Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography

Phosphoproteome studies are hampered by the lack of methods which allow a comprehensive and fast analysis of intact phosphoproteins. Here we describe an immobilized metal-affinity chromatography (IMAC)-based technique for the enrichment of phosphorylated proteins, which allows recovery of up to 90% of phosphoproteins. This technique is compatible with 2-DE and can be applied to cultured cells and tissues.     

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.     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme     

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.     

Development of immobilized Sn4+ affinity chromatography material for highly selective enrichment of phosphopeptides

In this work, we first immobilized tin(IV) ion on polydopamine‐coated magnetic graphene (magG@PDA) to synthesize Sn⁴⁺‐immobilized magG@PDA (magG@PDA‐Sn⁴⁺) and successfully applied the material to highly selective enrichment of phosphopeptides. The material gathered the advantages of large surface area of graphene, superparamagnetism of Fe₃O₄, good hydrophilicity and biocompatibility of polydopamine, and strong interaction between Sn⁴⁺ and phosphopeptides. The enrichment performance of magG@PDA‐Sn⁴⁺ toward phosphopeptides from digested β‐casein at different concentrations, with and without added digested BSA was investigated and compared with magG@PDA‐Ti⁴⁺. The results showed high selectivity and sensitivity of the Sn⁴⁺‐IMAC material toward phosphopeptides, as good as the Ti⁴⁺‐IMAC material. Finally, magG@PDA‐Sn⁴⁺ was applied to the analysis of endogenous phosphopeptides from a real sample, human saliva, with both MALDI‐TOF MS and nano‐LC‐ESI‐MS/MS. The results indicated that the as‐synthesized Sn⁴⁺‐IMAC material not only has good enrichment performance, but also could serve as a supplement to the Ti⁴⁺‐IMAC material and expand the phosphopeptide coverage enriched by the single Ti⁴⁺‐IMAC material, demonstrating the broad application prospects of magG@PDA‐Sn⁴⁺ in phosphoproteome research.     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Immobilized metal affinity chromatography in the presence of arginine

Arginine hydrochloride (ArgHCl) is a versatile solvent additive, as it suppresses protein aggregation. ArgHCl has been used for protein refolding and to solubilize proteins from loose inclusion bodies. Immobilized metal affinity chromatography (IMAC) is one of the most commonly used technologies for purification of recombinant proteins. Here we have evaluated compatibility of ArgHCl with IMAC purification for his-tag proteins. ArgHCl clearly interfered with protein binding to Ni-columns. Nevertheless, such interference was greatly reduced at ArgHCl concentration below 200 mM, demonstrating that IMAC purification can be done even in the presence of ArgHCl.     

Review: Multipoint binding and heterogeneity in immobilized metal affinity chromatography

Studies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal-coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple-site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. (c) 1995 John Wiley & Sons, Inc.     

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。     

Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography

While immobilized metal affinity chromatography (IMAC) has been widely used for affinity purification of phosphopeptides, the technique suffers from insufficient specificity. Therefore, there is an urgent need for IMAC optimization to yield the selectivity and sensitivity that is required for more challenging analyses. Recently, 2,5-dihydroxybenzoic acid (DHB) and phosphoric acid mixture has been reported as an efficient IMAC eluant. The disadvantage of DHB is that is not suitable for electrospray ionization-mass spectrometry. While further developing the IMAC elution protocol to overcome this problem, we noticed that DHB is not necessary and found a novel combination of phosphoric acid and acetonitrile to be more efficient. The purification efficacy of the novel protocol is superior to all previously described methods, while still being compatible with the most commonly used mass-spectrometric techniques in phosphoproteomics.     

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.     

Proteomic survey of copper-binding proteins in Arabidopsis roots by immobilized metal affinity chromatography and mass spectrometry

To plants, copper is vitally essential at low concentrations but extremely toxic at elevated concentrations. Plants have evolved a suite of mechanisms that modulate the uptake, distribution, and utilization of copper ions. These mechanisms require copper-interacting proteins for transporting, chelating, and sequestrating copper ions. In this study, we have systematically screened for copper-interacting proteins in Arabidopsis roots via copper-immobilized metal affinity chromatography (Cu-IMAC). We also compared Arabidopsis root metalloproteomes with affinity to Cu-IMAC and Zn-IMAC. From the identities of 38 protein spots with affinity to Cu-IMAC, 35 unique proteins were identified. Functional classification of these proteins includes redox/hydrolytic reactions, amino acid metabolism, glutathione metabolism, phosphorylation, translation machinery, membrane-associated proteins, and vegetative storage proteins. Potential copper-interacting motifs were predicted and scored. Six candidate motifs, H-(X)5 -H, H-(X)7 -H, H-(X)12 -H, H-(X)6 -M, M-(X)7 -H, and H-(X)3 -C, are present in Cu-IMAC-isolated proteins with higher frequency than in the whole Arabidopsis proteome.     

Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: separation of synthetic prion peptides

Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of nonporous polymeric beads with the goal of developing a new immobilized metal affinity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin-nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)(2), and (PHGGGWGQ)(4). Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q-->A substitution, or amide nitrogen methylation.     

Purification of penicillin G acylase using immobilized metal affinity membranes

The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.     

Facile preparation of magnetic graphene double‐sided mesoporous composites for the selective enrichment and analysis of endogenous peptides

In this work, magnetic graphene double‐sided mesoporous nanocomposites (mag‐graphene@mSiO₂) were synthesized by coating a layer of mesoporous silica materials on each side of magnetic grapheme. The surfactant (CTAB) mediated sol‐gel coating was performed using tetraethyl orthosilicate as the silica source. The as‐made magnetic graphene double‐sided mesoporous silica composites were treated with high‐temperature calcination to remove the hydroxyl on the surface. The novel double‐sided materials possess high surface area (167.8 cm²/g) and large pore volume (0.2 cm³/g). The highly open pore structure presents uniform pore size (3.2 nm) and structural stability. The hydrophobic interior pore walls could ensure an efficient adsorption of target molecules through hydrophobic–hydrophobic interaction. At the same time, the magnetic Fe₃O₄ particles on both sides of the materials could simplify the process of enrichment, which plays an important role in the treatment of complex biological samples. The magnetic graphene double‐sided nanocomposites were successfully applied to size‐selective and specific enrichment of peptides in standard peptide mixtures, protein digest solutions, and human urine samples. Finally, the novel material was applied to selective enrichment of endogenous peptides in mouse brain tissue. The enriched endogenous peptides were then analyzed by LC‐MS/MS, and 409 endogenous peptides were detected and identified. The results demonstrate that the as‐made mag‐graphene@mSiO₂ have powerful potential for peptidome research.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Purification of papain by immobilized metal affinity chromatography (IMAC) on chelating carboxymethyl cellulose

Chelating carboxymethyl cellulose was prepared in bead form by immobilizing iminodiacetic acid on carboxymethyl cellulose which was earlier crosslinked and activated by epichlorohydrin. The prepared matrix was used to purify papain by a factor of 2.6 from commercial papain, and by a factor of 4 from papaya latex by batch adsorption and immobilized metal affinity chromatography respectively. Purification factors obtained were equal in batch mode and double in column mode, to purifications obtained on Chelating Sepharose® Fast Flow. Flow rates up to 38 ml/cm2 h were easily possible on the prepared chelating carboxymethyl cellulose.     

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.     

固化金属离子亲和层析富集磷酸化多肽方法的选择及优化

在磷酸化蛋白质组学研究中,根据是否需要对待富集样品进行甲酯化处理,可将固化金属离子亲和层析（IMAC）方法分为两类,即需要甲酯化处理的IMAC方法（ME—IMAC）和不需要甲酯化处理的IMAC方法（Non—ME—IMAC）。要实现对磷酸化多肽的有效富集和鉴定,就必须对富集方法进行选择和优化。利用基质辅助激光解析离子化串联飞行时间质谱（MALDI—TOF—MS）对两种方法富集的磷酸化多肽进行了比较研究。结果表明,ME—IMAC方法容易发生样品丢失,质谱结果的分析也比较复杂,而Non—ME—IMAC方法则不仅操作简单而且富集效果理想。另外,优化了Non—ME—IMAC方法的实验条件,指出最佳的结合溶液是8％ACN／0．3％TFA,最佳的洗脱溶液是0．1mol／LEDTA（pH值8．0）,从而建立了一套完整而简单有效的磷酸化多肽富集方法。     

An oriented adsorption strategy for efficient solid phase PEGylation of recombinant staphylokinase by immobilized metal-ion affinity chromatography

Conjugation of truncated recombinant staphylokinase (trSak) with polyethylene glycol (PEG) is an effective way to overcome its short plasma half-life and enhance its therapeutic potential. However, conventional amine directed PEGylation chemistry inevitably led to modification at its functionally important N terminus, which resulted in a significantly reduced bioactivity of trSak. In this study, a novel solid phase PEGylation process was developed to shield the N-terminal region of the protein from PEGylation. The process was achieved by oriented adsorption of an N-terminally His-tagged trSak (His-trSak) onto an immobilized metal-ion affinity chromatography (IMAC). His-trSak was efficiently separated and retained on IMAC media before reaction with succinimidyl carbonate mPEG (SC-mPEG, 5, 10 or 20 kDa). The IMAC derived mono-PEGylated His-trSak showed structural and stability properties similar to the liquid phase derived conjugate. However, isoelectric focusing electrophoresis analysis revealed that mono-PEGylated His-trSaks via solid phase PEGylation were more homogeneous than those from liquid phase PEGylation. Moreover, tryptic peptide mapping analysis suggested that a complete N-terminal blockage of IMAC bound His-trSak from PEGylation with 10 kDa- and 20 kDa-SC-mPEG. In contrast, only partial protection of the N-terminal region was obtained for 5 kDa-SC-mPEG. Bioactivities of 10 kDa- and 20 kDa-PEG-His-trSak conjugates without N-terminal PEGylation were significantly higher than those of randomly PEGylated products. This further demonstrated the advantage of our new on-column PEGylation strategy.