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Tohru Koike - One of the best experts on this subject based on the ideXlab platform.
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phos tag based micropipette tip method for analysis of phosphomonoester type impurities in synthetic oligonucleotides
Journal of Chromatography A, 2020Co-Authors: Masaya Tsunehiro, Kenji Sasaki, Emiko Kinoshitakikuta, Eiji Kinoshita, Tohru KoikeAbstract:Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-Affinity micropipette tip (Phos-tag tip). All steps for the phosphate-Affinity Separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire Separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC–MS). Thus, a combination of the Phos-tag tip method and IP-RPLC–MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.
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a phos tag based micropipette tip method for rapid and selective enrichment of phosphopeptides
Electrophoresis, 2017Co-Authors: Elena Tianfei Yuan, Emiko Kinoshitakikuta, Eiji Kinoshita, Yoko Ino, Maho Kawaguchi, Yayoi Kimura, Hisashi Hirano, Tohru KoikeAbstract:Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the Separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-Affinity Separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire Separation protocol required less than 30 min per sample. By means of three independent Separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 μg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.
Zhen Liu - One of the best experts on this subject based on the ideXlab platform.
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glycan imprinted magnetic nanoparticle based selex for efficient screening of glycoprotein binding aptamers
ACS Applied Materials & Interfaces, 2018Co-Authors: Zhen LiuAbstract:Nucleic acid aptamers, as useful alternatives of antibodies, have found a large range of promising applications such as Affinity Separation and bioassays. The screening of aptamers is critical for their applications. Aptamers are often screened by an in vitro methodology called SELEX (systematic evolution of ligands by exponential enrichment). Although numerous SELEX methods have been established to facilitate the selection, new efficient selection methods are still much needed. Molecularly imprinted polymers, which are antibody alternatives at the material level and competitors of aptamers, have not been used as a platform for aptamer selection yet so far. In this study, a glycan-imprinted magnetic nanoparticles (MNPs)-based SELEX was developed to efficiently screen aptamers against glycoproteins. Glycan-imprinted MNPs were used as an Affinity interface to bind target glycoprotein, and then the target glycoprotein-bound MNPs were used as an Affinity substrate for aptamer selection. The glycan-imprinted M...
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Glycan-Imprinted Magnetic Nanoparticle-Based SELEX for Efficient Screening of Glycoprotein-Binding Aptamers
2018Co-Authors: Zhen LiuAbstract:Nucleic acid aptamers, as useful alternatives of antibodies, have found a large range of promising applications such as Affinity Separation and bioassays. The screening of aptamers is critical for their applications. Aptamers are often screened by an in vitro methodology called SELEX (systematic evolution of ligands by exponential enrichment). Although numerous SELEX methods have been established to facilitate the selection, new efficient selection methods are still much needed. Molecularly imprinted polymers, which are antibody alternatives at the material level and competitors of aptamers, have not been used as a platform for aptamer selection yet so far. In this study, a glycan-imprinted magnetic nanoparticles (MNPs)-based SELEX was developed to efficiently screen aptamers against glycoproteins. Glycan-imprinted MNPs were used as an Affinity interface to bind target glycoprotein, and then the target glycoprotein-bound MNPs were used as an Affinity substrate for aptamer selection. The glycan-imprinted MNPs were synthesized by a state-of-the-art imprinting approach called boronate Affinity controllable oriented surface imprinting. The glycan-imprinted MNPs exhibited high Affinity and specificity and therefore allowed preferential binding toward target glycoproteins while excluding unwanted species. Two representative glycoproteins, including RNase B and transferrin, were employed as target glycoproteins, and aptamers with high Affinity and specificity toward the two target glycoproteins were screened out in 3 rounds. This method exhibited some merits, such as high Affinity, fast speed, and avoiding negative screening. Therefore, the glycan-imprinted MNP-based SELEX approach holds great values for the efficient screening of high-performance aptamers
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preparation of molecularly imprinted polymers specific to glycoproteins glycans and monosaccharides via boronate Affinity controllable oriented surface imprinting
Nature Protocols, 2017Co-Authors: Rongrong Xing, Shuangshou Wang, Zijun Bie, Zhen LiuAbstract:Molecularly imprinted polymers (MIPs) are materials that are designed to be receptors for a template molecule (e.g., a protein). They are made by polymerizing the polymerizable reagents in the presence of the template; when the template is removed, the material can be used for many applications that would traditionally use antibodies. Thus, MIPs are biomimetic of antibodies and in this capacity have found wide applications, such as sensing, Separation and diagnosis. However, many imprinting approaches are uncontrollable, and facile imprinting approaches widely applicable to a large variety of templates remain limited. We developed an approach called boronate Affinity controllable-oriented surface imprinting, which allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans and monosaccharides. This approach relies on immobilization of a template (glycoprotein, glycan or monosaccharide) on a boronic-acid-functionalized substrate through boronate Affinity interaction, followed by self-polymerization of biocompatible monomer(s) to form an imprinting layer on the substrate with appropriate thickness. Imprinting in this approach is performed in a controllable manner, permitting the thickness of the imprinting layer to be fine-tuned according to the molecular size of the template by adjusting the imprinting time. This not only simplifies the imprinting procedure but also makes the approach widely applicable to a large range of sugar-containing biomolecules. MIPs prepared by this approach exhibit excellent binding properties and can be applied to complex real samples. The MIPs prepared by this protocol have been used in Affinity Separation, disease diagnosis and bioimaging. The entire protocol, including preparation, property characterization and performance evaluation, takes ∼3-8 d, depending on the type of substrate and template used.
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boronate Affinity materials for Separation and molecular recognition structure properties and applications
ChemInform, 2015Co-Authors: Yang Chen, Zhen LiuAbstract:Boronate Affinity materials, as unique sorbents, have emerged as important media for the selective Separation and molecular recognition of cis-diol-containing compounds. With the introduction of boronic acid functionality, boronate Affinity materials exhibit several significant advantages, including broad-spectrum selectivity, reversible covalent binding, pH-controlled capture/release, fast association/desorption kinetics, and good compatibility with mass spectrometry. Because cis-diol-containing biomolecules, including nucleosides, saccharides, glycans, glycoproteins and so on, are the important targets in current research frontiers such as metabolomics, glycomics and proteomics, boronate Affinity materials have gained rapid development and found increasing applications in the last decade. In this review, we critically survey recent advances in boronate Affinity materials. We focus on fundamental considerations as well as important progress and new boronate Affinity materials reported in the last decade. We particularly discuss on the effects of the structure of boronate ligands and supporting materials on the properties of boronate Affinity materials, such as binding pH, Affinity, selectivity, binding capacity, tolerance for interference and so on. A variety of promising applications, including Affinity Separation, proteomics, metabolomics, disease diagnostics and aptamer selection, are introduced with main emphasis on how boronate Affinity materials can solve the issues in the applications and what merits boronate Affinity materials can provide.
Shoichi Kusumoto - One of the best experts on this subject based on the ideXlab platform.
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oligosaccharide synthesis by Affinity Separation based on molecular recognition between podand ether and ammonium ion
Synlett, 2005Co-Authors: Koichi Fukase, Mamoru Takashina, Yumiko Hori, Daizo Tanaka, Katsunori Tanaka, Shoichi KusumotoAbstract:We previously reported a new synthetic methodology termed 'synthesis based on Affinity Separation' (SAS) in which the desired tagged compound was separated from the reaction mixture by solid-phase extraction using specific molecular recognition of the tag. The interaction between a crown ether tag and polymer-supported ammonium ion was initially employed for SAS. In the present study, a new SAS method using a podand-type tag, a pseudo-benzo-31-crown-10 structure, was elaborated for oligosaccharide synthesis. The podand tag was much easier to synthesize than the corresponding crown ether. The podand moiety was attached to the monosaccharide residue via appropriate linkers. After glycosylation of the tagged monosaccharide with a glycosyl donor, the reaction mixture was subjected to the Affinity Separation. The desired compounds possessing the podand tag were effectively separated by the Affinity between the podand and the ammonium ion. Continuous flow synthesis by integration of a microreactor and the present SAS system was applied for high throughput oligosaccharide synthesis.
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synthesis based on Affinity Separation sas Separation of products having barbituric acid tag from untagged compounds by using hydrogen bond interaction
Synlett, 2001Co-Authors: San Qi Zhang, Koichi Fukase, Minoru Izumi, Yoshiyuki Fukase, Shoichi KusumotoAbstract:A new method is described for Affinity purification of synthetic compounds based on molecular recognition between bis(2,6-diaminopyridine)amide of isophthalic acid and a barbituric acid derivative. The desired compounds possessing the barbituric acid derivative as a tag were readily isolated from the reaction mix- ture by the following procedure. After each reaction cycle, the reac- tion mixture was applied to the polystyrene column possessing bis(2,6-diaminopyridine)amide of isophthalic acid as an artificial receptor. The compound possessing the barbituric acid tag was se- lectively adsorbed on the column, whereas other impurities without the tag such as excess reagents and byproducts were washed off. Subsequent desorption with CH2Cl2-MeOH (1:1) afforded the de- sired compound with high purity. This new strategy was applied to the synthesis of a heterocycle, peptides, and oligosaccharides.
Sergey A Piletsky - One of the best experts on this subject based on the ideXlab platform.
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solid phase synthesis of molecularly imprinted nanoparticles
Nature Protocols, 2016Co-Authors: Francesco Canfarotta, Alessandro Poma, Antonio Guerreiro, Sergey A PiletskyAbstract:Molecularly imprinted polymers (MIPs) are synthetic materials, generally based on acrylic or methacrylic monomers, that are polymerized in the presence of a specific target molecule called the 'template' and capable of rebinding selectively to this target molecule. They have the potential to be low-cost and robust alternatives to biomolecules such as antibodies and receptors. When prepared by traditional synthetic methods (i.e., with free template in solution), their usefulness has been limited by high binding site heterogeneity, the presence of residual template and the fact that the production methods are complex and difficult to standardize. To overcome some of these limitations, we developed a method for the synthesis of MIP nanoparticles (nanoMIPs) using an innovative solid-phase approach, which relies on the covalent immobilization of the template molecules onto the surface of a solid support (glass beads). The obtained nanoMIPs are virtually free of template and demonstrate high Affinity for the target molecule (e.g., melamine and trypsin in our published work). Because of an Affinity Separation step performed on the solid phase after polymerization, poor binders and unproductive polymer are removed, so the final product has more uniform binding characteristics. The overall protocol, starting from the immobilization of the template onto the solid phase and including the purification and characterization of the nanoparticles, takes up to 1 week.
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solid phase synthesis of molecularly imprinted polymer nanoparticles with a reusable template plastic antibodies
Advanced Functional Materials, 2013Co-Authors: Alessandro Poma, Antonio Guerreiro, Michael J Whitcombe, Elena V Piletska, Anthony Turner, Sergey A PiletskyAbstract:Molecularly Imprinted Polymers (MIPs) are generic alternatives to antibodies in sensors, diagnostics and Separations. To displace biomolecules without radical changes in infrastructure in device manufacture, MIPs should share their characteristics (solubility, size, specificity and Affinity, localized binding domain) whilst maintaining the advantages of MIPs (low-cost, short development time and high stability) hence the interest in MIP nanoparticles. Herein we report a reusable solid-phase template approach (fully compatible with automation) for the synthesis of MIP nanoparticles and their precise manufacture using a prototype automated UV photochemical reactor. Batches of nanoparticles (30-400 nm) with narrow size distributions imprinted with: melamine (d = 60 nm, Kd = 6.3 × 10-8 m), vancomycin (d = 250 nm, Kd = 3.4 × 10-9 m), a peptide (d = 350 nm, Kd = 4.8 × 10-8 m) and proteins have been produced. Our instrument uses a column packed with glass beads, bearing the template. Process parameters are under computer control, requiring minimal manual intervention. For the first time we demonstrate the reliable re-use of molecular templates in the synthesis of MIPs (≥ 30 batches of nanoMIPs without loss of performance). NanoMIPs are produced template-free and the solid-phase acts both as template and Affinity Separation medium.
Eiji Kinoshita - One of the best experts on this subject based on the ideXlab platform.
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phos tag based micropipette tip method for analysis of phosphomonoester type impurities in synthetic oligonucleotides
Journal of Chromatography A, 2020Co-Authors: Masaya Tsunehiro, Kenji Sasaki, Emiko Kinoshitakikuta, Eiji Kinoshita, Tohru KoikeAbstract:Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-Affinity micropipette tip (Phos-tag tip). All steps for the phosphate-Affinity Separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire Separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC–MS). Thus, a combination of the Phos-tag tip method and IP-RPLC–MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.
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a phos tag based micropipette tip method for rapid and selective enrichment of phosphopeptides
Electrophoresis, 2017Co-Authors: Elena Tianfei Yuan, Emiko Kinoshitakikuta, Eiji Kinoshita, Yoko Ino, Maho Kawaguchi, Yayoi Kimura, Hisashi Hirano, Tohru KoikeAbstract:Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the Separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-Affinity Separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire Separation protocol required less than 30 min per sample. By means of three independent Separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 μg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.