Journal of Chromatography B (v.1021, #C)

Affinity-based separations in bioanalysis by Panagiotis Manesiotis; Georgios Theodoridis (1-2).

Antibodies as means for selective mass spectrometry by Tove Boström; Jenny Ottosson Takanen; Sophia Hober (3-13).
For protein analysis of biological samples, two major strategies are used today; mass spectrometry (MS) and antibody-based methods. Each strategy offers advantages and drawbacks. However, combining the two using an immunoenrichment step with MS analysis brings together the benefits of each method resulting in increased sensitivity, faster analysis and possibility of higher degrees of multiplexing. The immunoenrichment can be performed either on protein or peptide level and quantification standards can be added in order to enable determination of the absolute protein concentration in the sample. The combination of immunoenrichment and MS holds great promise for the future in both proteomics and clinical diagnostics. This review describes different setups of immunoenrichment coupled to mass spectrometry and how these can be utilized in various applications.
Keywords: Affinity enrichment; Antibody; Mass spectrometry; Immunoaffinity MS; Human Protein Atlas; Protein quantification; Review;

Over the years, analytical chemistry and immunology have contributed significantly to the field of clinical diagnosis by introducing quantitative techniques that can detect crucial and distinct chemical, biochemical and cellular biomarkers present in biosamples. Currently, quantitative two-dimensional hybrid immuno-analytical separation technologies are emerging as powerful tools for the sequential isolation, separation and detection of protein panels, including those with subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. One such technique to perform this challenging task is immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies and/or other affinity ligands as highly selective capture agents with the superior resolving power of capillary electrophoresis. Since affinity ligands can be polyreactive, i.e., binding and capturing more than one molecule, they may generate false positive results when tested under mono-dimensional procedures; one such application is enzyme-linked immunosorbent assay (ELISA). IACE, on the other hand, is a two-dimensional technique that captures (isolation and enrichment), releases, separates and detects (quantification, identification and characterization) a single or a panel of analytes from a sample, when coupled to one or more detectors simultaneously, without the presence of false positive or false negative data. This disruptive technique, capable of preconcentrate on-line results in enhanced sensitivity even in the analysis of complex matrices, may change the traditional system of testing biomarkers to obtain more accurate diagnosis of diseases, ideally before symptoms of a specific disease manifest.In this manuscript, we will present examples of the determination of biomarkers by IACE and the design of a miniaturized multi-dimensional IACE apparatus capable of improved sensitivity, specificity and throughput, with the potential of being used as a point-of-care instrument and holding promise for precision medicine and P4 medicine.
Keywords: Reviews; Bioanalysis; Biomarkers; Immunoaffinity capillary electrophoresis; Point-of-care instrumentation; Precision medicine; P4 medicine; Solid-phase extraction; Circulating tumor cells; Exosomes;

Molecular imprinting technology has been successfully applied to small molecular templates but a slow progress has been made in macromolecular imprinting owing to the challenges in natural properties of macromolecules, especially proteins. In this review, the macromolecular imprinting approaches are discussed with examples from recent publications. A new molecular imprinting strategy, microcontact imprinting is highlighted with its recent applications.
Keywords: Molecular imprinting; Macromolecular imprinting; Microcontact imprinting;

Affinity approaches in RNAi-based therapeutics purification by Patrícia Pereira; João A. Queiroz; Ana Figueiras; Fani Sousa (45-56).
The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.
Keywords: Affinity chromatography; Non-coding RNAs; Review; RNA purification;

Contributions of immunoaffinity chromatography to deep proteome profiling of human biofluids by Chaochao Wu; Jicheng Duan; Tao Liu; Richard D. Smith; Wei-Jun Qian (57-68).
Human biofluids, especially blood plasma or serum, hold great potential as the sources of candidate biomarkers for various diseases; however, the enormous dynamic range of protein concentrations in biofluids represents a significant analytical challenge for detecting promising low-abundance proteins. Over the last decade, various immunoaffinity chromatographic methods have been developed and routinely applied for separating low-abundance proteins from the high- and moderate-abundance proteins, thus enabling much more effective detection of low-abundance proteins. Herein, we review the advances of immunoaffinity separation methods and their contributions to the proteomic applications in human biofluids. The limitations and future perspectives of immunoaffinity separation methods are also discussed.
Keywords: Biofluid; Immunoaffinity chromatography; Plasma/Serum; Immunodepletion; Immunoenrichment; Biomarker discovery; Proteomics; Affinity proteomics;

The publications in macro-molecularly imprinted polymers have increased drastically in recent years with the development of water-based polymer systems. The macroporous structure of cryogels has allowed the use of these materials within different applications, particularly in affinity purification and molecular imprinting based methods. Due to their high selectivity, specificity, efficient mass transfer and good reproducibility, molecularly imprinted cryogels (MICs) have become attractive for researchers in the separation and purification of proteins. In this review, the recent developments in affinity based cryogels and molecularly imprinted cryogels in protein purification are reviewed comprehensively.
Keywords: Affinity purification; Molecularly imprinted cryogels; Biomacromolecules;

Discovery of bioactive compounds from complex mixtures is a challenge. In past decades, several strategies were developed and implemented for rapid and effective screening and characterization of bioactive components in complex matrices. This review mainly focused on the online strategies, which integrated the separation science, mass spectrometry, and bioactivity screening in a single platform, allowing simultaneous screening and characterization of active compounds from complex matrices, especially from the herbs. The online screening methodologies, including pre-column affinity-based screening and post-column bioassay, were discussed and their applied examples were also presented to illustrate the strengths and limitations of these approaches.
Keywords: Active components; Online screening; Chromatographic separation; Complex matrices; Herbs;

Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats by Erika L. Pfaunmiller; Jeanethe A. Anguizola; Mitchell L. Milanuk; NaTasha Carter; David S. Hage (91-100).
Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8–19 ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10 mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.
Keywords: Chromatographic immunoassay; Affinity microcolumn; Protein G; Competitive binding immunoassay; Human serum albumin;

Immunoaffinity chromatography (IAC) is an elegant and highly efficient method to isolate a particular compound from biological samples for measurement by mass spectrometry coupled to GC, CE, or LC. The utility of IAC for the quantitative determination of several prostaglandins including prostaglandin E2 (PGE2) by GC–MS/MS and LC–MS/MS has been demonstrated. The aim of the present work was to test whether the cross-reactivity of the antibody immobilized on an insoluble support can be utilized for the quantitative determination of biomolecules by stable-isotope dilution mass spectrometry. In this communication, we provide evidence that this is indeed possible for prostaglandin E1 (PGE1) in human plasma by GC–MS/MS using commercially available Sepharose 4-based IAC columns with immobilized mouse anti-PGE2 monoclonal antibody with a declared cross-reactivity of about 19% toward PGE1. Endogenous PGE1 and the internal standard [3,3′,4,4′-2H4]-PGE1 (d4-PGE1) externally added to human plasma samples were extracted by IAC, converted to their pentafluorobenzyl ester-methoxime-trimethylsilyl ether derivatives and analyzed by GC–MS/MS in the electron-capture negative-ion chemical ionization mode. Quantification was performed by selected-reaction monitoring of the mass transition m/z 526 →  m/z 258 for PGE1 and m/z 530 →  m/z 262 for d4-PGE1. By this method we measured PGE1 concentrations in EDTA plasma samples (1 mL) of six healthy volunteers in the range 10–25 pg/mL (29–72 pM). PGE1 plasma concentration showed a trend for positive correlation with plasma parameters such as low density lipoprotein (LDL)-cholesterol, total cholesterol and glucose. The method described here provides a novel tool to study the potential link of PGE1 formation to dyslipidemia, insulin resistance and related metabolic disorders.
Keywords: Cross-reactivity; Immunoaffinity column chromatography; Metabolic disorder; Stable isotopes; Tandem mass spectrometry;

The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5 μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane.
Keywords: Single-drop analysis; Captured-enzyme; Esterase; Lactate dehydrogenase; Micropurification;

In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein’s surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R 2  > 0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04–12 μM (R 2  > 0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40 nM and 132 nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX.
Keywords: AviTag; Camelid antibody; Immunoaffinity chromatography; Methotrexate; Oriented antibody immobilization;

In the present study, the feasibility of immunoaffinity chromatography (IAC) as a purification technology for the analysis of bioactive components in Traditional Chinese Medicine (TCM) was evaluated. IAC was used to analyze hesperidin (HP) and narirutin (NR) in TCM preparations containing Citri reticulatae Pericarpium (CRP, Chenpi in Chinese). An IAC column for the specific extraction and enrichment of HP and NR from TCM preparations containing CRP was developed and characterized. After HP reacted with carbonyl diimidazole and coupled to protein, it was used to immune mice for the generation of antibody. Through cell fusion, cloning and screening, monoclonal antibody was obtained. The IAC column was constructed by covalently coupling specific monoclonal antibody against HP and NR to CNBr-activated Sepharose 4B and packed into a common solid phase extraction cartridge. The extraction conditions including loading, washing and eluting, as well as flow rate for the extraction of HP and NR were optimized. Under optimal conditions, the maximum capacity, extraction recovery rate and stability of IAC column was also characterized. Results revealed that the maximum capacity of IAC column for HP and NR was approximately 16 μg and the relative binding capacity per 1 mL of the column volume was 27 μg. The extraction recovery rate of IAC column for HP and NR at three spiked levels was in the range of 94.05–109.15%. After the repeated application for 5 times, no significant loss of specific recognition was observed. Using high performance liquid chromatography (HPLC) as an effective analytic tool, HP and NR could be successfully separated via IAC column without the inference from impurities, suggesting that the extraction of HP and NR using the prepared IAC column is feasible. The application of IAC can solve the problem of quantitative analysis due to severe interference or low content. Furthermore, pretreatment methods in different matrixes can be unified. The IAC purification procedure can be used as an alternative effective analytical method for the pretreatment of bioactive components in TCM.
Keywords: Immunoaffinity chromatography; Hesperidin; Narirutin; Citri reticulatae Pericarpium; Traditional Chinese medicine; Purification technology;

Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking by Tushar S. Savane; Sanjit Kumar; Vignesh Narasimhan Janakiraman; Agamudi S. Kamalanathan; Mookambeswaran A. Vijayalakshmi (129-136).
Pseudobiospecific ligand l-histidine is an inexpensive, highly stable, non-toxic ligand explored successfully over the last twenty years for the purification of immunoglobulins in immobilised histidine ligand affinity chromatography. It is of great interest to know the molecular recognition sites of IgG to immobilized l-histidine. Here, we have used an in silico approach to explore the molecular recognition of l-histidine by IgG. We have assessed the feasible binding modes of histidine and its moieties at different sites of IgG and considered only those binding conformations which are exhibited via the imidazole ring NH group or any other OH donating group apart from the ones which are terminally conjugated with the support matrix. We categorised binding site into two categories; category I: inner binding groove and category II: surface binding groove and observed that the hinge region of IgG has most favourable binding pocket for l-histidine and histidyl moieties. Ser and Tyr residues on the hinge region make several significant interactions with l-histidine and histidyl moieties. In case of Fc region of IgG, l-histidine and histidyl moieties closely resemble the binding modes of Protein A, biomimetic ligand 22/8 and B domain of SpA to IgG. In addition to these we have also observed a significant binding site for l-histidine and histidyl moieties at Fab region of IgG.

Capsaicin and dihydrocapsaicin were selected as adulteration markers to authenticate vegetable oils. In this study, a method of immunoaffinity chromatography (IAC) combined with liquid chromatography–tandem mass spectrometry was established for the determination of capsaicin and dihydrocapsaicin in vegetable oils. In this method, immunosorbents were obtained by covalently coupling highly specific capsaicinoid polyclonal antibodieswith CNBr-activated Sepharose 4B, and then packed into a polyethylene column. In this paper, the major parameters affecting IAC extraction efficiency, including loading, washing and eluting conditions, were also investigated. The IAC column displayed high selectivity for capsaicin and dihydrocapsaicin with the maximum capacity of 240 ng. The limit of detection (LOD) and limit of quantification (LOQ) for capsaicin were calculated as 0.02 and 0.08 μg kg−1, and for dihydrocapsaicin were 0.03 and 0.10 μg kg−1. The recoveries of capsaicin and dihydrocapsaicin in oil samples were in the range of 87.3–95.2% with the relative standard deviation (RSD) of less than 6.1%. The results indicated that capsaicinoid compounds could not be found in edible vegetable oils. Therefore, the proposed method is simple, reliable and adequate for routine monitoring of capsaicinoid compounds in vegetable oils and has an excellent potential for detection of adulteration with inedible waste oil.
Keywords: Capsaicinoids; Immunoaffinity chromatography; Isotope dilution internal standard; LC–MS/MS; Inedible waste oil;

Mycotoxins usually found in agricultural products such as peanut, corn, and wheat, are a serious threat to human health and their detection requires multiplexed and sensitive analysis methods. Herein, a simultaneous determination for aflatoxin B1, B2, G1, G2, ochratoxin A, zearalanone and T-2 toxin was investigated using high performance liquid chromatography coupled with tandem mass spectrometry in a single run via a home-made multiple immunoaffinity column. Four monoclonal antibodies were produced in our lab against aflatoxins, ochratoxin A, zearalanone and T-2 toxin, respectively, then combined as a pool and bound to Sepharose-4B for affinity chromatography. Seven mycotoxins were effectively extracted from the agricultural product samples by using acetonitrile/water/acetic acid (80:19:1, v/v/v) Then, the extraction was cleanup by multiple immunoaffinity column. This method demonstrated a considerable linear range of 0.30–25, 0.12–20, 0.30–20, 0.12–20, 0.60–30, 0.30–25, and 1.2–40 μg kg−1and lower limits of detection at 0.1, 0.04, 0.1, 0.04, 0.2, 0.1 and 0.4 μg kg−1 for AFB1, AFB2, AFG1, AFG2, OTA, ZEN and T-2, respectively, in comparison with previously reported methods, as well as excellent recoveries. The mIAC capacity for AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and T-2 were 187, 181, 153, 151, 105, 130, 88 ng, respectively. It was found that all of the 7 mycotoxins were present in 90 agricultural product samples. The proposed method meets the requirements for rapid sample preparation and highly sensitive identification of multiple mycotoxins in agricultural product and food safety. This method provides a promising alternative with high throughput and high sensitivity for rapid analysis of seven mycotoxins in the monitoring of food safety.
Keywords: Multiple immunoaffinity columns; Multiple mycotoxins; High performance liquid chromatography coupled with tandem mass spectrometry;

Expedient screening for HIV-1 protease inhibitors using a simplified immunochromatographic assay by Kuntida Kitidee; Wannisa Khamaikawin; Weeraya Thongkum; Yardpiroon Tawon; Tim R. Cressey; Rachaneekorn Jevprasesphant; Watchara Kasinrerk; Chatchai Tayapiwatana (153-158).
A colloidal gold-based immunochromatographic (IC) strip test was developed and validated for the detection of HIV-1 protease (HIV-PR) activity and inhibitory effect of HIV-PR inhibitors (PIs). It is a unique ‘two-step’ process requiring the combination of proteolysis of HIV-PR and an immunochromatographic reaction. Monoclonal antibodies to the free C-terminus of HIV matrix protein (HIV-MA) conjugated to gold particles and a monoclonal antibody against intact and cleaved forms of the HIV-MA are immobilized on the ‘Test’-line of the IC strip. Using lopinavir, a potent HIV protease inhibitor, the IC-strip was optimized to detect inhibitory activity against HIV-protease. At a lopinavir concentration of 1000 ng/mL (its suggested minimum effective concentration), a HIV-PRH6 concentration of 6 mg/mL and incubation period of 60 min were the optimal conditions. A preliminary comparison between a validated high-performance liquid chromatography assay and the IC-strip to semi-quantify HIV protease inhibitor concentrations (lopinavir and atazanavir) demonstrated good agreement. This simplified method is suitable for the rapid screening of novel protease inhibitors for future therapeutic use. Moreover, the IC strip could also be optimized to semi-quantify PIs concentrations in plasma samples.
Keywords: HIV-1 protease inhibitor; Immunochromatographic strip; Semi-quantification; Rapid screening platform;

Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130 mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100 mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9 mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.
Keywords: Protein A chromatography; Dynamic binding capacity; Ligand coupling; Spacer technology; mAb adsorption;

Coenzyme-like ligands for affinity isolation of cholesterol oxidase by Yu Xin; Liushen Lu; Qing Wang; Ling Zhang; Yanjun Tong; Wu Wang (169-174).
Keywords: Cholesterol oxidase; Coenzyme-like ligand; Affinity isolation;

The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1–2 mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute–protein interactions in complex systems.
Keywords: Human serum albumin; Glycation; Fatty acids; Drug–protein binding; High-performance affinity chromatography;

Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100 ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally.
Keywords: Immobilized metal affinity chromatography; IMAC; Protein purification; 6xHis-tag; Recombinant protein; SDS; Denaturing conditions;

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies by Cong Bi; Abby Jackson; John Vargas-Badilla; Rong Li; Giana Rada; Jeanethe Anguizola; Erika Pfaunmiller; David S. Hage (188-196).
A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka ) measured by frontal analysis at pH 7.4 and 37 °C for carbamazepine with AGP was found to be 1.0 (±0.5) × 105  M−1, which agreed with a previously reported value of 1.0 (±0.1) × 105  M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.
Keywords: Alpha1-acid glycoprotein; High-performance affinity chromatography; Drug-protein binding; Entrapment; Human serum albumin;

Stoichiometric molecularly imprinted polymers for the recognition of anti-cancer pro-drug tegafur by Paula Mattos dos Santos; Andrew J. Hall; Panagiotis Manesiotis (197-203).
Molecularly imprinted polymers (MIPs) targeting tegafur, an anti-cancer 5-fluorouracil pro-drug, have been prepared by stoichiometric imprinting using 2,6-bis(acrylamido)pyridine (BAAPy) as the functional monomer. Solution association between tegafur and BAAPy was studied by 1H NMR titration, which confirmed the formation of 1:1 complexes with an affinity constant of 574 ± 15 M−1 in CDCl3. Evaluation of the synthesised materials by HPLC and equilibrium rebinding experiments revealed high selectivity of the imprinted polymer for the pro-drug vs. 5-fluorouracil and other competing analytes, with maximum imprinting factors of 25.3 and a binding capacity of 45.1 μmol g−1. The synthesised imprinted polymer was employed in solid-phase extraction of the pro-drug using an optimised protocol that included a simple wash with the porogen used in the preparation of the material. Tegafur recoveries of up to 96% were achieved from aqueous samples and 92% from urine samples spiked with the template and three competing analytes. The results demonstrate the potential of the prepared polymers in the pre-concentration of tegafur from biological samples, which could be an invaluable tool in the monitoring of patient compliance and drug uptake and excretion.
Keywords: Tegafur; 5-Fluorouracil; Bioanalysis; Molecularly imprinted polymers; Solid-phase extraction;

Monolithic macroporous cryogel imprinted with protein G was prepared using a functional co-monomer of N-methacryloyl-l-phenylalanine and 2-hydroxyethyl methacrylate. The chemical structure of the cryogel prepared was studied by FTIR-spectroscopy and its porosity was analysed using scanning electron microscopy. The cryogel was used to purify protein G from recombinant Escherichia coli cell lysate and the effect of pH, temperature, ionic strength, flow rate, etc on the adsorption of protein G to the monolithic column have been investigated. The selectivity of the imprinted cryogel was studied using protein A and myoglobin. It was possible to capture about 9 mg of Protein G per g of the cryogel.
Keywords: Macroporous; Antibodies; Molecular printing; Recombinant protein G;

Multiplexed MALDI-MS arrays for screening of MIP solid phase extraction materials by Kishore Kumar Jagadeesan; Celina Wierzbicka; Thomas Laurell; Börje Sellergren; Sudhirkumar Shinde; Simon Ekström (213-220).
Technology that facilitates rapid investigation of solid phase extraction protocols using very small amounts of sorbent can save both time and money. The microfabricated ISET (Integrated Selective Enrichment Target) interfaced with MALDI mass spectrometry is able to provide an efficient, economic and generic optimization process for SPE sample preparation. The SPE is performed in a rapid and parallel fashion, with a processing time off only 2 h per ISET with 96 samples. Each of the 96 wells on the ISET can hold 600 nL of SPE sorbent. The ability to work with small amounts of sorbent and samples in the ISET platform provides a big advantage when developing affinity sorbents, such as molecularly imprinted polymers (MIPs). Here it is demonstrated that an amount of 25 mg phosphoserine imprinted MIP (pS-MIP) sorbent can allow for analysis of more than 500 ISET nanovials using a multitude of different conditions. In the presented case, the multiplexed experiments allowed for early discovery of unspecific interactions and subsequent minimization of these, resulting in a protocol that provided improved enrichment of phosphopeptides.
Keywords: Solid-phase extraction; Mass spectrometry; Proteomics; MALDI MS; ISET;

Determination of roxithromycin from human plasma samples based on magnetic surface molecularly imprinted polymers followed by liquid chromatography-tandem mass spectromer. by Jie Ding; Fengshuang Zhang; Xiaopan Zhang; Long Wang; Changjia Wang; Qi Zhao; Yang Xu; Lan Ding; Nanqi Ren (221-228).
In this paper, a simple and reproducible method for the determination of roxithromycin in human plasma samples is proposed. The surface magnetic molecularly imprinted polymers (MMIPs) were utilized as sorbent. Roxithromycin was used as imprinted compound. The experimental results showed that the MMIPs had high affinity and selectivity toward roxithromycin. The extraction process was carried out in a single step by mixing the extraction solvent, MMIPs and human plasma samples by vortex. When the extraction process was completed, the MMIPs adsorbed the analyte were separated from the sample matrix by an external magnet due to the magnetism. The analyte eluted from the MMIPs was analysed by liquid chromatography-tandem mass spectrometry. Some main factors affecting the extraction of roxithromycin such as the amount of MMIPs, extraction solvent, extraction time, washing and elution conditions were optimized in this study. The calibration curve obtained by analyzing matrix-matched standards showed excellent linear relationship (r 2  = 0.9997) in the concentration range of 10–1000 ng mL−1. The limit of detection and quantification obtained were 3.8 and 9.8 ng mL−1, respectively. The relative standard deviations of intra- and inter-day obtained were in the range of 3.9 %–5.5 % and 2.9 %–4.6 % with the recoveries ranging from 86.5 % to 91.5 %.
Keywords: Magnetic molecularly imprinted polymers; Surface molecularly imprinted polymers; Roxithromycin; Human plasma; Liquid chromatography–tandem mass spectrometry;