Immobilized Metal Affinity Chromatography Resin Research Articles

Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes.

Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (GstCeuE and PthCeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with CjCeuE, the analogous protein of Campylobacter jejuni. Upon immobilization, the GstCeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, while retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles, a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilized protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilized ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilization of redox-reversible ArMs facilitates their "catch-and-release" assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that ‘soft’ proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.

Use of tandem affinity-buffer exchange chromatography online with native mass spectrometry for optimizing overexpression and purification of recombinant proteins.

Purification of recombinant proteins typically entails overexpression in heterologous systems and subsequent chromatography-based isolation. While denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is routinely used to screen a variety of overexpression conditions (e.g., host, medium, inducer concentration, post-induction temperature and/or incubation time) and to assess the purity of the final product, its limitations, including aberrant protein migration due to compositional eccentricities or incomplete denaturation, often preclude firm conclusions regarding the extent of overexpression and/or purification. Therefore, we recently reported an automated liquid chromatography-mass spectrometry-based strategy that couples immobilized metal affinity chromatography (IMAC) with size exclusion-based online buffer exchange (OBE) and native mass spectrometry (nMS) to directly analyze cell lysates for the presence of target proteins. IMAC-OBE-nMS can be used to assess whether target proteins (1) are overexpressed in soluble form, (2) bind and elute from an IMAC resin, (3) oligomerize, and (4) have the expected mass. Here, we use four poly-His-tagged proteins to demonstrate the potential of IMAC-OBE-nMS for expedient optimization of overexpression and purification conditions for recombinant protein production.

Purification of Polyhistidine-Tagged Proteins by Immobilized Metal Affinity Chromatography.

Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni2+) immobilized as transition metal chelate complexes on a chromatography resin. The main protocol here is optimized for use of Ni2+-NTA resin to purify soluble 6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity flow for washes and elution. This protocol does not require any specialized equipment other than a simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems are designed specifically for the chromatographic separations of proteins and other biomolecules. These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity meter, pH meter, fraction collector, and other options that allow for the simultaneous purification, analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC can become a high-precision, automated instrument that separates proteins at a high resolution. An alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC. Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and, finally, considerations for storing purified proteins are discussed.

Purification of Lumbricus terrestris erythrocruorin (LtEc) with anion exchange chromatography

The naturally extracellular hemoglobin (erythrocruorin) of the Canadian nightcrawler, Lumbricus terrestris (LtEc), is a unique oxygen transport protein that may be an effective substitute for donated human blood. Indeed, this ultra-high molecular weight (~3.6 MDa) hemoglobin has already been shown to avoid the side effects associated with previous hemoglobin-based oxygen carriers and its high thermal stability (Tm = 56°C) and resistance to heme oxidation (kox = 0.04 hr−1 × 103 at 20°C) allow it to be stored for long periods of time without refrigeration. However, before it can be tested in human clinical trials, an effective and scalable purification process for LtEc must be developed. We have previously purified LtEc for animal studies with tangential flow filtration (TFF), which allows rapid and scalable purification of LtEc based on its relatively large size, but that type of size-based purification may not be able to specifically remove some impurities and high MW (>500 kDa) contaminants like endotoxin (MW = ~1–4 MDa). Anion exchange (AEX) and immobilized metal affinity chromatography (IMAC) are two purification methods that have been previously used to purify mammalian hemoglobins, but they have not yet been used to purify large invertebrate hemoglobins like LtEc. Therefore, the goal of this study was to determine if AEX and IMAC resins could successfully purify LtEc from crude earthworm homogenate, while also preserving its macromolecular structure and function. Both processes were able to produce purified LtEc with low levels of endotoxin, but IMAC purification induced significantly higher levels of heme oxidation and subunit dissociation than AEX. In addition, the IMAC process required an additional desalting step to enable LtEc binding. In contrast, AEX produced highly pure LtEc that was not dissociated. LtEc purified by AEX also exhibits similar oxygen binding characteristics (P50 = 27.33 ± 1.82 mm Hg, n = 1.58 ± 0.17) to TFF-purified LtEc (P50 = 28.84 ± 0.40 mm Hg, n = 1.93 ± 0.02). Therefore, AEX appears to be the optimal method for LtEc purification.

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography.

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

Purification of Tag-Free Chlamydia trachomatis Scc4 for Structural Studies Using Sarkosyl-Assisted on-Column Complex Dissociation

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes the most common sexually transmitted bacterial disease in the world. The bacterium has a unique biphasic developmental cycle with a type III secretion system (T3SS) to invade host cells. Scc4 is a class I T3SS chaperone forming a heterodimer complex with Scc1 to chaperone the essential virulence effector, CopN. Scc4 also functions as an RNA polymerase binding protein to regulate σ66-dependent transcription. Aggregation and low solubility of 6X-histidine-tagged Scc4 and the insolubility of 6X-histidine and FLAG-tagged Scc1 expressed in Escherichia coli have hindered the high-resolution nuclear magnetic resonance (NMR) structure determination of these proteins and motivated the development of an on-column complex dissociation method to produce tag-free Scc4 and soluble FLAG-tagged Scc1. By utilizing a 6X-histidine-tag on one protein, the coexpressed Scc4-Scc1 complex was captured on nickel-charged immobilized metal affinity chromatography resin, and the nondenaturing detergent, sodium N-lauroylsarcosine (sarkosyl), was used to dissociate and elute the non-6X-histidine-tagged protein. Tag-free Scc4 was produced in a higher yield and had better NMR spectral characteristics compared to 6X-histidine-tagged Scc4, and soluble FLAG-tagged Scc1 was purified for the first time in a high yield. The backbone structure of Scc4 after exposure to sarkosyl was validated using NMR spectroscopy, demonstrating the usefulness of the method to produce proteins for structural and functional studies. The sarkosyl-assisted on-column complex dissociation method is generally applicable to protein complexes with high affinity and is particularly useful when affinity tags alter the protein's biophysical properties or when coexpression is necessary for solubility.

Phosphopeptide enrichment with cross-linked Os(II)(dmebpy)2 Cl-derivatized acrylamide and vinylimidazole copolymer.

Reversible phosphorylation of proteins catalyzed by kinases and phosphatases plays a key regulatory role in intracellular biological processes. Protein phosphorylation profiling is still a challenge due to its low stoichiometry, diversity of phosphorylated protein forms, and dynamic nature of phosphorylation states. Mass spectrometry (MS) has been widely used for the characterization of protein phosphorylation, due to its high sensitivity and MS/MS sequencing capability. However, the low abundance and ionization efficiency of phosphorylated peptides and interference from their non-phosphorylated counterparts and other peptides in the enzymatic digests of proteins complicate the localization of phosphorylation sites in liquid chromatography (LC)/MS analysis. So the enrichment of phosphopeptides from the digests is often required before LC/MS. Immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) are the two most commonly used enrichment techniques for phosphopeptides prior to MS analysis. Cross-linked Os(II)(4,4'-dimethyl-2,2'-bipyridine)2 Cl-derivatized acrylamide and vinylimidazole copolymer were applied for the enrichment of phosphopeptides. Under neutral loading buffer conditions phosphopeptides bind on the Os-polymer without nonspecific binding of acidic peptides. Differential binding of monophosphorylated and multiply phosphorylated peptides can be achieved under different concentrations of imidazole. Sequential elution of bound phosphopeptides can be obtained with elution buffers of different pH values below 3. The loading buffers with imidazole can be aqueous or 7/3 H2 O/ACN. Once phosphopeptides bind onto the Os-polymer, washing with water, 0.1% acetic acid (pH~3) or 1/1 H2 O/ACN 0.05% acetic acid (pH ~3) does not elute phosphopeptides. The Os-polymer does not show bias of binding and elution toward phosphopeptide standards with singly, doubly and triply phosphorylated sites. Cross-linked Os(II)(dimethylbipyridine)2 Cl-derivatized poly(acrylamide)-poly(vinylimidazole) copolymer is proven to be a new efficient IMAC resin for phosphopeptide enrichment and shows some unique properties for differential binding and sequential elution of phosphopeptides. It could become a better alternative to traditional IMAC and TiO2 for phosphopeptide enrichment.

Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.

Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.

The impact of immobilized metal affinity chromatography (IMAC) resins on DNA aptamer selection

DNA aptamers are single-stranded oligonucleotides which can form various secondary and tertiary structures. They can recognize a broad range of targets ranging from small molecules, such as ions, vitamins, antibiotics, to high molecular weight structures, including enzymes and antibodies. DNA aptamers are extensively studied as a potential source of new pharmaceutical drugs due to their inexpensive synthesis, low immunogenicity, and high specificity. The commonly used aptamer selection procedure is systematic evolution of ligands by exponential enrichment (SELEX) where the target molecule is immobilized on an appropriate chromatography resin. For peptide/protein targets, immobilized metal affinity chromatography (IMAC) resins are frequently used. There is a broad range of commercially available resins which can be used for IMAC. They are characterized by different metal ions, linker types, and bead materials. In this study, we tested the impact of different IMAC resins on the DNA aptamer selection process during eight SELEX cycles. A histidine-tagged 29 amino acid peptide corresponding to the interdomain connecting loop of human proliferating cell nuclear antigen was used as a selection target. Different resin materials containing the same metal ion (Co2+) were tested. Simultaneously, agarose resins containing identical linkers, but different metal ions (Co2+, Cu2+, Ni2+, and Zn2+) were analyzed. The results of this study clearly demonstrated the impact of the metal ion and resin material on the DNA aptamer selection progress. The presented data indicate that for successful IMAC resin-based SELEX, the determination of the optimal resin might be crucial.

Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

Correction to Identification of c-Type Heme-Containing Peptides Using Nonactivated Immobilized Metal Affinity Chromatography Resin Enrichment and Higher-Energy Collisional Dissociation

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionCorrection to Identification of c-Type Heme-Containing Peptides Using Nonactivated Immobilized Metal Affinity Chromatography Resin Enrichment and Higher-Energy Collisional DissociationHaizhen Zhang, Feng Yang, Wei-Jun Qian, Roslyn N. Brown, Yuexi Wang, Eric D. Merkley, Jea H. Park, Matthew E. Monroe, Samuel O. Purvine, Ronald J. Moore, Liang Shi, James K. Fredrickson, Ljiljana Paša-Tolić, Richard D. Smith, and Mary S. Lipton*Cite this: Anal. Chem. 2012, 84, 17, 7591Publication Date (Web):September 4, 2012Publication History Published online4 September 2012Published inissue 4 September 2012https://doi.org/10.1021/ac301396bCopyright © 2012 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views248Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (106 KB) Get e-Alerts Get e-Alerts

Teaching biochemistry and molecular biology using dihydrofolate reductase as an expression system

The enzyme dihydrofolate reductase (DHFR) converts dihydrofolic acid to tetrahydrofolic acid, thereby regulating the synthesis of purine and certain amino acids. Since DHFR plays such an important role in cell proliferation and growth, it was the first enzyme targeted for chemotherapy. The objective of this project is to teach molecular and biochemical techniques by having students induce gene expression, purify and enzymatically assay for DHFR activity. During this semester‐long project, students induce expression in recombinant bacteria and purify the DHFR protein using IMAC (immobilized metal affinity chromatography) resin. Students check purity via SDS‐PAGE electrophoresis and identify the fraction (insoluble vs. soluble) containing DHFR. Additionally, they design PCR primers for site‐directed mutagenesis, verify PCR success via restriction enzyme digestion and agarose gel electrophoresis, transform into expression competent cells, and confirm the amino acid mutation by DNA sequencing. Students then examine specific activity of the wildtype DHFR protein and compare it with the mutant DHFR protein. They also determine protein concentrations using the Bradford assay and perform Western blot analysis to detect glutathione S‐transferase (GST) and histidine (His) tags. All student groups successfully purified DHFR and detected both GST and His tags by Western blot analysis. Site‐directed mutagenesis showed a decrease in specific activity ranging from 32–122% compared to the wild‐type protein. This project provides students with the opportunity to gain hands‐on experience with a multitude of laboratory techniques as well as the opportunity to design and implement cutting edge research approaches.

Recombinant expression of His-tagged saposin B and pH-dependent binding to the lipid coenzyme Q10

The use of coenzyme Q10 (CoQ10) has been increasing rapidly during recent years due to its postulated beneficial properties in human health, providing energy and antioxidant protection. There are no known negative side effects of CoQ10 even at very high levels. Recently, native saposin B (sapB) has been shown to bind CoQ10 and subsequently be excreted. It is thought that this interaction between sapB and CoQ10 could be a mechanism to avoid any possible CoQ10 toxicity. The interaction between sapB and CoQ10 is poorly understood. Here we present an increased fermentative yield of recombinant sapB and demonstrate that recombinant sapB will bind CoQ10 in a pH-dependent manner similar to sapB binding with other lipids. SapB was coated onto an IMAC (immobilized metal affinity chromatography) resin and successfully bound CoQ10 at pH 5.0 with release of the CoQ10 at pH 9.0.

Use of IMAC magnetic beads for simplified detergent screening for optimization of purification work flows of histidine‐tagged membrane proteins

Affinity purification is a powerful tool that has become a prime choice as capture step in membrane protein purification. Recombinant membrane proteins are commonly purified by use of polyhistidine tags and immobilized metal affinity chromatography (IMAC).The combination of magnetic beads and IMAC resins (pre‐charged with Ni2+‐ions) allows for an easy‐to‐use format for rapid, small‐scale, screening of conditions optimal for purification of membrane proteins.Screening for optimal purification conditions for a number of integral membrane proteins was performed. Vital parameters as choice of detergent, wash and elution buffer concentrations etc were addressed. All experiments were done using Ni2+‐charged chromatography media on magnetic beadsThe magnetic bead format simplified and shortened the handling procedures through‐out the screening workflow. Obtained results from screening for several detergents gave reliable results that could be fed into further scale‐up experiments.

Enrichment of Phosphopeptides Using Biphasic Immobilized Metal Affinity-Reversed Phase Microcolumns

Immobilized metal affinity chromatography (IMAC) based on Fe (3+) or Ga (3+) chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of beta-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of beta-casein monophosphorylated peptide in 20 microL of a solution of human serum protein digest (from 200 microg total serum protein after digestion and desalting).

Fe 3O 4@Al 2O 3 magnetic core–shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe 3O 4@Al 2O 3 magnetic core–shell microspheres with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe 3O 4@Al 2O 3 magnetic core–shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe 3O 4@Al 2O 3 magnetic core–shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins β-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe 3O 4@Al 2O 3 magnetic core–shell microspheres over Fe 3+-immobilized magnetic silica microspheres, commercial Fe 3+–IMAC (immobilized metal affinity chromatography) resin, and TiO 2 beads. Finally, the Al 2O 3 coated Fe 3O 4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe 3O 4@Al 2O 3 magnetic core–shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.

Automated protein purification using His‐tagged affinity chromatography with the Profinia protein purification system.

Immobilized Metal Affinity Chromatography (IMAC) has become one of the most popular technologies for purifying recombinant proteins. Using high-capacity IMAC resins, His-tagged proteins can be obtained at high yields from crude extracts without prior removal of other cellular components. An appealing feature of IMAC is that a standardized protocol can be applied to a diverse range of His-tagged proteins, which allows efficient purification for proteomics research and protein structural studies. Difficulties commonly encountered in His-tagged protein expression and purification includes low expression level and low solubility of over expressed proteins. The Profinia protein purification system, an automated low pressure chromatography system, has been used to enrich both poorly expressed and low solubility proteins, resulting in up to 7.5 mg of target protein that is more than 90 % pure from 500 ml of bacterial culture. Profinia comes preprogrammed with standard IMAC methods, but is also customizable for optimizing purification protocols. Optimization of IMAC purification parameters for a challenging protein, and representative yield and purity data for multiple proteins will be presented.

Noncompetitive immunoassay of small analytes at the femtomolar level by affinity probe capillary electrophoresis: direct analysis of digoxin using a uniform-labeled scFv immunoreagent.

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.