. H12 (gamma 400-411) inhibited the binding of an Arg-Gly-Asp-containing peptide to platelets with similar dose response to inhibition of fibronectin binding. We have previously reported that GPIIb-IIIa binds to immobilized Arg-Gly-Asp peptides and can be eluted by Arg-Gly-Asp-containing peptides in solution. Both H12 and L10 (gamma 402-411) completely eluted GPIIb-IIIa bound to immobilized Arg-Gly-Asp peptides. Conversely, when GPIIb-IIIa was bound to immobilized L10, either L10 or an Arg-Gly-Asp peptide could elute it. Peptide specificity was established by the failure of Gly-Arg-Gly-Glu-Ser-Pro or acetylated L10 to elute GPIIb-IIIa from the immobilized peptides. These results indicate that the two peptide sets interact with the same receptor which contains GPIIb-IIIa.     

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.     

The interaction of bioactive peptides with an immobilized phosphatidylcholine monolayer.

The interaction of three bioactive peptides, bombesin, beta-endorphin, and glucagon with a phosphatidylcholine monolayer that was immobilized to porous silica particles and packed into a stainless steel column cartridge, has been studied using dynamic elution techniques. This immobilized lipid monolayer provides a biophysical model system with which to study the binding of peptides to a lipid membrane. In particular, the influence of temperature and methanol concentration on the affinity of each peptide for the immobilized lipid surface was assessed. For all test peptides, nonlinear retention plots were observed at all temperatures that contrasted sharply with the simple linear plots observed for the small unstructured control molecules N-acetyltryptophanamide and diphenylalanine. An analysis of the thermodynamics of the interaction of peptides with the immobilized monolayer was also carried out. The results revealed that while the peptides interacted with the monolayer predominantly through hydrophobic interactions, the relative contribution of DeltaH(assoc)(O) and DeltaS(assoc)(O) to the overall free energy of association was dependent on the temperature and methanol concentration. In particular, it was evident that under most conditions, the binding of the peptides to the immobilized lipid monolayer was enthalpy-driven, i.e., mediated by nonclassical hydrophobic interactions. Significant band-broadening and asymmetric and split peaks were also observed for bombesin, beta-endorphin, and glucagon at different temperatures and methanol concentrations. These changes in affinity and peak shape are consistent with the formation of multiple conformational species during the interaction of these peptides with the lipid monolayer. In addition, the binding behavior of the three test peptides on an n-octylsilica surface that lacked the phospho headgroups of the phospholipid was significantly different from that observed with the immobilized phosphatidylcholine surface, indicating a specificity of interaction between the peptides and the lipid surface. Overall, these experimental results demonstrate that the biomimetic phosphatidylcholine monolayer provides a stable and sensitive system with which to explore the molecular mechanism of peptide conformational changes during membrane interactions.     

Comparison of three quantitative phosphoproteomic strategies to study receptor tyrosine kinase signaling

There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.     

Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.     

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.     

Recognition properties of antisense peptides to Arg8-vasopressin/bovine neurophysin II biosynthetic precursor sequences

We studied the interaction properties of synthetic antisense (AS) peptides encoded in the antisense strand of DNA corresponding to the N-terminal 20-residue sequence of the biosynthetic precursor of Arg8-vasopressin (AVP) and its binding protein bovine neurophysin II (BNPII). Binding affinities of sense polypeptides AVP and BNPII with AS peptides were measured by analytical affinity chromatography, in each case by the extent of chromatographic retardation of a soluble polypeptide interactor on an affinity matrix containing the other interactor as the immobilized species. Chromatographically calculated dissociation constants ranged from 10(-3) to 10(-6) M. Experiments were carried out to define the selectivity and underlying forces involved in the AS peptide interactions. For AS peptide elutions on sense peptide affinity supports, reduced binding affinity with increasing 1-propanol concentration and ionic strength suggested the presence of both ionic and hydrophobic contributions to AS peptide/immobilized sense peptide recognition. This same conclusion was reached with the antisense peptides as the immobilized species and measurement of elution of sequence-simplified, truncated, and charge-depleted forms of sense peptides. Immobilized AS 20-mer affinity matrix differentially retarded AVP versus oxytocin (OT) and BNPII versus BNPI (the neurophysin related biosynthetically to OT) and was used to separate these polypeptides from acid extracts of bovine posterior pituitaries. In addition, immobilized AS 12-mer corresponding to AVP-Gly-Lys-Arg could be used to separate AVP from OT. The results confirm that antisense peptides recognize sense peptides with significant selectivity in the AVP/BNPII precursor case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multifunctional poly(ethylene glycol) semi-interpenetrating polymer networks as highly selective adhesive substrates for bioadhesive peptide grafting

Novel artificial extracellular matrices were synthesized in the form of semi-interpenetrating polymer networks containing copolymers of poly(ethylene glycol) and acrylic acid (PEG-co-AA) grafted with synthetic bioadhesive peptides onto exposed carboxylic acid moieties. These substrates were very resistant to cell adhesion, but when they were grafted with adhesive peptides they were highly biospecific in their ability to support cell adhesion. Extensive preadsorption of adhesive proteins or peptides did not render these materials cell adhesive; yet covalent grafting of adhesive peptides did render these materials highly cell adhesive even in the absence of serum proteins. Polymer networks containing immobilized PEG-co-AA were grafted with peptides at densities of 475 +/- 40 pmol/cm(2). Polymer networks containing immobilized PEG-co-AA N-terminally grafted with GRGDS supported cell adhesion efficiencies of 42 +/- 4% 4 h after seeding and became confluent after 12 h. These cells displayed cell spreading and cytoskeletal grafted with inactive control peptides (GRDGS, GRGES, or no peptide) supported cell adhesion efficiencies of 0 +/- 0%, even when challenged with high seeding densities (to 100,000 cell/cm(2)) over 14 days. These polymer networks are suitable substrates to investigate in vitro cell-surface interactions in the presence of serum proteins without nonspecific protein adsorption adhesion signals other than those immobilized for study.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.     

Hydroxyapatite affinity chromatography for the highly selective enrichment of mono‐ and multi‐phosphorylated peptides in phosphoproteome analysis

The most challenging analytical task facing phosphoproteome determination requires the isolation of phosphorylated peptides from the myriad of unphosphorylated species. In the past, several strategies for phosphopeptide isolation have been proposed in combination with subsequent mass spectrometric investigations. Among these techniques, immobilized metal affinity chromatography and titanium dioxide have been recognized as the most effective. Here, we present an alternative method for the enrichment of phosphopeptides based on hydroxyapatite (HAP) chromatography. By taking advantage of the strong interaction of HAP with phosphate and calcium ions, we developed an efficient method for the selective separation and fractionation of phosphorylated peptides. The effectiveness and efficiency of recovery for this procedure was assayed using tryptic digests of standard phosphorylated protein mixtures. Based on the higher affinity of multi‐phosphorylated peptides for HAP surfaces, the introduction of a phosphate buffer gradient for stepwise peptide elution resulted in the separation of mono‐, di‐, tri‐, and multi‐phosphorylated peptides. Thus, we demonstrated that this technique is highly selective and independent of the degree of peptide phosphorylation.     

Affinity chromatography on immobilized anhydrotrypsin: general utility for selective isolation of C-terminal peptides from protease digests of proteins

Recently we have succeeded in the efficient isolation of the C-terminal peptides from tryptic digests of the tail sheath protein (with C-terminal Gly) and the tube protein (with C-terminal Glu) of bacteriophage T4, by taking advantage of a unique property of immobilized anhydrotrypsin, that is, a strong specific affinity for peptides containing Arg or Lys residues at their C-termini. In this study, the utility of affinity chromatography on immobilized anhydrotrypsin was further demonstrated in the cases of Streptomyces subtilisin inhibitor (as a reduced and S-carboxymethylated form, with C-terminal Phe) and alpha 1-antitrypsin (with C-terminal Lys). By subjecting a tryptic digest of the former protein and a chymotryptic digest of the latter protein to the affinity chromatography, the C-terminal peptides were specifically recovered in the breakthrough fraction and in the adsorbed fraction, respectively. It was further shown that immobilized anhydrotrypsin can also adsorb peptides with C-terminal S-aminoethyl-Cys residues and exerts adsorptive ability even toward the peptides in solution containing urea at a high concentration if appropriate precautions are taken. These findings suggest the general utility of this simple method for C-terminal peptide isolation, which is extremely helpful for studies to confirm amino acid sequences deduced from nucleotide sequences of the cDNA (or genomic DNA) of proteins.     

Active Peptide Sequences in the Cell Binding Domain of Thrombospondins

Methods for measuring the attachment of viable cells to immobilized peptides to identify sequences possessing cell binding activity are described. The 212-residue C-terminal cell binding domain (CBD) of human thrombospondin-1 (TS1 or TS) was thought to contain a site or sites for the attachment of a number of cell types based on inhibition of cell attachment to TS1 with monoclonal antibodies. The presence of such a site was confirmed by expression of this region of TS1 in Escherichia coli and demonstration of its cell binding activity independent of other known cell attachment sites in TS1. To further define active sequences within the CBD, a set of eight overlapping peptides that spanned the 212-residue domain was synthesized with standard techniques. Several of these long (30-mer) peptides were found to be insoluble in aqueous buffers; thus, they could not be tested as soluble inhibitors of the attachment of cells to TS1, a standard approach to identification of active cell binding sequences. Using chemically derivatized plates prepared by Costar Corp., we devised assays to test the direct attachment of cells to the peptides immobilized on plastic wells. We also found that the peptides were adsorbed to underivatized plastic when solubilized in 6 M guanidine hydrochloride. Even rather short peptides (5 residues) were efficiently immobilized with these methods. Peptides immobilized directly on plastic gave higher levels of cell attachment than the same peptides conjugated to a carrier protein and then immobilized. Using these methods, two of the 30-mers were found to support cell attachment. The active sequences within the 30-mers were then narrowed down to a hexamer in one case and a pentamer, both containing the tripeptide VVM. Shorter, soluble peptides containing these active sequences act synergistically to inhibit the attachment of cells to TS1 and its recombinant CBD.     

Surface immobilization chemistry influences peptide‐based detection of lipopolysaccharide and lipoteichoic acid

Antimicrobial peptides (AMPs) have recently gained attention as potentially valuable diagnostic and therapeutic agents. The utilization of these peptides for diagnostic purposes relies on the ability to immobilize them on the surface of a detection platform in a predictable and reliable manner that facilitates target binding. The method for attachment of peptides to a solid support is guided by peptide length, amino acid composition, secondary structure, and the nature of the underlying substrate. While immobilization methods that target amine groups of amino acid sequences are widely used, they can result in heterogeneous conjugation at multiple sites on a peptide and have direct implications for peptide presentation and function. Using two types of commercial amine‐reactive microtiter plates, we described the effects of analogous immobilization chemistries on the surface attachment of AMPs and their differential binding interaction with Gram‐specific bacterial biomarkers, lipopolysaccharide and lipoteichoic acid. As might be expected, differences in overall binding affinities were noted when comparing AMPs immobilized on the two types of plates. However, the two‐amine‐targeted linking chemistries also affected the specificity of the attached peptides; lipopolysaccharide generally demonstrated a preference for peptides immobilized on one type of plate, while (when observed at all) lipoteichoic acid bound preferentially to AMPs immobilized on the other type of plate. These results demonstrate the potential for tuning not only the binding affinities but also the specificities of immobilized AMPs by simple alterations in linking strategy. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Peptide and ester synthesis in organic solvents catalyzed by seryl proteases linked to alumina

Trypsin and alpha-chymotrypsin were immobilized to alumina-phosphocolamine complex, activated by glutaraldehyde. The immobilized enzymes show a great stability toward organic solvents miscible or immiscible with water. In the presence of a low concentration of water, the immobilized enzymes catalyzed transesterification reactions as well as peptide synthesis. The synthesized peptides were stable toward the immobilized enzymes.     

Enzymatic nanolithography of FRET peptide layer using V8 protease-immobilized AFM probe

In our study, a method based on Enzymatic nanolithography was successfully performed in a buffered solution using Staphylococcal serine V8 protease and AFM. To estimate the lithographing activity of the protease immobilized on the AFM tip to peptides immobilized on a substrate, we designed fluorescence resonance energy transfer (FRET) peptides as reporter peptides that showed enzymatic action specific to the V8 protease. When the protease digested the reporter peptide a quencher residue was released from the peptide and resulted in the appearance of fluorescence. In the designed 9-mer peptides, TAMRA functioned as a good quencher for FAM. When the fluorescence resonance energy transfer peptides immobilized on a glass substrate were hydrolyzed by V8 protease at the C-terminal of glutamic acid, fluorescence of a reporter dye was observed because of the release of a quencher from the substrate. After contacting and lateral scanning of the protease-immobilized AFM tip to the reporter peptide layer, a fluorescent area was observed by imaging using total internal refection fluorescence microscopy (TIRFM). The increment of fluorescence intensity of the digested peptide indicates the performance of lithography. Lithographing rates increased in inverse relation to scanning rates of the probe. The maximum limit of the scanning rate, i.e., that was too fast to permit cutting of the peptide on the substrate, and the lithographing performance are discussed in this study.     

Antisense peptide recognition of sense peptides: sequence simplification and evaluation of forces underlying the interaction

Structural principles were studied which underlie the recognition of sense peptides (sense DNA encoded) by synthetic peptides encoded in the corresponding antisense strand of DNA. The direct-readout antisense peptides corresponding to ribonuclease S-peptide bind to an affinity matrix containing immobilized S-peptide with significant selectivity and with dissociation constants in the range of 10(-6) M as judged by analytical affinity chromatography. Synthetic, sequence-modified forms of antisense peptides also exhibit substantial binding affinity, including a "scrambled" peptide in which the order of residue positions is changed while the overall residue composition is retained. The antisense mutants, as the original antisense peptides, bind at saturation with greater than 1:1 stoichiometry to immobilized S-peptide. The data suggest significant sequence degeneracy in the interaction of antisense with sense peptide. In contrast, selectivity was confirmed by the inability of several control peptides to bind to immobilized S-peptide. The idea was tested that the hydropathic pattern of the amino acid sequence serves to induce antisense peptide recognition. A hydropathically sequence-simplified mutant of antisense peptide was made in which all strongly hydrophilic (charged) residues were replaced by Lys, all strongly hydrophobic residues by Leu, and all weakly hydrophilic and hydrophobic residues by Ala, except Gly which was unchanged. This "KLAG" mutant also binds to immobilized S-peptide, with an affinity only an order of magnitude less than that with the original antisense peptide and with multiple stoichiometry. Mutants of the KLAG model, in which the hydropathic pattern was changed substantially, exhibited a lower binding affinity for S-peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance immobilized metal ion affinity chromatography of peptides: analytical separation of biologically active synthetic peptides

The separation of more than 30 biologically active synthetic peptides and their analogs on a high-performance immobilized metal ion affinity chromatography column is described. The metal chelating gel (TSK gel chelate-5PW) contains iminodiacetic acid (IDA) covalently coupled to a hydrophilic, resin-based matrix with a bead diameter of 10 micron. The retention of the peptides on Cu(II), Ni(II), and Zn(II) ions immobilized on the chelating gel showed that some of them can be separated by isocratic elution while the majority of them are retained and are separated into distinct fractions by elution with a linear imidazole gradient or with a continuously decreasing pH gradient. Of the three immobilized metal ions investigated here, the IDA-Cu(II) chelate column gave the best resolution irrespective of the type of gradient used. This is amply illustrated by the resolution of angiotensins I and II and their seven synthetic analogs. The results obtained here serve as guidelines for the future exploitation of this separation method for the efficient fractionation of a wide variety of peptides on an analytical or preparative scale.