Optimized operating parameters for the displacement separation of biomolecules in immobilized metal ion affinity chromatography

The ideal immobilized metal ion affinity chromatography (IMAC) model was employed to investigate the effect of operating parameters change on the displacement separation of biomolecules. By combining a lower initial mobile phase modifier (MPM) concentration and a higher final MPM concentration, the displacement chromatographic separation produced both higher concentration of feeds and better throughput in IMAC displacement separating systems.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

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.     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

Immobilized metal ion affinity chromatography

This article describes the technique of immobilized metal ion affinity chromatography (1MAC). The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The number of stationary phases that can be synthesized for efficient chclation of metal ions is unlimited, but the critical consideration is that there is enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. The versatility of IMAC is one of its greatest assets. An important contribution to the correct use of IMAC for protein purification is a simplified presentation of the various sample elution procedures.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.     

Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography

Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.     

Preparative separation of small molecular weight peptides from casein hydrolysate using gel filtration and immobilized metal ion affinity chromatography

A two step method consisting of a gel filtration step, followed by a Immobilized Metal Affinity Chromatography (IMAC) step using a IDA-Cu coupled Sephadex G-25 column, on a preparative scale is described for the group separation of peptides from a casein hydrolysate. The 48 groups of peptides thus separated are further characterised by RP-HPLC and amino acid analysis. Some peptides after the analytical RP-HPLC step are further characterised by sequencing. An insight into the mechanism of retention on IMAC of the peptides is attempted. In such complex mixtures as casein hydrolysate, the peptide-peptide interaction can mask the potential sites of interactions in a single peptide. The results obtained using volatile buffers as eluents show the possibility of using IMAC step as an alternative to obtain gram quantities of group of peptides free of salts from complex protein hydrolysates.     

Immobilized metal ion affinity chromatography.

The introduction of immobilized metal ion affinity chromatography, directed toward specific protein side chains, has opened a new dimension in protein purification. This review covers the principles and practice of IMAC that can be performed under very mild, nondenaturing conditions. IMAC is particularly suitable for preparative group fractionation of complex extracts and biofluids, but can also be used in high-performance mode: "HP-IMAC." Single-step purifications of 1000-fold or more may allow isolation of a particular protein from crude extracts on a milligram or gram scale. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography, and the immobilized metal ion ligand complexes are more likely to withstand wear and tear than are antibodies or enzymes. The enormous potential of IMAC and related metal affinity techniques is only in the initial stages of being explored and exploited. Synthesis of IMA adsorbents, and various modes of performing IMAC are discussed and exemplified with selected applications. Advantages and disadvantages are listed. Effective means of counteracting the few undesirable effects that can occur are suggested.     

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.     

Nucleic acid separations utilizing immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is widely used for protein purification, e.g., in the isolation of proteins bearing the well-known hexahistidine affinity tag. We report that IMAC matrixes can also adsorb single-stranded nucleic acids through metal ion interactions with aromatic base nitrogens and propose that metal affinity technologies may find widespread application in nucleic acid technology. Oligonucleotide duplexes, plasmid, and genomic DNA show low IMAC binding affinity, while RNA and single-stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose. The affinity of yeast RNA for IDA-chelated metal ions decreases in the following order: Cu(II), Ni(II), Zn(II), and Co(II). Adsorption isotherms for 20-mer oligonucleotide homopolymers show that purines are strongly favored over pyrimidines and that double-stranded duplexes are not bound. IMAC columns have been used to purify plasmid DNA from E. coli alkaline lysates, to purify a ribozyme, to remove primers and imperfect products from PCR reactions, and to separate 20-mer oligonucleotide duplexes containing centered single-base mismatches. Potential further applications include SNP scoring, hybridization assays, and the isolation of polyadenylated messenger RNA.     

Separation of pure and immunoreactive virus-like particles using gel filtration chromatography following immobilized metal ion affinity chromatography

A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni(2+)) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm(3). However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus.     

Selective separation of caseinophosphopeptides through immobilized metal ion affinity chromatography: effects of pH values, salt concentrations and degree of phosphorylation

A method based on immobilized metal ion affinity chromatography (IMAC) using a CS-IDA-Cu(II) gel is described for the separation of caseinophosphopeptides (CPPs) from a casein hydrolysate. According to the amino acid sequence and the degree of phosphorylation, operating parameters, such as pH values, salt concentrations for selective purification of different CPPs can be designed.     

Polymer versus monomer as displacer in immobilized metal affinity chromatography

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu²⁺-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48°C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)₆-tag [(His)₆-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu²⁺ ion. Complete elution of bound (His)₆-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)₆-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.     

Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications

Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions.     

Anti-free-radical properties of the peptide fractions isolated from string bean by immobilized metal ion affinity chromatography

In this study, peptides and proteins extracted from string bean with 1% TCA were separated by chromatography on columns with copper ions immobilized through IDA and o-phosphoserine OPS. Protein and peptide concentrations and the anti-free-radical properties of the isolated fractions were determined. Identification was obtained using mass spectrometry. The anti-free- radical activity of all analyzed samples was determined using the DPPH test, and was found to depend on reaction time, choice of chelating agent and the order in which the fractions were eluted from the column.     

Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H(+)-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.