Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography

Electrospray ionization mass spectrometry (ESI/MS) affords a rapid and sensitive technique for determining peptides produced by the enzymatic digestion of phosphoroteins. When coupled with on-line immobilized metal-ion affinity chromatography (IMAC), the combmation allows separation and mass spectrometric identification of phosphorylated and nonphosphorylated peptides. In this study, the feasibility and general applicability of on-line IMAC/ESI/MS is investigated by using immobilized ferric ions for selective chelation of several phosphotyrosine and phosphoserine peptides. The sensitivity and practicality of the technique for phosphoproteins are demonstrated via the analysis of 30 pmol (～0.7 μg) of bovine β-casein purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, and digested in situ with trypsin. It is observed that on-line IMAC/ESI/MS suffers less from sample losses than experiments performed off-line, suggesting that the limiting factors in sensitivity for this technique are the purification procedures and sample handling rather than the IMAC and mass spectrometry. Thus, the ability to inject the tryptic digest of an electroblotted protein directly onto the column without buffer exchange and to analyze the eluent directly via on-line coupling of the IMAC column to the mass spectrometer greatly reduces sample losses incurred through sample handling and provides a convenient method for analyzing phosphopeptides at low levels.     

Immobilized metal-ion affinity chromatography of recombinant Fab protein OPG C11 in the presence of EDTA-Mg(II)

Undesired adsorption of host cell proteins poses a big challenge for immobilized metal-ion affinity chromatography (IMAC) purification. In this study, by using His6-tagged protein Fab OPG C11 from Escherichia coli fermentation as a model, we found that the presence of low concentrations of EDTA-Mg2+ in feed streams weakens the adsorption but makes it more specific towards polyhistidine tag. By combining EDTA-Mg2+ treatment and periplasmic extraction, we developed a one-step purification procedure for His6-tagged recombinant Fab OPG C11 using Ni-IDA (iminodiacetic acid) chromatography. This procedure eliminated the buffer exchange step after periplasmic extraction, which is usually required before IMAC in order to remove EDTA. In addition to savings on time and cost, this procedure eliminates undesired adsorption of most host cell proteins thus significantly improves the purity of polyhistidine-tagged recombinant proteins. The strategy of EDTA-Mg2+ treatment may have general application potentials.     

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

Purification of alpha-L-fucosidase from various sources by affinity chromatography.

An affinity column for alpha-L-fucosidases was constructed by linking p-amino-phenyl 1-thio-alpha-L-fucopyranoside to Sepharose 4B through linkers of succinyl 3,3'-diamino-dipropylamine. Excellent purification of alpha-L-fucosidase from rat epididymis, Clostridium perfringens and Limulus polyphemus (horse shoecrab) could be effected inone step with good yield. An affinity column purification step can be introduced at any point in published purification procedures. The purified enzyme is essentially free of other glycosidases and proteolytic enzymes. The column material is stable and can be reused for at least two years.     

Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.     

Microcalorimetric Study of the Effect of Hexa-histidine Tag and Denaturant on the Interaction Mechanism between Protein and Metal-Chelating Gel

A recombinant protein, Schistosoma japonicum glutathione-S-transferase (SjGST), was fused with a C-terminal hexa-histidine tag to obtain SjGST/His. Both proteins were used to probe the interaction mechanisms with the metal ions immobilized on chromatography gels. Isothermal titration calorimetry was used to directly measure the adsorption enthalpies (DeltaH(ads)) of both proteins with Ni-NTA and TALON (Co(2+)) commercial affinity resins, under the conditions of with and without the presence of a denaturant. The result reveals that SjGST/His had a lower DeltaH(ads) value with Ni-NTA than did SjGST, mainly attributed to the formation of more coordination bonds with or a stronger binding with Ni-NTA. Furthermore, the difference between the DeltaH(ads) values of SjGST/His onto TALON under the nature and denaturing conditions were insignificant, implying that the binding topography of the hexa-histidine tail with immobilized Co(2+) was not significantly changed with the presence of a denaturant. In addition, this study shows that the proposed binding models and the directly measured adsorption heat can be combined to elucidate the difference in the interaction mechanisms of SjGST/His adsorption onto those two adsorbents from a thermodynamic perspective. Copyright 2001 Academic Press.     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes

Three anthraquinone glutathionyl-biomimetic dye ligands, comprising as terminal biomimetic moiety glutathione analogues (glutathionesulfonic acid, S-methyl-glutathione and glutathione) were synthesised and characterised. The biomimetic ligands were immobilised on agarose gel and the affinity adsorbents, together with a nonbiomimetic adsorbent bearing Cibacron Blue 3GA, were studied for their purifying ability for the glutathione-recognising enzymes, NAD+-dependent formaldehyde dehydrogenase (FaDH) from Candida boidinii, NAD(P)+-dependent glutathione reductase from S. cerevisiae (GSHR) and recombinant maize glutathione S-transferase I (GSTI). All biomimetic adsorbents showed higher purifying ability for the target enzymes compared to the nonbiomimetic adsorbent, thus demonstrating their superior effectiveness as affinity chromatography materials. In particular, the affinity adsorbent comprising as terminal biomimetic moiety glutathionesulfonic acid (BM1), exhibited the highest purifying ability for FaDH and GSTI, whereas, the affinity adsorbent comprising as terminal biomimetic moiety methyl-glutathione (BM2) exhibited the highest purifying ability for GSHR. The BM1 adsorbent was integrated in a facile two-step purification procedure for FaDH. The purified enzyme showed a specific activity equal to 79 U/mg and a single band after sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Molecular modelling was employed to visualise the binding of BM1 with FaDH, indicating favourable positioning of the key structural features of the biomimetic dye. The anthraquinone moiety provides the driving force for the correct positioning of the glutathionyl-biomimetic moiety in the binding site. It is located deep in the active site cleft forming many favourable hydrophobic contacts with hydrophobic residues of the enzyme. The positioning of the glutathione-like biomimetic moiety is primarily achieved by the strong ionic interactions with the Zn2+ ion of FaDH and Arg 114, and by the hydrophobic contacts made with Tyr 92 and Met 140. Molecular models were also produced for the binding of BM1 and BM3 (glutathione-substituted) to GSTI. In both cases the biomimetic dye forms multiple hydrophobic interactions with the enzyme through binding to a surface pocket. While the glutathioine moiety of BM3 is predicted to bind in the crystallographically observed way, an alternative, more favourable mode seems to be responsible for the better purification results achieved with BM1.     

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.     

Purification of copper-zinc-superoxide dismutase and catalase from human erythrocytes by copper-chelate affinity chromatography

A relatively simple and reproducible procedure involving copper-chelate affinity chromatography for the isolation of copper-zinc-superoxide dismutase (E.C. 1.15.1.1) and catalase (E.C. 1.11.1.6) from human erythrocytes has been developed. Using this method, the two enzymes were easily and highly purified and appeared to be homogeneous as judged by polyacrylamide gel electrophoresis.     

Purification of soluble acetylcholinesterase from sheep liver by affinity chromatography

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium’s pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A–Sepharose 4B and edrophonium–Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5 ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21 U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04 kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5 kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.     

Comparative affinity chromatography of acetylcholinesterases from five vertebrate species

The efficacy of N-methylacridinium affinity chromatography in the purification of acetylcholinesterases from chicken, rat, calf and human brain and from the electric organ of the electric fish Torpedo marmorata has been investigated. Retention of the enzymes on the N-methylacridinium columns exceeded 90% in all instances except for the chicken enzyme, where 40-80% retention was observed depending on the acridinium concentration. Sucrose density gradient centrifugation profiles revealed no difference between the distribution of molecular forms in the crude extracts and in the partially purified fractions eluted from the columns by decamethonium iodide.     

Immobilized metal affinity chromatography as a means of fractionating microsomal cytochrome P-450 isozymes.

Fractionation of microsomal cytochrome P-450s is usually done by chromatography on ion-exchange resins and hydroxyapatite. The resolution of the great number of similar P-450 isozymes, however, requires additional methods based on different separation parameters. For this purpose immobilized-metal affinity chromatography (IMAC) was applied to the separation of P-450 isozymes. The method in its application to rat liver microsomes is described in detail. For method optimization and for the reproducibility of analytical fractionations a completely automatic fast protein liquid chromatographic system especially designed for IMAC is presented. Optimization is done with respect to the choice of the immobilized metal ion and the elution conditions. The chromatographic resolution is markedly enhanced by using segmented vs. linear gradients. The efficiency of P-450 resolution is demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, verifying the different retention behaviours of the isozymes. However, for all the isozymes analysed so far, reactivity with one particular polyclonal antibody is observed with more than two IMAC fractions of a single run. This may be explained in part by the occurrence of isozymic forms distinguishable by the pattern of chymotryptic peptides. Hence IMAC appears to be suitable for the separation of closely related isozyme forms.     

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.     

High-Speed Purification of Recombinant Interleukin-11 by IMAC with Rigid Biporous Beads

Rigid biporous beads were prepared and modified by iminodiacetic acid (IDA) for application in immobilized metal affinity chromatography of proteins. The retention behavior of four model proteins on the metal chelate columns loaded with copper (II) and nickel (II) ions were studied. The separation of the four proteins by the Ni-IDA column at 40 cm.min⁻¹ was realized within 2 min. His6-interluekin-11 (His6-IL-11) was also purified by the Ni-IDA column at 40 cm.min⁻¹. The collected His6-IL-11 fraction showed a purity of about 80%. The results indicate that the IMAC with the biporous medium is promising for high-speed protein purification.     

Isolation of naturally occurring aluminium ligands using immobilized metal affinity chromatography for analysis by ESI-MS

Aluminium (iii) is one of the most abundant metal ions found in soil. Typically, Al(+3) is bound to minerals, but its bioavailability and toxicity toward vascular plants increases with increasing soil acidity. Ectomycorrhizal fungi, which live symbiotically on the roots of numerous woody plants, often confer Al(+3) resistance to host plants by reducing metal availability to the plant by unknown mechanisms. A potential mechanism of detoxification is binding of the Al(+3) by organic compounds that are exuded by the fungi into the surrounding soil and solution. A novel method has been developed to purify and characterize Al(+3) binding ligands from Pisolithus tinctorius exudate solutions using Al(+3) immobilized metal affinity chromatography (IMAC), reversed phase chromatography, and mass spectrometry. Fungal exudates produced by P. tinctorius exhibit a strong binding capacity for Al(+3), allowing their selective enrichment and collection using this IMAC method. Elution of the ligands requires the use of high pH. RP-HPLC separation and elemental analysis of the IMAC elutent indicates that the Al(+3) and the exudate ligands both elute from the column but are not bound in a complex. Thus, reversed phase HPLC at pH 10 is used for separation of the ligands and Al(+3) prior to MS analysis. The strongest binding IMAC fraction is analyzed by electrospray ionization mass spectrometry in positive and negative ion modes. This report provides new methods for the direct purification and analysis of naturally occurring ligands that bind hard metal ions.     

Trypsin and affinity chromatography.

Affinity adsorbents for trypsin which were prepared by immobilizing product-type ligands, that is, peptides having C-terminal arginine, proved to be effective not only for preparative purposes but also for basic research on molecular recognition. The properties of the binding site of trypsin were revealed by chromatographic experiments. Quantitative analysis based on the theory of frontal affinity chromatography proved to be extremely effective. As an extension of the product-type ligands, peptide argininals were also used and information on the mechanism of action of these inhibitors was obtained. Anhydrotrypsin, which lost the hydroxyl group of Ser183, was found to gain increased binding ability for product-type compounds. This inactivated enzyme was also used as an immobilized ligand and the unique affinity adsorbent thus prepared proved to be extremely effective for the separation of peptides and recombinant proteins based on their C-terminal structures. High-performance affinity chromatography of trypsin and related enzymes using a polymer-based support was also developed.     

Thermosensitive copolymers of N-vinylimidazole as displacers of proteins in immobilised metal affinity chromatography

Synthetic copolymers of N-vinylcaprolactam (VCL) and N-vinylimidazole (VI) were studied as thermosensitive, reusable displacers for immobilised metal affinity chromatography (IMAC) of proteins. The copolymer with weight-average molecular mass of 11700 g/mol prepared by free radical polymerisation at a 9:1 monomer molar ratio was separated into several fractions by IMAC and thermal precipitation. The fraction with an average VI content of 8.5% was most efficient as a reusable displacer for IMAC of ovalbumin, lysozyme and other proteins of egg white on Cu2+-IDA-Sepharose. The displacer exhibited a sharp breakthrough curve and binding capacity of 16-20 mg/ml gel, depending on the flow-rate. The recovery of egg white proteins in the course of displacement chromatography was >95%. The displacer could be removed quantitatively from the protein fractions by thermal precipitation at 48 degrees C. Co-precipitation of lysozyme with the displacer was minimal in the presence of 3% (v/v) acetonitrile, while the lysozyme enzymatic activity in the supernatant was completely retained. Addition of free imidazole to the mobile phase increased the rate of protein desorption and allowed better separation of egg white proteins and the displacer in the course of chromatography. The displacement profile of the egg white extract consisted of three zones with different distributions of individual proteins characterised by SDS-PAGE. Regeneration of the column was easily performed with 0.02 M EDTA in 0.15 M sodium chloride, pH 8.0, followed by washing with distilled water and reloading with Cu2+. The displacer could also be regenerated by thermal precipitation at 48 degrees C and subsequent dialysis against dilute hydrochloric acid (pH 2.5).