Combinatorial optimization of the DNA cleaving Ni(II) x Xaa-Xaa-His metallotripeptide domain

A positional-scanning combinatorial protocol was employed to optimize the deoxyribose-based cleavage of B-form DNA by Ni(II) x Xaa-Xaa-His metallopeptides. This procedure employed 18 naturally occurring amino acids (excluding Cys and Trp) to generate two libraries in which the first and second positions of the peptide ligand were varied. Increased direct DNA cleavage relative to Ni(II) x Gly-Gly-His was observed when (1) the amino-terminal peptide position contained Pro, Met, Arg, or Lys (with Pro exhibiting the greatest activity) and (2) the second peptide position contained Lys, Arg, Met, Ser, or Thr (with Lys exhibiting the greatest activity); the optimized metallopeptide, Ni(II) x Pro-Lys-His, was found to cleave DNA an order of magnitude better than Ni(II) x Gly-Gly-His. While metal complexation and the A/T-rich site selectivity of the optimized metallopeptides were not altered, DNA binding affinity was slightly increased relative to Ni(II) x Gly-Gly-His, however, not to an extent necessary to account for the observed increase in reactivity. Examination of molecular models of Ni(II) x Pro-Lys-His bound to the minor groove of DNA via hydrogen bonding of the His N3 imidazole hydrogen to the N3 of adenine or O2 of thymine suggests that the Pro residue can make hydrophobic contacts with the sugars lining the walls of the groove while the Lys residue is able to form a salt bridge with a proximal phosphate; with these interactions, the metal center is poised to abstract the C4'-H of an adjacent nucleotide suggesting that noncovalent interactions result in a positioning which contributes to increased DNA cleavage activity.     

Chelating peptide-immobilized metal ion affinity chromatography. A new concept in affinity chromatography for recombinant proteins

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Construction of a high sensitive Escherichia coli alkaline phosphatase reporter system for screening affinity peptides

An enzyme-linker-peptide fusion protein reporter system was constructed for sensitive analysis of affinity of peptide ligands to their receptor. An E. coli alkaline phosphatase (EAP) mutant enzyme with high catalytic activity was selected as the reporter protein. Interaction of affinity peptide and streptavidin was applied as demonstration of the method. Three affinity peptides, strep-tag I (SI), strep-tag II (SII) and streptavidin binding peptide (SBP) were genetically fused to the C-terminal of EAP respectively, with an insertion of a flexible linker peptide in between. The enzyme activity of the EAP fusions showed no obvious change. After expression and purification, the EAP-affinity peptide fusions were applied to the streptavidin modified surface. Binding of the fusions to the surface through interaction of affinity peptides to streptavidin was indicated by color generated from conversion of the substrate by EAP. The relative affinity and specificity of each affinity peptides to the immobilized streptavidin were then evaluated with high sensitivity and broad detection range. This method may be used for effective high-throughput screening of high affinity peptide from the peptide pool.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Recombinant human insulin. VIII. Isolation of fusion protein--S-sulfonate, biotechnological precursor of human insulin, from the biomass of transformed Escherichia coli cells

Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure.This structure causes highly efficient fusion protein folding.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

The non-specificity of dog serum albumin and the N-terminal model peptide glycylglycyl-L-tyrosine N-methylamide for nickel is due to the lack of histidine in the third position.

Equilibrium dialysis of dog serum albumin (DSA) against Ni(II) in 0.1 M-N-ethylmorpholine/HCl, pH 7.53, demonstrates the absence of a specific Ni(II)-binding site in DSA. To evaluate at the molecular level the influence of the genetic substitution of L-tyrosine for L-histidine at the N-terminal of DSA, a simple model tripeptide of the N-terminal residues, glycylglycyl-L-tyrosine N-methylamide, was synthesized and its Ni(II)-binding properties studied. A comparison of the visible absorption characteristics of Ni(II)-DSA with those of Ni(II)-glycylglycyl-L-tyrosine N-methylamide reveals a similar change from octahedral to planar co-ordination as the pH is increased. Both systems exhibit a low Ni(II)-binding affinity at physiological pH, with DSA binding a greater percentage of Ni(II) owing to the availability of at least two binding sites of similar affinities. The complex equilibria between Ni(II) and glycylglycyl-L-tyrosine N-methylamide were studied by analytical potentiometry (0.15 M-NaCl, 25 degrees C). Four major complex species, MHA, MH-1A2, MH-2A2 and MH-3A [where M and A represent Ni(II) ion and anionic peptide respectively], were detected, MHA being the single species at physiological pH. There is no evidence for the involvement of the phenolic hydroxy group in the octahedral MHA complex, or within the plane of co-ordination in the high-pH species. The results provide direct evidence that the low Ni(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the N-terminal region of the protein.     

Effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor (Cys17Ser) in aqueous two-phase systems containing chelated metal ions

High-level expression of recombinant proteins in Escherichia coli frequently leads to the formation of insoluble protein aggregates, termed inclusion bodies. In order to recover a native protein from inclusion bodies, various protein refolding techniques have been developed. Column-based refolding methods and refolding in aqueous two-phase systems are often an attractive alternative to dilution refolding due to simultaneous purification and improved refolding yields. In this work, the effect of surface histidine mutations and their number on the partitioning and refolding of recombinant human granulocyte-colony stimulating factor Cys17Ser variant (rhG-CSF (C17S)) from solubilized inclusion bodies in aqueous two-phase systems polyethylene glycol (PEG)-dextran, containing metal ions, chelated by dye Light Resistant Yellow 2KT (LR Yellow 2KT)-PEG derivative, was investigated. Human G-CSF is a growth factor that regulates the production of mature neutrophilic granulocytes from the precursor cells. Initially, the role of His156 and His170 residues in the interaction of rhG-CSF (C17S) with Cu(II), Ni(II) and Hg(II) ions, chelated by LR Yellow 2KT-PEG, was investigated at pH 7.0 by means of affinity partitioning of purified, correctly folded rhG-CSF (C17S) mutants. It was determined that both His156 and His170 mutations reduced the affinity of rhG-CSF (C17S) for chelated Cu(II) ions at pH 7.0. His170 mutation significantly reduced the affinity of protein for chelated Ni(II) ions. However, histidine mutations had only a small effect on the affinity of protein for Hg(II) ions. The influence of His156 and His170 mutations on the refolding of rhG-CSF (C17S) from solubilized inclusion bodies in aqueous two-phase systems PEG-dextran, containing chelated Ni(II) and Hg(II) ions, was investigated. Reversible interaction of protein mutants with chelated metal ions was used for refolding in aqueous two-phase systems. Both histidine mutations resulted in a significant decrease of protein refolding efficiency in two-phase systems containing chelated Ni(II) ions, while in the presence of chelated Hg(II) ions their effect on protein refolding was negligible. Refolding studies of rhG-CSF variants with different number of histidine mutations revealed that a direct correlation exists between the number of surface histidine residues and refolding efficiency of rhG-CSF variant in two-phase systems containing chelated Ni(II) ions. This method of protein refolding in aqueous two-phase systems containing chelated metal ions should be applicable to other recombinant proteins that contain accessible histidine residues.     

Ni(II) and Cu(II) binding with a 14-aminoacid sequence of Cap43 protein, TRSRSHTSEGTRSR

The tetradecapeptide containing the 10 aminoacid repeated sequence on the C-terminus of the Ni(II)-induced Cap43 protein, was analyzed for Ni(II) and Cu(II) binding. A combined pH-metric and spectroscopic UV-VIS, EPR, CD and NMR study of Ni(II) and Cu(II) binding to the blocked CH3CO-Thr-Arg-Ser-Arg-Ser-His-Thr-Ser-Glu-Gly-Thr-Arg-Ser-Arg-NH2 (Ac-TRSRSHTSEGTRSR-Am) peptide, modeling a part of the C-terminal sequence of the Cap43 protein, revealed the formation of octahedral complexes involving imidazole nitrogen of histidine, at pH 5.5 and pH 7 for Cu(II) and Ni(II), respectively; a major square planar 4N-Ni(II) complex (about 100% at pH 9, log K* = -28.16) involving imidazole nitrogen of histidine and three deprotonated amide nitrogens of the backbone of the peptide was revealed; a 3N-Cu(II) complex (maximum about 70% at pH 7, log K*=-13.91) and a series of 4N-Cu(II) complexes starting at pH 5.5 (maximum about 90% at pH 8.7, log K* = -21.39 for CuH(-3)L), were revealed. This work supports the existence of a metal binding site at the COOH-terminal part of the Cap43 peptide.     

Sequence-specific Ni(II)-dependent peptide bond hydrolysis in a peptide containing threonine and histidine residues

Previously we demonstrated that Ni(II) complexes of Ac-Thr-Glu-Ser-His-His-Lys-NH2 hexapeptide, representing residues 120-125 of human histone H2A, and some of its analogs undergo E-S peptide bond hydrolysis. In this work we demonstrate a similar coordination and reactivity pattern in Ni(II) complexes of Ac-Thr-Glu-Thr-His-His-Lys-NH2, its threonine analogue, studied using potentiometry, electronic absorption spectroscopy and HPLC. For the first time we present the detailed temperature and pH dependence of such Ni(II)-dependent hydrolysis reactions. The temperature dependence of the rate of hydrolysis yielded activation energy E(a) = 92.0 kJ mol(-1) and activation entropy DeltaS# = 208 J mol(-1) K(-1). The pH profile of the reaction rate coincided with the formation of the four-nitrogen square-planar Ni(II) complex of Ac-Thr-Glu-Thr-His-His-Lys-NH2. These results expand the range of protein sequences susceptible to Ni(II) dependent cleavage by those containing threonine residues and permit predictions of the course of this reaction at various temperatures and pH values.     

Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins

The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The difficulty in obtaining this enzyme led us to look for an optimal method for its use. In this work, we produced both the wild-type and the S219V mutant TEV proteases fused to the Streptag II affinity sequence (Streptag II-TEV(WT), and Streptag II-TEV(S219V), respectively). The two enzymes were affinity immobilized on a streptavidin-agarose matrix and compared to their respective free forms. Both immobilized Streptag II-TEV(WT) and Streptag II-TEV(S219V) were active on the 74-kDa Streptag II substrate with a retained activity of 83.5% and 81%, respectively compared to their free corresponding forms. The slight enzyme activity decrease caused by the immobilization was balanced by the enhanced stability and the successful repetitive use of the proteolytic columns. Thus, the wild-type and the mutant immobilized proteases were used, during a period of 18 months, for nine batch reactions with retention of 38% and 51% of their initial activities, respectively. The present results demonstrate that immobilized TEV protease on streptavidin-agarose is an attractive and efficient tool for fusion protein cleavage, especially when the target protein is fused to a streptagged fusion partner. Using this strategy, the total process can be shortened by performing the cleavage and the recovery of the purified target protein in one step.     

Microbial bio-production of a recombinant stimuli-responsive biosurfactant

Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states.     

Synthesis of peptide gels for the investigation of oligopeptide-oligonucleotide interactions

Peptide gels usable as protein model systems have been synthesized by a cross-linking copolymerization of acryloyl substituted peptides with 1,4-tetramethylene dimethacrylate. A specially adapted approach to peptide synthesis allows the removal of the amino terminal Cbo group at the end of the peptide synthesis, followed by the introduction of an acryloyl group. The polymerizable peptide monomers obtained can be transferred into insoluble peptide gels by radical copoylmerization with cross-linking agents. After cleavage of the protecting groups of the side chains, these peptide gels can be used both as protein model systems for investigating peptide–oligonucleotide interaction and as sorbents for affinity chromatography. The preparation and characterization of the peptide gels Ala-Lys-Glu-Lys-Ala-OMe (I), Ala-Arg-Glu-Arg-Ala-OMe (II), Ala-Arg-Glu-Lys-Ala-OMe (III), and Ala-Arg-Ala-Lys-Ala-OMe (IV) as well as the conditions for the removal of the protecting groups is presented. Gel III contains the natural peptide sequence Arg-Glu-Lys while the other gels are analogs of this sequence.     

Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.     

A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation

The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (approximately 95 kDa) at pH approximately 7, allowing a rapid and clean separation from the carrier thioredoxin (approximately 14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 degrees C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.     

Activation of soluble guanylyl cyclase by four-coordinate metalloporphyrins: evidence for a role for porphyrin conformation

Four-coordinate metalloporphyrins activate soluble guanylyl cyclase. Ni(II)PPIX and Cu(II)PPIX are high affinity activators, with activation constants of 24 and 17 nM, respectively. Both metalloporphyrins remain stably bound to the enzyme, enabling spectroscopic characterization of the Ni(II)- and Cu(II)-reconstituted protein. Electronic absorption and resonance Raman spectroscopy reveal that Ni(II)PPIX remains four coordinate when bound to soluble guanylyl cyclase. Analysis of the vibrational frequencies of the Ni(II)-reconstituted enzyme suggests that the protein imposes a constraining force on the porphyrin, favoring a planar conformation. Spectroscopic data for the Cu(II)-substituted protein are also consistent with four coordination. The intensification of the vibrational modes of the peripheral vinyl groups in both Ni(II)- and Cu(II)-reconstituted soluble guanylyl cyclase are consistent with a substantial influence of the protein on the porphyrin environment. Together these data support a model where activation of soluble guanylyl cyclase correlates with the absence of a metal-to-proximal histidine bond and with decreased conformational freedom for the tetrapyrrole in the activated state.     

Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.

A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag. This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein). A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification. Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage. The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column. This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine. We have constructed general cloning vectors and demonstrated single-column purification of several proteins. In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity.     

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.