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Sample records for affinity chromatography method

  1. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  2. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

  3. Mixed-bed affinity chromatography: principles and methods.

    PubMed

    Boschetti, Egisto; Righetti, Pier Giorgio

    2015-01-01

    Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. Other interesting preparative applications of mixed-bed chromatography have since been developed. In this chapter the basic concepts first and then detailed application recipes are described for (1) the reduction of protein dynamic concentration range, (2) the removal of impurity traces at the last stage of a biopurification process, and (3) the selection and use of sorbents as mixed bed in protein purification.

  4. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

  5. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  6. [Affinity chromatography and proteomic screening as the effective method for S100A4 new protein targets discovery].

    PubMed

    Koshelev, Iu A

    2014-01-01

    Affinity chromatography followed by a selective binding proteins identification can be using as effective method for a biological impotent interactions discovery. The molecular structure and their surface charge as and conformational regulation possibilities, which change their surface hydrophobic properties, all they should to taken in account during method optimization process. With the same' method we had identify some new S100A4 target proteins such as cytoskeleton proteins Sept2, Sept7, Sept11 and this interaction would can to highlight as S100A4 would regulate cell motility. Even we had identify the transcription cofactor Ddx5 and through such complex formation a S100A4 protein would can to regulate E-cadherin, p21 Waf1/Cip1), Bnip3 gene expression. The same protocol can be using for a target proteins search with another S100 protein family members, because their molecules demonstrate a high homology level in amino aside sequences and 3D structures.

  7. Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.

    PubMed

    Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N

    2011-05-25

    Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

  8. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

  9. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

  10. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

  11. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  12. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  13. [Separation of osteoclasts by lectin affinity chromatography].

    PubMed

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  14. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

    2013-12-01

    The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

  15. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  16. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  17. A general method for fractionation of plasma proteins. Dye-ligand affinity chromatography on immobilized Cibacron blue F3-GA.

    PubMed

    Gianazza, E; Arnaud, P

    1982-01-01

    The chromatographic behaviour of 27 different plasma proteins on fractionation of human plasma on immobilized Cibacron Blue F3-GA was studied. The column was eluted by using a three-step procedure. First, a low-molarity buffer (30 mM-H3PO4/Na3PO4, pH 7.0, I0.053) was used, then a linear salt gradient (0-1 M-NaCl in the buffer above) was applied, followed by a wash with two bed volumes of 1.0 M-NaCl. Finally, bound proteins were 'stripped' with 0.5 M-NaSCN. Up to 1 ml of whole plasma could be loaded per 5 ml bed volume. No denaturation of proteinase inhibitors or complement fractions was observed. The recovery of individual proteins ranged between 52 and greater than 95%. Enrichment of four individual plasma components (alpha 1-antitrypsin, caeruloplasmin, antithrombin III and haemopexin) was between 10-fold and 75-fold. These results indicate that chromatography on immobilized Cibacron Blue F3-GA can be a useful initial step in the purification of plasma proteins.

  18. Purification of cytochrome c oxidase by lysine-affinity chromatography.

    PubMed

    Felsch, J; Kotake, S; Copeland, R A

    1992-02-01

    A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.

  19. Purification of baculovirus vectors using heparin affinity chromatography

    PubMed Central

    Nasimuzzaman, Md; Lynn, Danielle; van der Loo, Johannes CM; Malik, Punam

    2016-01-01

    Baculoviruses are commonly used for recombinant protein and vaccine production. Baculoviruses are nonpathogenic to vertebrates, have a large packaging capacity, display broad host and cell type tropism, infect both dividing and nondividing cells, and do not elicit strong immune or allergic responses in vivo. Hence, their use as gene delivery vehicles has become increasingly popular in recent years. Moreover, baculovirus vectors carrying mammalian regulatory elements can efficiently transduce and express transgenes in mammalian cells. Based on the finding that heparan sulfate, which is structurally similar to heparin, is an attachment receptor for baculovirus, we developed a novel scalable baculovirus purification method using heparin-affinity chromatography. Baculovirus supernatants were loaded onto a POROS heparin column, washed to remove unbound materials, and eluted with 1.5 mol/l NaCl, which yielded a recovery of purified baculovirus of 85%. After ultracentrifugation, baculovirus titers increased from 200- to 700-fold with overall yields of 26–29%. We further show that baculovirus particles were infectious, normal in morphology and size, despite high-salt elution and shear forces used during purification and concentration. Our chromatography-based purification method is scalable and, together with ultracentrifugation and/or tangential flow filtration, will be suitable for large-scale manufacturing of baculovirus stocks for protein and vaccine production and in gene therapy applications. PMID:27933303

  20. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  1. Extension of the selection of protein chromatography and the rate model to affinity chromatography.

    PubMed

    Sandoval, G; Shene, C; Andrews, B A; Asenjo, J A

    2010-01-01

    The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

  2. Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

    PubMed

    London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

    2015-04-01

    Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis.

  3. Affinity monolith chromatography: A review of principles and recent analytical applications

    PubMed Central

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  4. Cross-linked leucaena seed gum matrix: an affinity chromatography tool for galactose-specific lectins.

    PubMed

    Seshagirirao, Kottapalli; Leelavathi, Chaganti; Sasidhar, Vemula

    2005-05-31

    A cross-linked leucaena (Leucaena leucocephala) seed gum (CLLSG) matrix was prepared for the isolation of galactose-specific lectins by affinity chromatography. The matrix was evaluated for affinity with a known galactose-specific lectin from the seeds of snake gourd (Trichosanthes anguina). The matrix preparation was simple and inexpensive when compared to commercial galactose-specific matrices (i.e. about 1.5 US dollars/100 ml of matrix). The current method is also useful for the demonstration of the affinity chromatography technique in laboratories. Since leucaena seeds are abundant and inexpensive, and the matrix preparation is easy, CLLSG appears to be a promising tool for the separation of galactose-specific lectins.

  5. Virus inactivation by protein denaturants used in affinity chromatography.

    PubMed

    Roberts, Peter L; Lloyd, David

    2007-10-01

    Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.

  6. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column.

  7. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

    PubMed

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-11-14

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

  8. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling*

    PubMed Central

    Kennedy, Jacob J.; Yan, Ping; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Moore, Heather D.; Lin, Chenwei; Pogosova-Agadjanyan, Era L.; Stirewalt, Derek L.; Reding, Kerryn W.; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  9. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    PubMed

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  10. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  11. Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

    PubMed Central

    Chung, Jinhwa A.; Wollack, James W.; Hovlid, Marisa L.; Okesli, Ayse; Chen, Yan; Mueller, Joachim D.; Distefano, Mark D.; Taton, T. Andrew

    2009-01-01

    Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation. PMID:18834849

  12. A recombinant envelope protein from Dengue virus purified by IMAC is bioequivalent with its immune-affinity chromatography purified counterpart.

    PubMed

    Hermida, L; Rodríguez, R; Lazo, L; López, C; Márquez, G; Páez, R; Suárez, C; Espinosa, R; García, J; Guzmán, G; Guillén, G

    2002-03-28

    Semi-purified DEN-4 envelope protein, obtained in Pichia pastoris, was capable of generating neutralising and protecting antibodies after immunisation in mice. Here we compared two purification processes of this recombinant protein using two chromatographic steps: immune-affinity chromatography and immobilised metal ion adsorption chromatography (IMAC). The protein purified by both methods produced functional antibodies reflected by titres of haemagglutination inhibition and neutralisation. IMAC could be used as an alternative for high scale purification.

  13. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  14. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  15. A new affinity approach to isolate Escherichia coli 6S RNA with histidine-chromatography.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2010-01-01

    6S RNA is an abundant non-coding RNA in Escherichia coli (E. coli), but its function has not been discovered until recently. The first advance on 6S RNA function was the demonstration of its ability to bind the σ(70)-holoenzyme form of RNA polymerase, inhibiting its activity and consequently the transcription process. The growing interest in the investigation of non-coding small RNAs (sRNA) calls for the development of new methods for isolation and purification of RNA. This work presents an optimized RNA extraction procedure and describes a new affinity chromatography method using a histidine support to specifically purify 6S RNA from other E. coli sRNA species. The RNA extraction procedure was optimized, and a high yield was obtained in the separation of sRNA and ribosomal RNA (rRNA) from total RNA (RNAt). This improved method takes advantage of its simplicity and significant cost reduction, since some complex operations have been eliminated. A purification strategy was also developed to separate 6S RNA from an sRNA mixture. Pure RNA can be advantageously obtained using the histidine-affinity chromatography method, aiming at its application to structural or functional studies.

  16. Proton Affinity Calculations with High Level Methods.

    PubMed

    Kolboe, Stein

    2014-08-12

    Proton affinities, stretching from small reference compounds, up to the methylbenzenes and naphthalene and anthracene, have been calculated with high accuracy computational methods, viz. W1BD, G4, G3B3, CBS-QB3, and M06-2X. Computed and the currently accepted reference proton affinities are generally in excellent accord, but there are deviations. The literature value for propene appears to be 6-7 kJ/mol too high. Reported proton affinities for the methylbenzenes seem 4-5 kJ/mol too high. G4 and G3 computations generally give results in good accord with the high level W1BD. Proton affinity values computed with the CBS-QB3 scheme are too low, and the error increases with increasing molecule size, reaching nearly 10 kJ/mol for the xylenes. The functional M06-2X fails markedly for some of the small reference compounds, in particular, for CO and ketene, but calculates methylbenzene proton affinities with high accuracy.

  17. Fractionation of the genetic variants of human alpha 1-acid glycoprotein in the native form by chromatography on an immobilized copper(II) affinity adsorbent. Heterogeneity of the separate variants by isoelectrofocusing and by concanavalin A affinity chromatography.

    PubMed

    Hervé, F; Gomas, E; Duché, J C; Tillement, J P

    1993-05-19

    Fractionation of the three main genetic variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.

  18. Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein.

    PubMed

    Uhlik, Ondrej; Kamlar, Marek; Kohout, Ladislav; Jezek, Rudolf; Harmatha, Juraj; Macek, Tomas

    2008-12-22

    The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.

  19. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.

  20. Mullerian inhibiting substance fractionation by dye affinity chromatography.

    PubMed

    Budzik, G P; Powell, S M; Kamagata, S; Donahoe, P K

    1983-08-01

    Mullerian inhibiting substance (MIS), a large glycoprotein secreted by the fetal and neonatal testis, is responsible for regression of the Mullerian ducts in the male embryo. This fetal growth regulator has been purified more than 2000-fold from crude testicular incubation medium following fractionation on a triazinyl dye affinity support. A high yield of 60% recovered activity was achieved in the absence of exogenous carrier protein by stabilizing MIS with 2-mercaptoethanol, EDTA, and Nonidet-P40 and eliminating losses in the handling and concentration of MIS fractions. Although affinity elution with nucleotides has proved successful in other systems, MIS could not be eluted with ATP, GTP, or AMP, with or without divalent metal ions. Nucleotide elution, however, does remove contaminating proteins prior to MIS recovery with high ionic strength. The 2000-fold-purified MIS fraction, although not homogeneous, shows a reduction-sensitive band after SDS-gel electrophoresis that has been proposed to be the MIS dimer.

  1. Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

    PubMed

    Fleminger, G; Neufeld, T; Star-Weinstock, M; Litvak, M; Solomon, B

    1992-04-24

    The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.

  2. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  3. Glycan-specific whole cell affinity chromatography: a versatile microbial adhesion platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a C-glycoside ketohydrazide affinity chromatography resin that interacts with viable whole-cell microbial populations with biologically appropriate stereo-specificity in a carbohydrate-defined manner. It readily allows for the quantification, selection, and manipulation of target...

  4. Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions.

    PubMed

    Hirabayashi, J; Arata, Y; Kasai, K

    2000-08-25

    Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.

  5. [Prospects of application of the chitin-binding domains to isolation and purification of recombinant proteins by affinity chromatography: a review].

    PubMed

    Kurek, D V; Lopatin, S A; Varlamov, V P

    2009-01-01

    Properties of substrate-binding domains, some parameters of affinity sorbents, and a number of other special features that were necessary to take into account during creation of chromatographic system for isolation and purification of proteins with incorporated chitin-binding domain were discussed in this review. This method was shown to be successfully used along with metal-chelate affinity chromatography. The metal-chelate affinity chromatography with the use of polyhistidine peptides as affinity labels is successfully applied to isolation, purification, and investigation of recombinant proteins. However, this system had some disadvantages. At present, scientists attracted more and more attention to substrate-binding domains, including those chitin-binding, because they had a number of advantages being used as affinity label.

  6. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-08

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  7. Hemoglobin Ypsilanti: a high-oxygen-affinity hemoglobin demonstrated by two automated high-pressure liquid chromatography systems.

    PubMed

    Mais, Daniel D; Boxer, Laurence A; Gulbranson, Ronald D; Keren, David F

    2007-11-01

    Hemoglobin (Hb) Ypsilanti is a rare high-oxygen-affinity hemoglobin. Like other high-oxygen-affinity hemoglobins, Hb Ypsilanti manifests as erythrocytosis. Because the migration of many high-oxygen-affinity variants on alkaline and acid gels does not differ from that of HbA, oxygen-hemoglobin dissociation studies are often used to document their presence. Hb Ypsilanti is a notable exception because its electrophoresis pattern on alkaline gel is highly characteristic, exemplifying the phenomenon of hybrid formation in variant hemoglobins. In the past few years, several laboratories have begun to use high-pressure liquid chromatography (HPLC) as a screen for hemoglobinopathies. We demonstrate the elution profile of Hb Ypsilanti on the 2 most widely used HPLC methods.

  8. Affinity chromatography approaches to overcome the challenges of purifying plasmid DNA.

    PubMed

    Sousa, Fani; Prazeres, Duarte M F; Queiroz, João A

    2008-09-01

    The diversity of biomolecules present in plasmid DNA (pDNA)-containing extracts and the structural and chemical similarities between pDNA and impurities are some of the main challenges of improving or establishing novel purification procedures. In view of the unequalled specificity of affinity purification, this technique has recently begun to be applied in downstream processing of plasmids. This paper discusses the progress and importance of affinity chromatography (AC) for the purification of pDNA-based therapeutic products. Several affinity approaches have already been successfully developed for a variety of applications, and we will focus here on highlighting their possible contributions to the pDNA purification challenge. Diverse affinity applications and their advantages and disadvantages are discussed, as well as the most significant results and improvements in the challenging task of purifying plasmids.

  9. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    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 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/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.

  10. Affinity chromatography of Band 3, the anion transport protein of erythrocyte membranes.

    PubMed

    Pimplikar, S W; Reithmeier, R A

    1986-07-25

    Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.

  11. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  12. Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand.

    PubMed

    Novotná, Lenka; Hrubý, Martin; Benes, Milan J; Kucerová, Zdenka

    2005-08-19

    Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.

  13. Identification of potential cellular targets of aloisine A by affinity chromatography.

    PubMed

    Corbel, Caroline; Haddoub, Rose; Guiffant, Damien; Lozach, Olivier; Gueyrard, David; Lemoine, Jérôme; Ratin, Morgane; Meijer, Laurent; Bach, Stéphane; Goekjian, Peter

    2009-08-01

    Affinity chromatography was used to identify potential cellular targets of aloisine A (7-n-butyl-6-(4'-hydroxyphenyl)-5H-pyrrolo[2,3b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine.

  14. A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.

    PubMed

    Huang, Yongdong; Zhang, Rongyue; Li, Juan; Li, Qiang; Su, Zhiguo; Ma, Guanghui

    2014-03-01

    Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100-500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins.

  15. Preparation of adsorbents for affinity chromatography using TSKgel Tresyl-Toyopearl 650M.

    PubMed

    Nakamura, K; Toyoda, K; Kato, Y; Shimura, K; Kasai, K

    1989-09-08

    The optimum conditions for the coupling of proteins were investigated using TSKgel Tresyl-Toyopearl 650M. They were dependent on the proteins coupled. For example, when soybean trypsin inhibitor was coupled at pH 8 the coupling was completed within 1 h and the subsequent adsorption capacity for trypsin was maximal. Longer coupling times decreased the adsorption capacity due to multi-point attachment. The adsorbents obtained were successfully used for affinity chromatography in a short time.

  16. Optimising the design and operation of semi-continuous affinity chromatography for clinical and commercial manufacture.

    PubMed

    Pollock, James; Bolton, Glen; Coffman, Jon; Ho, Sa V; Bracewell, Daniel G; Farid, Suzanne S

    2013-04-05

    This paper presents an integrated experimental and modelling approach to evaluate the potential of semi-continuous chromatography for the capture of monoclonal antibodies (mAb) in clinical and commercial manufacture. Small-scale single-column experimental breakthrough studies were used to derive design equations for the semi-continuous affinity chromatography system. Verification runs with the semi-continuous 3-column and 4-column periodic counter current (PCC) chromatography system indicated the robustness of the design approach. The product quality profiles and step yields (after wash step optimisation) achieved were comparable to the standard batch process. The experimentally-derived design equations were incorporated into a decisional tool comprising dynamic simulation, process economics and sizing optimisation. The decisional tool was used to evaluate the economic and operational feasibility of whole mAb bioprocesses employing PCC affinity capture chromatography versus standard batch chromatography across a product's lifecycle from clinical to commercial manufacture. The tool predicted that PCC capture chromatography would offer more significant savings in direct costs for early-stage clinical manufacture (proof-of-concept) (∼30%) than for late-stage clinical (∼10-15%) or commercial (∼5%) manufacture. The evaluation also highlighted the potential facility fit issues that could arise with a capture resin (MabSelect) that experiences losses in binding capacity when operated in continuous mode over lengthy commercial campaigns. Consequently, the analysis explored the scenario of adopting the PCC system for clinical manufacture and switching to the standard batch process following product launch. The tool determined the PCC system design required to operate at commercial scale without facility fit issues and with similar costs to the standard batch process whilst pursuing a process change application. A retrofitting analysis established that the direct cost

  17. Polystyrene as an affinity chromatography matrix for the purification of antibodies.

    PubMed

    Staak, C; Salchow, F; Clausen, P H; Luge, E

    1996-08-14

    Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

  18. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-04

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  19. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa.

  20. Purification of human immunoglobulin G autoantibodies to tumor necrosis factor using affinity chromatography and magnetic separation.

    PubMed

    Sennikov, S V; Golikova, E A; Kireev, F D; Lopatnikova, J A

    2013-04-30

    Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.

  1. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%.

  2. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    PubMed

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

  3. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high performance affinity chromatography.

    PubMed

    Li, Haiyan; Ge, Jingwen; Guo, Tao; Yang, Shuo; He, Zhonggui; York, Peter; Sun, Lixin; Xu, Xu; Zhang, Jiwen

    2013-08-30

    It is challenging and extremely difficult to measure the kinetics of supramolecular systems with extensive, weak binding (Ka<10(5)M(-1)), and fast dissociation, such as those composed of cyclodextrins and drugs. In this study, a modified peak profiling method based on high performance affinity chromatography (HPAC) was established to determine the dissociation rate constant of cyclodextrin supramolecular systems. The interactions of β-cyclodextrin with acetaminophen and sertraline were used to exemplify the method. The retention times, variances and the plate heights of the peaks for acetaminophen or sertraline, conventional non-retained substance (H2O) on the β-cyclodextrin bonded column and a control column were determined at four flow rates under linear elution conditions. Then, plate heights for the theoretical non-retained substance were estimated by the modified HPAC method, in consideration of the diffusion and stagnant mobile phase mass transfer. As a result, apparent dissociation rate constants of 1.82 (±0.01)s(-1) and 3.55 (±0.37)s(-1) were estimated for acetaminophen and sertraline respectively at pH 6.8 and 25°C with multiple flow rates. Following subtraction of the non-specific binding with the support, dissociation rate constants were estimated as 1.78 (±0.00) and 1.91 (±0.02)s(-1) for acetaminophen and sertraline, respectively. These results for acetaminophen and sertraline were in good agreement with the magnitude of the rate constants for other drugs determined by capillary electrophoresis reported in the literature and the peak fitting method we performed. The method described in this work is thought to be suitable for other supramolecules, with relatively weak, fast and extensive interactions.

  4. A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

    SciTech Connect

    Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F

    2005-08-25

    Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend

  5. G-quadruplex on oligo affinity support (G4-OAS): an easy affinity chromatography-based assay for the screening of G-quadruplex ligands.

    PubMed

    Musumeci, Domenica; Amato, Jussara; Randazzo, Antonio; Novellino, Ettore; Giancola, Concetta; Montesarchio, Daniela; Pagano, Bruno

    2014-05-06

    A simple, cheap, and highly reproducible affinity chromatography-based method has been developed for the screening of G-quadruplex binders. The tested compounds were flowed through a polystyrene resin functionalized with an oligonucleotide able to form, in proper conditions, a G-quadruplex structure. Upon cation-induced control of the folding/unfolding processes of the immobilized G-quadruplex-forming sequence, small molecules specifically interacting with the oligonucleotide structure were first captured and then released depending on the used working solution. This protocol, first optimized for different kinds of known G-quadruplex ligands and then applied to a set of putative ligands, has allowed one to fully reuse the same functionalized resin batch, recycled for several tens of experiments without loss in efficiency and reproducibility.

  6. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  7. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  8. Separation and analysis of cis-diol-containing compounds by boronate affinity-assisted micellar electrokinetic chromatography.

    PubMed

    Wang, Heye; Lü, Chenchen; Li, Hengye; Chen, Yang; Zhou, Min; Ouyang, Jian; Liu, Zhen

    2013-10-01

    Cis-diol-containing compounds (CDCCs) are usually highly hydrophilic compounds and are therefore difficult to separate by conventional reversed-phase-based micellar electrokinetic chromatography (MEKC) due to poor selectivity. Here, we report a new method, called boronate affinity-assisted micellar electrokinetic chromatography (BAA-MEKC), to solve this issue. A boronic acid with a hydrophobic alkyl chain was added to the background electrolyte, which acted as a modifier to adjust the selectivity. CDCCs can covalently react with the boronic acid to form negatively charged surfactant-like complexes, which can partition into micelles formed with a cationic surfactant. Thus, CDCCs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase. To verify this method, eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCCs. The effects of experimental conditions on the separation were investigated. Finally, the BAA-MEKC method was applied to the separation and analysis of nucleosides extracted from human urine. BAA-MEKC exhibited better selectivity and improved separation as compared with conventional MEKC and CZE. Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.

  9. Isolation and partial characterization of Bromelia hemisphaerica protease by affinity chromatography.

    PubMed

    Ochoa, N; Agundis, C; Córdoba, F

    1987-01-01

    Hemisphaericin, the protease from Bromelia hemisphaerica fruit juice was isolated by affinity chromatography in one step, using a mercurial sepharose derivative. The enzyme behaves as a single component in immunodifussion, immunoelectrophoresis and polyacrylamide electrophoresis in the presence of SDS and 2-mercaptoethanol. Association and dissociation of active components were evidenced in electrophoresis at pH 3.6 and at pH 8.6. Immunoelectrophoresis analyses also disclosed a certain degree of internal immunological heterogeneity. The results are explained by the presence of an enzyme subunit, of about 8000 daltons, endowed with polymeric properties induced by the pH and oxidative environment.

  10. Rapid and Complete Purification of Acetylcholinesterases of Electric Eel and Erythrocyte by Affinity Chromatography

    PubMed Central

    Berman, Jonathan Dembitz; Young, Michael

    1971-01-01

    Affinity chromatography has been used to purify acetylcholinesterase both from the electric tissue of Electrophorus electricus and from bovine erythrocyte membranes. For this purpose, several specific enzymic inhibitors of each protein were synthesized and joined covalently to an insoluble support resin. AchE is selectively retained by such inhibitor-resins when highly impure solutions are chromatographed upon them. After removal from the resin, both enzymes are electrophoretically homogeneous and they may be recovered in yields of 75% or more. Images PMID:5277092

  11. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

  12. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  13. Partial purification of the microsomal rat liver iodothyronine deiodinase. II. Affinity chromatography.

    PubMed

    Mol, J A; van den Berg, T P; Visser, T J

    1988-02-01

    Iodothyronine deiodinase has been solubilized and purified approximately 2400 times from liver microsomal fractions of male Wistar rats pretreated with thyroxine. The deiodinase was solubilized with 1% cholate, and stripped of adhering phospholipids by ammonium sulfate precipitation followed by solubilization with the non-ionic detergent Emulgen 911. The enzyme was further purified by successive ion-exchange chromatography on DEAE-Sephacel and Cellex-P and affinity chromatography on 3,3',5-triiodothyronine-Sepharose. Finally, the deiodinase was reacted with 6-propionyl-2-thiouracil-Sepharose, a derivative of the mechanism-based inhibitor 6-propyl-2-thiouracil. Covalent binding was observed only in the presence of substrate in agreement with the proposed mechanism of deiodination. The deiodinase was eluted from the affinity column by reduction of the enzyme-propylthiouracil mixed disulfide with 50 mM dithiothreitol. The enzyme was approximately 50% pure as judged by SDS-PAGE, exhibiting a subunit molecular weight of 25,000. This preparation was equally enriched in outer ring and inner ring deiodinase activities in keeping with the view that both are intrinsic to a single, type I deiodinase.

  14. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  15. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  16. Freeze chromatography method and apparatus

    DOEpatents

    Scott, C.D.

    1987-04-16

    A freeze chromatography method and apparatus are provided which enable separation of the solutes contained in a sample. The apparatus includes an annular column construction comprising cylindrical inner and outer surfaces defining an annular passage therebetween. One of the surfaces is heated and the other cooled while passing an eluent through the annular passageway so that the eluent in contact with the cooled surface freezes and forms a frozen eluent layer thereon. A mixture of solutes dissolved in eluent is passed through the annular passageway in contact with the frozen layer so that the sample solutes in the mixture will tend to migrate either toward or away the frozen layer. The rate at which the mixture flows through the annular passageway is controlled so that the distribution of the sample solutes approaches that at equilibrium and thus a separation between the sample solutes occurs. 3 figs.

  17. Application of Frontal Affinity Chromatography to Study the Biomolecular Interactions with Trypsin.

    PubMed

    Hu, YuanYuan; Qian, Junqing; Guo, Hui; Jiang, ShengLan; Zhang, Zheng

    2015-07-01

    Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

  18. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  19. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme.

  20. Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH.

    PubMed

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Yamada, Atsushi; Endo, Mika; Koike, Tohru

    2006-10-01

    While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

  1. Selective isolation of β-glucan from corn pericarp hemicelluloses by affinity chromatography on cellulose column.

    PubMed

    Yoshida, Tomoki; Honda, Yoichi; Tsujimoto, Takashi; Uyama, Hiroshi; Azuma, Jun-ichi

    2014-10-13

    A combination of anion-exchange chromatography and affinity chromatography on a cellulose column was found to be effective for the isolation of β-(1,3;1,4)-glucan (BG) from corn pericarp hemicelluloses (CPHs). CPHs containing 6.6% BG were extracted from corn pericarp with 6M urea-2 wt% NaOH solution and initially fractionated into neutral and acidic parts by anion exchange chromatography to remove acidic arabinoxylan consisting of arabinose (35.6%) and xylose (50.9%). The neutral fraction (yield; 10.1% on the basis of CPHs) consisting of 1.0% arabinose, 10.1% xylose and 80.3% glucose containing 28.4% BG was then applied to a cellulose column of Whatman CF-11. BG could be recovered from the adsorbed fraction on the cellulose column by elution with 2% NaOH in a yield of 2.6% on the basis of CPHs with a purity of 84.7%. The chemical structure of the isolated corn pericarp BG was confirmed by (13)C NMR spectroscopic, methylation and lichenase treatment analyses. The results indicate that the ratios of (1,4)/(1,3) linkage and cellotriosyl/cellotetraosyl segments of the BG were 2.60 and 2.5, respectively.

  2. Some general methods of preparing affinity columns

    PubMed Central

    Sundaram, P.V.

    1974-01-01

    Some general methods of covalent coupling of nucleotides, especially derivatized nucleotides, polynucleotides and cofactors to insoluble polymers are described in this paper. Wherever necessary individual methods also carry some information on the binding of enzymes to the same polymers to serve as a guide to the efficiency of the coupling methods. PMID:10793714

  3. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

  4. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  5. Purification and characterization of a new type lactose-binding Ulex europaeus lectin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

  6. Identification of the Cardiac Beta-Adrenergic Receptor Protein: Solubilization and Purification by Affinity Chromatography

    PubMed Central

    Lefkowitz, Robert J.; Haber, Edgar; O'Hara, Donald

    1972-01-01

    A protein that binds catecholamines with a specificity parallel to that of their in vivo effects on cardiac contractility (isoproterenol > epinephrine or norepinephrine > dopamine > dihydroxyphenylalanine) was solubilized from a microsomal fraction of canine ventricular myocardium. The binding protein was purified 500 to 800-fold by solubilization and subsequent affinity chromatography with conjugates of norepinephrine linked to agarose beads. Purified β-adrenergic binding protein exists in two forms, corresponding to molecular weights of 40,000 and 160,000. The purified material has a single association constant, 2.3 × 105 liters/mol (as compared to two association constants, 107 and 106 liters/mol, for the binding protein in particulate form) but retains the identical binding specificity for β-adrenergic drugs and antagonists. Images PMID:4507606

  7. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  8. Contamination of ribosome inactivating proteins with ribonucleases, separated by affinity chromatography on red sepharose.

    PubMed

    Wang, H X; Ng, T B; Cheng, C H K; Fong, W P

    2003-05-01

    Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

  9. Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.

    PubMed

    Wiley, J P; Hughes, K A; Kaiser, R J; Kesicki, E A; Lund, K P; Stolowitz, M L

    2001-01-01

    Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

  10. Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography

    PubMed Central

    Kuehne, Christian; Wedepohl, Stefanie; Dernedde, Jens

    2017-01-01

    l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin. PMID:28125045

  11. Purification of peroxidase from red cabbage (Brassica oleracea var. capitata f. rubra) by affinity chromatography.

    PubMed

    Somtürk, Burcu; Kalın, Ramazan; Özdemir, Nalan

    2014-08-01

    Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9% from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K m and V max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC50 and K i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702±0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.

  12. Using affinity chromatography to engineer and characterize pH-dependent protein switches.

    PubMed

    Sagermann, Martin; Chapleau, Richard R; DeLorimier, Elaine; Lei, Margarida

    2009-01-01

    Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.

  13. Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

    PubMed Central

    Dean, N; Berk, A J

    1987-01-01

    Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay. Images PMID:3697084

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

    PubMed

    Melissis, S C; Rigden, D J; Clonis, Y D

    2001-05-11

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

  15. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form.

  16. Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.

    PubMed

    Zhuang, Ran; Zhang, Yuan; Zhang, Rui; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2008-05-01

    GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.

  17. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  18. Comparative study of glycated hemoglobin by ion exchange chromatography and affinity binding nycocard reader in type 2 diabetes mellitus.

    PubMed

    Gautam, N; Dubey, R K; Jayan, A; Nepaune, Y; Padmavathi, P; Chaudhary, S; Jha, S K; Sinha, A K

    2014-12-01

    The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus (DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n = 100) who visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa, Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their fasting (164.46 ± 45.33 mg/dl) and random (187.93 ± 78.02 mg/dl) serum glucose level along with clinical history highly suggestive of type 2 DM. The HbA1c values of (7.8 ± 1.9%) and (8.0 ± 2.2%) were found in DM patients as estimated by those two different methods respectively. The highest frequency was observed in HbA1c > 8.0% indicating maximum cases were under very poor glycemic control. However, there were no significant differences observed in HbA1c value showing both methods are comparable in nature and can be used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c determination, the availability of report at the time of the patient visit can be made easy by using Nycocard Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control, clinical decision-making and changes in treatment regimen.

  19. Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

    PubMed Central

    Liang, Aiye; Thakkar, Jay N.; Hindle, Michael; Desai, Umesh R.

    2013-01-01

    Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4 m/z, which most probably arise from variably functionalized (β-O4—β-β-linked trimers and/or a β-O4—β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants. PMID:23122400

  20. Purification and characterization of a Cytisus-type Ulex europeus hemagglutinin II by affinity chromatography.

    PubMed

    Konami, Y; Tsuji, T; Matsumoto, I; Osawa, T

    1981-07-01

    Ulex europeus hemagglutinin II [Cytisus-type anti-H(O) hemagglutinin] inhibited most by di-N-acetylchitobiose has been purified by affinity chromatography on a column of chitobiose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. The purified hemagglutinin was homogeneous by ultracentrifugal analysis and gave a single band by electrophoresis on polyacrylamide gel, and had a molecular weight of 105 000 by sedimentation equilibrium and an isoelectric point of pH 6.66. This hemagglutinin was found to be composed of four, apparently identical, subunits of a molecular weight of 25 000 +/- 2 000 by dodecyl sulphate-polyacrylamide gel electrophoresis, and to contain 10.3% carbohydrate in which mannose (3.7%) was the predominant sugar, with smaller amounts of glucose, glucosamine, xylose, fucose and galactose. Amino acid analysis of the purified hemagglutinin II showed a large amount of aspartic acid and serine, but as little as 0.1 mol/100 mol of cystine or methionine could be detected.

  1. NMR screening of new carbocyanine dyes as ligands for affinity chromatography.

    PubMed

    Cruz, Carla; Boto, Renato E F; Drzazga, Anna K; Almeida, Paulo; Queiroz, João A

    2014-04-01

    Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N-carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α-chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un-substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α-chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α-chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD-NMR technique was successfully used to screen cyanine-protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography.

  2. Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography.

    PubMed

    Jaiswal, Nivedita; Pandey, Veda P; Dwivedi, Upendra N

    2015-01-01

    A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70 °C) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold purification of laccase with a specific activity of 41.3 units mg(-1) and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ∼ 260 kDa. The purified enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate (ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable up to 70 °C such that it retained ∼ 80% activity upon 30 min incubation at 70 °C. The Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol(-1). The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (K(cat)/K(m)) in the order of 7.25>0.67>0.27 mM(-1) min(-1) for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn(2+), Cd(2+), Ca(2+), Na(+), Fe(2+), Co(2+) and Cu(2+) while weakly inhibited by Hg(2+). The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising candidate enzyme for industrial exploitation.

  3. Characterization of minor site probes for human serum albumin by high-performance affinity chromatography.

    PubMed

    Sengupta, A; Hage, D S

    1999-09-01

    This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cisor trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole-benzodiazepine site. However, phenol red did bind at the warfarin-azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the general relationship between these binding regions on HSA. This information should be useful in future studies that employ HPAC for characterizing the binding of HSA to other drugs or clinical agents.

  4. Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Alves, Nathan J; Bilgicer, Basar

    2013-05-21

    This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

  5. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

  6. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  7. Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

    PubMed

    Zacharis, Constantinos K; Kalaitzantonakis, Eftichios A; Podgornik, Ales; Theodoridis, Georgios

    2007-03-09

    In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

  8. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-15

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

  9. Purification and characterization of two types of Cytisus multiflorus hemagglutinin by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Tsuji, T; Matsumoto, I; Osawa, T

    1983-10-01

    Two hemagglutinins were separated from extracts of Cytisus multiflorus seeds by successive affinity chromatographies on columns of galactose- and di- N-acetylchitobiose-Sepharose 4B. One was found to be inhibited by di- N-acetylchitobiose or tri- N-acetylchitotriose and shown to possess anti-H(O) activity [Cytisus-type anti-H(O) hemagglutinin designated as Cytisus multiflorus hemagglutinin I]. The other, which was not a blood group-specific hemagglutinin, was inhibited by galactose or lactose (hemagglutinin II). Hemagglutinins I and II were further purified by gel filtration on Sephacryl S-300. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular weights of the purified hemagglutinins I and II were found to be 86000 by sedimentation equilibrium analysis and 80000 by gel filtration. On disc gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, both hemagglutinins gave a single component of a molecular weight of 42000 +/- 2000, suggesting that these hemagglutinins are dimeric proteins of two identical subunits. Hemagglutinins I and II contain 2.7% and 1.5% carbohydrate, respectively, and only very small amounts of cystine and methionine were detected, but they are rich in aspartic acid and serine. Treatment of human O erythrocytes with a purified H-decomposing enzyme (alpha-L-fucosidase from Bacillus fulminans abolished the agglutinability of the cells with hemagglutinin I. This indicates that the L-fucosyl residue is important even for the H-specificity detected by this di-N-acetylchitobiose-specific hemagglutinin I.

  10. Affinity-based methods in drug-target discovery.

    PubMed

    Rylova, Gabriela; Ozdian, Tomas; Varanasi, Lakshman; Soural, Miroslav; Hlavac, Jan; Holub, Dusan; Dzubak, Petr; Hajduch, Marian

    2015-01-01

    Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the "bench" scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.

  11. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  12. Liquid chromatography detection unit, system, and method

    DOEpatents

    Derenzo, Stephen E.; Moses, William W.

    2015-10-27

    An embodiment of a liquid chromatography detection unit includes a fluid channel and a radiation detector. The radiation detector is operable to image a distribution of a radiolabeled compound as the distribution travels along the fluid channel. An embodiment of a liquid chromatography system includes an injector, a separation column, and a radiation detector. The injector is operable to inject a sample that includes a radiolabeled compound into a solvent stream. The position sensitive radiation detector is operable to image a distribution of the radiolabeled compound as the distribution travels along a fluid channel. An embodiment of a method of liquid chromatography includes injecting a sample that comprises radiolabeled compounds into a solvent. The radiolabeled compounds are then separated. A position sensitive radiation detector is employed to image distributions of the radiolabeled compounds as the radiolabeled compounds travel along a fluid channel.

  13. Robust activation method for negative electron affinity photocathodes

    DOEpatents

    Mulhollan, Gregory A.; Bierman, John C.

    2011-09-13

    A method by which photocathodes(201), single crystal, amorphous, or otherwise ordered, can be surface modified to a robust state of lowered and in best cases negative, electron affinity has been discovered. Conventional methods employ the use of Cs(203) and an oxidizing agent(207), typically carried by diatomic oxygen or by more complex molecules, for example nitrogen trifluoride, to achieve a lowered electron affinity(404). In the improved activation method, a second alkali, other than Cs(205), is introduced onto the surface during the activation process, either by co-deposition, yo-yo, or sporadic or intermittent application. Best effect for GaAs photocathodes has been found through the use of Li(402) as the second alkali, though nearly the same effect can be found by employing Na(406). Suitable photocathodes are those which are grown, cut from boules, implanted, rolled, deposited or otherwise fabricated in a fashion and shape desired for test or manufacture independently supported or atop a support structure or within a framework or otherwise affixed or suspended in the place and position required for use.

  14. Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography.

    PubMed

    Robichon, Carine; Luo, Jianying; Causey, Thomas B; Benner, Jack S; Samuelson, James C

    2011-07-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

  15. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  16. Copper(II)-based metal affinity chromatography for the isolation of the anticancer agent bleomycin from Streptomyces verticillus culture.

    PubMed

    Gu, Jiesi; Codd, Rachel

    2012-10-01

    The glycopeptide-based bleomycins are structurally complex natural products produced by Streptomyces verticillus used in combination therapy against testicular and other cancers. Bleomycin has a high affinity towards a range of transition metal ions with the 1:1 Fe(II) complex relevant to its mechanism of action in vivo and the 1:1 Cu(II) complex relevant to its production from culture. The affinity between Cu(II) and bleomycin was the underlying principle for using Cu(II)-based metal affinity chromatography in this work to selectively capture bleomycin from crude S. verticillus culture. A solution of standard bleomycin was retained at a binding capacity of 300 nmol mL(-1) on a 1-mL bed volume of Cu(II)-loaded iminodiacetate (IDA) resin at pH 9 via the formation of the heteroleptic immobilized complex [Cu(IDA)(bleomycin)]. Bleomycin was eluted from the resin at pH 5 as the metal-free ligand under conditions where pK(a) (IDA)affinity chromatography as a green chemistry platform for streamlined access to this high-value therapeutic agent.

  17. Rapid purification of mitochondrial hexokinase from rat brain by a single affinity chromatography step on Affi-Gel blue.

    PubMed

    Wilson, J E

    1989-01-01

    The mitochondrial hexokinase from rat brain, selectively released from mitochondria by the action of glucose 6-phosphate, can be purified to greater than 90% homogeneity by a single affinity chromatography step on Affi-Gel Blue; the Cibacron Blue F3GA ligand bound to this matrix serves as an analog of ATP, the normal substrate for the enzyme, and selective elution is accomplished using glucose 6-phosphate which is a competitive ligand vs. ATP. With this and other modifications to the previously described procedure highly purified enzyme is readily obtained in good yield and with retention of the ability to rebind to mitochondria.

  18. The antigenicity in guinea pigs and monkeys of three mycobacterial polysaccharides purified by affinity chromatography with concanavalin A.

    PubMed

    Daniel, T M

    1975-06-01

    The antigenicity of 3 polysaccharides purified from culture filtrates of Mycobacterim tuberculosis by affinity chromatography using a concanavalin A-agarose absorbent was studied. All 3 purified polysaccharides were found to be potent elicitors of delayed skin test reactions in sensitized guinea pigs and in a tuberculos monkey. This antigenicity could not be attributed to contaminating protein. Small dermal reactions were also observed in control guinea pigs. All 3 polysaccharides reacted with precipitating antibody in guinea pig sera, the antigenic specificity observed with the guinea pig sera differing from that demonstrated with reference goat antiserum. The 3 polysaccharides were also demonstrated to contain hemagglutination antigenic sites.

  19. Methods for determining the genetic affinity of microorganisms and viruses

    NASA Technical Reports Server (NTRS)

    Fox, George E. (Inventor); Willson, III, Richard C. (Inventor); Zhang, Zhengdong (Inventor)

    2012-01-01

    Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

  20. Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.

    PubMed

    Merkley, Eric D; Anderson, Brian J; Park, Jea; Belchik, Sara M; Shi, Liang; Monroe, Matthew E; Smith, Richard D; Lipton, Mary S

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide-spectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1×10(-4) was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  1. Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

    SciTech Connect

    Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.; Belchik, Sara M.; Shi, Liang; Monroe, Matthew E.; Smith, Richard D.; Lipton, Mary S.

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  2. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

    PubMed

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E; Yates, John R

    2011-11-04

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

  3. Immobilized fusion protein affinity chromatography combined with HPLC-ESI-Q-TOF-MS/MS for rapid screening of PPARγ ligands from natural products.

    PubMed

    Zhu, Junfeng; Yi, Xiaojiao; Liu, Wenhui; Xu, Yingchun; Chen, Shuqing; Wu, Yongjiang

    2017-04-01

    Screening agonists of peroxisome proliferator-activated receptor-γ (PPARγ) from natural products is particularly motivated by the need for effective anti-diabetic agents. However, method for direct identification of PPARγ ligands from a complex sample is rarely reported. Here we propose a novel immobilized fusion protein affinity chromatography (IFPAC) to achieve rapid multicomponent screening. First, functional human PPARγ ligand binding domain (hPPARγLBD) was bacterially produced by fusion to glutathione S-transferase (GST). The unpurified GST-hPPARγLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose. Due to the strong affinity between GST and glutathione, the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization, which was rapidly purified under non-denaturing conditions. Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds. The proposed approach was applied in the high-throughput screening of PPARγ ligands from natural products, followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS. Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARγ ligand. Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.

  4. Synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide), a conjugate useful for affinity chromatography and for testosterone immunoassays.

    PubMed

    Luppa, P; Hauck, S; Schwab, I; Birkmayer, C; Hauptmann, H

    1996-01-01

    We describe the synthesis of 17 beta-hydroxyandrost-4-en-3-one-7 alpha-(biotinyl-6-N-hexylamide) from 17 beta-hydroxyandrost-4-en-3-one (testosterone) via copper-catalyzed 1,6 Michael addition of a 6-(tertbutyldimethylsilyloxyhexyl) chain to 6-dehydrotestosterone 17 beta-acetate. After chromatographic separation of the 7 alpha-isomer from the alpha / beta mixture and cleavage of the silyl ether, the alcohol was oxidized to the 6-hexanal side chain and then subjected to reductive amination. The resulting primary amine is easily biotinylated using biotinyl-N-hydroxysuccinimide ester. The overall yield for the epimeric 7 alpha-end product was 30%. The absolute configurations of the epimers were investigated by 1H NMR studies by the nuclear Overhauser effect. We introduced a biotin label to the testosterone molecule at ring position 7 in compliance with Landsteiner's principle, which states that antibody specificity is directed primarily at that portion of the hapten furthest from the functional group linking it to the carrier protein. Thus, this negligible alteration in comparison to the structure of the respective testosterone hapten used to elicit antibodies offers the feasibility of applying the testosterone derivative as an optimal immunoadsorbent in affinity chromatography. The 7 alpha-biotinylated testosterone was used to obtain active antitestosterone antibodies from a specific antiserum by affinity chromatography. This was achieved by attaching the biotinylated testosterone to agarose-coupled streptavidin beads. Accordingly, a 3H-testosterone-binding test demonstrated a 20-fold increase in affinity of the purified antibody to the steroid compared to the original antiserum, and a recovery of > 80% could be obtained. The antitestosterone antibody, obtained by that method, is an effective component for use in a competitive immunoassay for testosterone in human sera. An assay configuration is conceivable with the same 7 alpha-biotinylated testosterone employed as

  5. Chromatography on DEAE ion-exchange and Protein G affinity columns in tandem for the separation and purification of proteins.

    PubMed

    Qi, Y; Yan, Z; Huang, J

    2001-10-30

    A high-performance liquid-chromatographic method based on coupled DEAE anion-exchange and Protein G affinity columns has been developed for the simultaneous separation and purification of immunoglobulin G and albumin from mouse serum. The diluted mouse serum was injected directly into this system, and the proteins were eluted separately from the DEAE and Protein G columns, coupled in series, by the column-switching technique. The advantages of this method are that IgG and albumin can be separated and purified simultaneously, the expensive affinity column is protected from contamination by the impurities in the mouse serum, and it is fast, selective, robust, and reproducible.

  6. Analysis of Free Drug Fractions in Serum by Ultrafast Affinity Extraction and Two-Dimensional Affinity Chromatography using α1-Acid Glycoprotein Microcolumns

    PubMed Central

    Bi, Cong; Zheng, Xiwei; Hage, David S.

    2016-01-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830 ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20 min when using only 3–10 µL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  7. Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine.

    PubMed

    Yang, Xiaoting; Hu, Yufei; Li, Gongke

    2014-05-16

    Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N'-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17-243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06-0.80μg/L), good linearity (with R(2) between 0.9979 and 0.9993). The recoveries in urine samples were 81.0-105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1-8.2% and 3.7-10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample.

  8. High performance affinity chromatography (HPAC) as a high-throughput screening tool in drug discovery to study drug-plasma protein interactions.

    PubMed

    Vuignier, Karine; Guillarme, Davy; Veuthey, Jean-Luc; Carrupt, Pierre-Alain; Schappler, Julie

    2013-02-23

    Drug-plasma protein binding is an important parameter that, together with other physicochemical properties such as lipophilicity and pK(a), greatly influences drug absorption, distribution, metabolism, and excretion (ADME). Therefore, it is important for pharmaceutical companies to develop a rapid screening assay to examine plasma protein binding during the early stages of the drug discovery process. Human serum albumin (HSA) and α(1)-acid glycoprotein (AGP) are the most important plasma proteins that are capable of binding drugs. In this work, an automated and high-throughput (<3 min/compound) strategy was developed using high performance affinity chromatography (HPAC) with commercial HSA and AGP columns to evaluate drug-plasma protein interactions for drug screening. A generic gradient was used throughout the study to separate drugs that were weakly and tightly bound to HSA and AGP. To accelerate the analysis time, the system was calibrated in a single run by pooling reference compounds without overloading the column. For both HSA and AGP studies, the developed methods were successfully transferred from HPAC-UV to HPAC-MS with single quadrupole MS detection and ammonium acetate, pH 7.0 as a volatile mobile phase. The MS detection enhanced the sensitivity, selectivity, and throughput of the method by pooling unknown compounds. For HSA analyses, the binding percentages obtained using HPAC were well correlated with the binding percentages from the literature. This method was also able to rank compounds based on their affinity for HSA. Concerning the AGP analyses, the quality of the correlation between the binding percentages obtained in HPAC and those from the literature was weaker. However, the method was able to classify compounds into weak, medium, and strong binders and rank compounds based on their affinity for AGP.

  9. Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose.

    PubMed

    Perła, Joanna; Undas, Anetta; Twardowski, Tomasz; Jakubowski, Hieronim

    2004-08-05

    Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.

  10. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    PubMed Central

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-01-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  11. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    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 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally.

  12. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins.

  13. Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

    2007-04-10

    In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

  14. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  15. A feasible dual affine scaling steepest descent method for the linear semidefinite programming problem

    NASA Astrophysics Data System (ADS)

    Zhadan, V. G.

    2016-07-01

    The linear semidefinite programming problem is considered. The dual affine scaling method in which all current iterations belong to the feasible set is proposed for its solution. Moreover, the boundaries of the feasible set may be reached. This method is a generalization of a version of the affine scaling method that was earlier developed for linear programs to the case of semidefinite programming.

  16. Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

    PubMed

    Teng, S F; Sproule, K; Husain, A; Lowe, C R

    2000-03-31

    A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

  17. Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment.

    PubMed

    Xia, Hui; Murray, Kermit; Soper, Steven; Feng, June

    2012-02-01

    Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

  18. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13.

  19. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  20. Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration.

    PubMed

    Robinson, F Darlene; Moxley, Robert A; Jarrett, Harry W

    2004-01-23

    C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred. After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.

  1. Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis.

    PubMed

    Song, Yufeng; Zhang, Hongxiao; Chen, Chen; Wang, Guiping; Zhuang, Kai; Cui, Jin; Shen, Zhenguo

    2014-04-01

    Copper (Cu) is an essential micronutrient required for plant growth and development. However, excess Cu can inactivate and disturb protein structure as a result of unavoidable binding to proteins. To understand better the mechanisms involved in Cu toxicity and tolerance in plants, we developed a new immobilized metal affinity chromatography (IMAC) method for the separation and isolation of Cu-binding proteins extracted from roots of rice seedling exposed to excess Cu. In our method, IDA-Sepharose or EDDS-Sepharose column (referred as pre-chromatography) and Cu-IDA-Sepharose column (referred as Cu-IMAC) were connected in tandem. Namely, protein samples were pre-chromatographed with IDA-Sepharose column to removal metal ions, then protein solution was flowed into Cu-IMAC column for enriching Cu-binding proteins in vitro. Compared with the control (Cu-IMAC without any pre-chromatography), IDA-Sepharose pre-chromatography method markedly increased yield of the Cu-IMAC-binding proteins, and number of protein spots and the abundance of 40 protein spots on two-dimensional electrophoresis (2-DE) gels. Thirteen protein spots randomly selected from 2-DE gel and 11 proteins were identified using MALDI-TOF-TOF MS. These putative Cu-binding proteins included those involved in antioxidant defense, carbohydrate metabolism, nucleic acid metabolism, protein folding and stabilization, protein transport and cell wall synthesis. Ten proteins contained one or more of nine putative metal-binding motifs reported by Smith et al. (J Proteome Res 3:834-840, 2004) and seven proteins contained one or two of top six motifs reported by Kung et al. (Proteomics 6:2746-2758, 2006). Results demonstrated that more proteins specifically bound with Cu-IMAC could be enriched through removal of metal ions from samples by IDA-Sepharose pre-chromatography. Further studies are needed on metal-binding characteristics of these proteins in vivo and the relationship between Cu ions and protein biological

  2. Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.

    PubMed Central

    Srivastava, P N; Farooqui, A A

    1979-01-01

    Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C. Images Fig. 4. PMID:540029

  3. An illustration of the clinical relevance of detecting human antimouse antibody interference by affinity chromatography.

    PubMed

    Koper, N P; Massuger, L F; Thomas, C M; Beyer, C; Crooy, M J

    1999-10-01

    Elevated Cancer antigen 125 (CA 125) serum concentrations (up to 221 kU/1) were measured in a 39 year old woman with a positive family history of breast cancer. The serum determinations were performed with the automated Immulite OM-MA chemiluminescent enzyme immunoassay system (Diagnostic Products). Laparoscopic evaluation of the ovaries did not reveal any abnormalities. CA 125 measurements in the same patient using the automated IMx immunoassay system (Abbott) demonstrated normal serum levels. Using a previously reported chromatography procedure IgG type human antimouse antibody activity was found to be present in the serum samples explaining the falsely elevated levels. To prevent this interference the manufacturer modified the assay system by replacing the monoclonal M11 detection antibody with a rabbit polyclonal antibody. Using the modified OM-MA CA 125 assay results were comparable with the IMx values.

  4. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  5. Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

    PubMed

    Baieli, María F; Urtasun, Nicolás; Martinez, María J; Hirsch, Daniela B; Pilosof, Ana M R; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

    2017-01-01

    Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

  6. New approach for separating Bacillus subtilis metalloprotease and alpha-amylase by affinity chromatography and for purifying neutral protease by hydrophobic chromatography.

    PubMed

    Lauer, I; Bonnewitz, B; Meunier, A; Beverini, M

    2000-01-14

    Proteases are commonly used in the biscuit and cracker industry as processing aids. They cause moderate hydrolysis of gluten proteins and improve dough rheology to better control product texture and crunchiness. Commercial bacterial proteases are derived from Bacillus fermentation broth. As filtration and ultrafiltration are carried out as the only recovery steps, these preparations contain also alpha-amylase and beta-glucanase as the main side activities. The aim of this study is to purify and characterize the Bacillus subtilis metalloprotease from a commercial preparation, in order to study separately the impact of the protease activity with regards to its functionality on biscuit properties. Purification was achieved by means of affinity chromatography on Cibacron Blue and HIC as a polishing step. Affinity appeared to be the most appropriate matrix for large scale purification while ion exchange chromatography was inefficient in terms of recovery yields. The crude product was first loaded on a Hi Trap Blue column (34 microm, Pharmacia Biotech); elution was carried out with a gradient of NaCl in the presence of 1 mM ZnCl2. This step was only efficient in the presence of Zn cations, because this salt promoted both protease stabilization resulting in high recovery yields and also complexation of amylase units into dimers resulting in amylase retention on the column and a better separation of the 3 activities. Beta-glucanase was mostly non retained on the column and a part was coeluted with the protease. This protease fraction was then loaded on a Resource Phe column (15 microm, Pharmacia Biotech) in a last step of polishing. Elution was carried out with a linear gradient of 100-0% ammonium sulfate 1.3 M; protease was eluted at the beginning of the gradient and well separated from amylase and glucanase trace impurities. The homogeneity of the purified protease was confirmed by SDS-PAGE, which showed that its MW was about 38. pH and temperature optima were also

  7. Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

    PubMed Central

    Callaghan, J M; Toh, B H; Simpson, R J; Baldwin, G S; Gleeson, P A

    1992-01-01

    We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized

  8. Isolation of two molecular populations of human complement factor H by hydrophobic affinity chromatography.

    PubMed Central

    Ripoche, J; Al Salihi, A; Rousseaux, J; Fontaine, M

    1984-01-01

    Human complement factor H was prepared in highly purified form from fresh serum by euglobulin precipitation, DEAE-Sephacel chromatography and Sephacryl S-300 gel filtration. This preparation allowed the recovery of 37% of the initial factor H. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that factor H was homogeneous both in reduced and non-reduced media and exhibited a molecular mass of 150 kDa. Charge-shift experiments clearly showed the presence of hydrophobic sites in the factor H molecule. Charge shifts were observed with two detergent systems (Triton/sodium deoxycholate and Triton/cetyltrimethylammonium bromide). Factor H was able to bind to phenyl-Sepharose. This property allowed us to study two populations of factor H. These two populations exhibited the same physicochemical parameters, but revealed differences in their ability to aggregate in low- and iso-ionic-strength media. The molecular basis and biological significance of this heterogeneity are discussed. Images Fig. 3. Fig. 4. Fig. 5. PMID:6235808

  9. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  10. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  11. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  12. Purification of a lectin from M. rubra leaves using immobilized metal ion affinity chromatography and its characterization.

    PubMed

    Sureshkumar, Thavamani; Priya, Sulochana

    2012-12-01

    Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, D-galactosamine and N-acetyl-D-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4-11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe(2+), Ca(2+), Zn(2+), Ni(2+), Mn(2+), Na(+) and K(+), HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3(+) and Al(3+)) and Cu(2+) did not affect the activity. In the presence of Mg(2+) and Hg(2+), only 25 % of HA activity remained.

  13. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography.

    PubMed

    Machado, Gleyce Alves; Oliveira, Heliana Batista de; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-05-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

  14. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

    SciTech Connect

    Niture, Suryakant K.; Doneanu, Catalin E.; Velu, Chinavenmeni S.; Bailey, Nathan I.; Srivenugopal, Kalkunte S. . E-mail: Kalkunte.srivenugopal@ttuhsc.edu

    2005-12-02

    Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

  15. Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

    PubMed Central

    Gilbert, H J; Lowe, C R; Drabble, W T

    1979-01-01

    Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. PMID:44191

  16. Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer

    PubMed Central

    Ambrose, Sarah R.; Gordon, Naheema S.; Goldsmith, James C.; Wei, Wenbin; Zeegers, Maurice P.; James, Nicholas D.; Knowles, Margaret A.; Bryan, Richard T.; Ward, Douglas G.

    2015-01-01

    Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers. PMID:28248271

  17. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

    NASA Astrophysics Data System (ADS)

    Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

  18. Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1

    PubMed Central

    Bonza, Cristina; Carnelli, Antonella; De Michelis, Maria Ida; Rasi-Caldogno, Franca

    1998-01-01

    The Ca2+-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thaliana chloroplast envelope. PMID:9490776

  19. Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

    NASA Astrophysics Data System (ADS)

    Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

    2016-10-01

    Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

  20. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

    PubMed Central

    Machado, Gleyce Alves; de Oliveira, Heliana Batista; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-01-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (Junbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJunbound) and aqueous (AJunbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for Junbound, 92.5% and 93.5% for DJunboundand 82.5% and 82.6% for AJunbound. By immunoblot, the DJunboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJunboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot. PMID:23778661

  1. LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.

    PubMed

    Song, Ehwang; Zhu, Rui; Hammoud, Zane T; Mechref, Yehia

    2014-11-07

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC-MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC-ESI-MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts.

  2. Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane.

    PubMed

    Zhao, Yingxin; Zhang, Wei; White, Michael A; Zhao, Yingming

    2003-08-01

    Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.

  3. Minibodies and Multimodal Chromatography Methods: A Convergence of Challenge and Opportunity.

    PubMed

    Gagnon, Pete; Cheung, Chia-Wei; Lepin, Eric J; Wu, Anna M; Sherman, Mark A; Raubitschek, Andrew A; Yazaki, Paul J

    2010-02-01

    This case study describes early phase purification process development for a recombinant anticancer minibody produced in mammalian cell culture. The minibody did not bind to protein A. Cation-exchange, anion-exchange, hydrophobic-interaction, and hydroxyapatite (eluted by phosphate gradient) chromatographic methods were scouted, but the minibody coeluted with BSA to a substantial degree on each. Hydroxyapatite eluted with a sodium chloride gradient separated BSA and also removed a dimeric contaminant, but BSA consumed so much binding capacity that this proved impractical as a capture tool. Capto MMC media proved capable of supporting adequate capture and significant dimer removal, although both loading and elution selectivity varied dramatically with the amount of supernatant applied to the column. An anion-exchange step was included to fortify overall virus and DNA removal. These results illustrate the value of multimodal chromatography methods when affinity chromatography methods are lacking and conventional alternatives prove inadequate.

  4. Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

    PubMed

    Regnault, V; Rivat, C; Vallar, L; Geschier, C; Stolz, J F

    1992-12-11

    The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

  5. Enantioseparation of nuarimol by affinity electrokinetic chromatography-partial filling technique using human serum albumin as chiral selector.

    PubMed

    Martínez-Gómez, Maria Amparo; Escuder-Gilabert, Laura; Villanueva-Camañas, Rosa M; Sagrado, Salvador; Medina-Hernández, Maria J

    2008-10-01

    The present paper deals with the enantiomeric separation of nuarimol enantiomers by affinity EKC-partial filling technique using HSA as chiral selector. Firstly, a study of nuarimol interactions with HSA by CE-frontal analysis was performed. The binding parameters obtained for the first site of interaction were n(1) = 0.84; K(1) = 9.7 +/- 0.3x10(3 )M(-1) and the protein binding percentage of nuarimol at physiological concentration of HSA was 75.2 +/- 0.2%. Due to the moderate affinity of nuarimol towards HSA the possibility of using this protein as chiral selector for the separation of nuarimol using the partial filling technique was evaluated. A multivariate optimization approach of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length was carried out. Separation of nuarimol enantiomers was obtained under the following selected conditions: electrophoretic buffer composed of 50 mM Tris at pH 7.3; 160 muM HSA solution applied at 50 mbar for 156 s as chiral selector; nuarimol solutions in the range of 2-8x10(-4) M injected hydrodynamically at 30 mbar for 2 s and the electrophoretic runs performed at 30 degrees C applying 15 kV voltage. Resolution, accuracy, reproducibility speed and cost of the proposed method make it suitable for quality control of the enantiomeric composition of nuarimol in formulations and for further toxicological studies. The results showed a different affinity between nuarimol enantiomers towards HSA.

  6. An in depth proteomic analysis based on ProteoMiner, affinity chromatography and nano-HPLC-MS/MS to explain the potential health benefits of bovine colostrum.

    PubMed

    Altomare, Alessandra; Fasoli, Elisa; Colzani, Mara; Parra, Ximena Maria Paredes; Ferrari, Marina; Cilurzo, Francesco; Rumio, Cristiano; Cannizzaro, Luca; Carini, Marina; Righetti, Pier Giorgio; Aldini, Giancarlo

    2016-03-20

    Bovine colostrum (BC), the initial milk secreted by the mammary gland immediately after parturition, is widely used for several health applications. We here propose an off-target method based on proteomic analysis to explain at molecular level the potential health benefits of BC. The method is based on the set-up of an exhaustive protein data bank of bovine colostrum, including the minor protein components, followed by a bioinformatic functional analysis. The proteomic approach based on ProteoMiner technology combined to a highly selective affinity chromatography approach for the immunoglobulins depletion, identified 1786 proteins (medium confidence; 634 when setting high confidence), which were then clustered on the basis of their biological function. Protein networks were then created on the basis of the biological functions or health claims as input. A set of 93 proteins involved in the wound healing process was identified. Such an approach also permits the exploration of novel biological functions of BC by searching in the database the presence of proteins characterized by innovative functions. In conclusion an advanced approach based on an in depth proteomic analysis is reported which permits an explanation of the wound healing effect of bovine colostrum at molecular level and allows the search of novel potential beneficial effects.

  7. Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Aryal, Uma K; Krochko, Joan E; Ross, Andrew R S

    2012-01-01

    Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.

  8. Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection.

    PubMed

    Yari, Sh; Hadizadeh Tasbiti, A; Fateh, A; Karimi, A; Yari, F; Sakhai, F; Ghazanfari, M; Bahrmand, A

    2011-11-01

    Tuberculosis has been declared a global emergency. The mainstay for its control is the rapid and accurate identification of infected individual. Antibodies to A60, one of the macromolecular antigen complexes of mycobacteria were commonly used in the rapid detection of Mycobacterium tuberculosis. The aim of this study was to prepare specific antibodies against A60 for detection of tuberculosis infection. Specific polyclonal antibodies against A60, (A60-Ab) were prepared in rabbits using 2 boosted injections of the antigen (A60). The antibodies were purified and treated with normal oral flora to remove any non-specific and cross-reactive antibodies. These antibodies were conjugated to CNBr-activated Sepharose 4B and used to isolate subunits of A60 with more specificity for M. tuberculosis. A new affinity column was designed to prepare modified (purified) A60 antigen. Purified A60 antigen (PA60-Ag) was used to develop antibody production by Immunoaffinity chromatography. 113 patients with a confirmed diagnosis of pulmonary TB at Pasteur Institute were selected for the study. The specificity of the results was analyzed with TB-rapid test by using PA60-antibodies. TB-rapid test revealed that normal oral flora-absorbed antibodies could lead to more specific results than that of the non-absorbed antibodies. The developed, modified A60 antibodies, (PA60-Ab)-rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. Moreover, PA60-Ag showed only two protein bands of molecular weight 45 and 66kDa in SDS-PAGE while untreated A60 showed multiple bands. Thus, our study helped in the purification of a novel and well characterized A60 antigen and good diagnostic potential for detecting tuberculosis infection.

  9. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  10. Purification and characterization of two types of Cytisus sessilifolius anti-H(O) lectins by affinity chromatography.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T

    1991-02-01

    Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.

  11. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  12. The purification of human enterokinase by affinity chromatography and immunoadsorption. Some observations on its molecular characteristics and comparisons with the pig enzyme.

    PubMed Central

    Grant, D A; Hermon-Taylor, J

    1976-01-01

    A method is described for the purification of human enterokinase from accumulated duodenal fluid by affinity chromatography using p-aminobenzamidine as the ligand. Resolution was greatest when glycylglycine was substituted as the spacer arm. Purification was not a one-step procedure, and some contamination, principally by the alpha-glucosidases, remained. Their removal was completed by immunoadsorption using antisera raised to enterokinase-free material containing these enzymes, prepared as a by-product of the purification procedure. The final preparation had an activity of 4260 nmol of trypsin/min per mg and was free of other enzymic activity tested. Amino acid and sugar analyses of the highly purified enzyme indicated an acidic glycoprotein containing 57% sugar (neutral sugars 47%, amino sugars 10%). The apparent mol.wts. and Stokes radii of human and pig enterokinase were 296 000 and 316 000, and 5.65 and 5.78 nm respectively. Two isoenzymes were identified for human enterokinase and three for the pig enzyme. Human enterokinase demonstrated a resistance to reduction of disulphide linkages and to sodium dodecyl sulphate binding, which may be related to the need for it to retain its integrity in the digestive environment of the upper small intestine. Antisera to highly purified pig and human enterokinases specifically inhibited enterokinase activity. Immuno-inhibition of intestinal aminopeptidase, maltase and glucoamylase by homologous antisera was not observed. Images PLATE 1 PMID:945736

  13. Enantioseparation of phenotiazines by affinity electrokinetic chromatography using human serum albumin as chiral selector: application to enantiomeric quality control in pharmaceutical formulations.

    PubMed

    Martínez-Gómez, María Amparo; Sagrado, Salvador; Villanueva-Camañas, Rosa María; Medina-Hernández, Maria José

    2007-01-23

    Nowadays, there is a special interest within the pharmaceutical laboratories to develop single enantiomer formulations and consequently a need for analytical methods to determine the enantiomeric purity of drugs. The present paper deals with the enantiomeric separation of promethazine and trimeprazine enantiomers by affinity electrokinetic chromatography (AEKC)-partial filling technique using human serum albumin (HSA) as chiral selector. A multivariate optimization of the most critical experimental variables in enantioresolution, running pH, HSA concentration and plug length, is carried out to obtain enantioresolution of promethazine and trimeprazine. The estimated maximum and optimum resolution of trimeprazine and prometazine enantiomers (Rs=1.74 and 2.01, respectively) corresponded to the following experimental conditions: pH 7.5; [HSA] 170 microM and plug length 190 s and pH 7.6; [HSA] 170 microM and plug length 170 s, for trimeprazine and prometazine, respectively. The developed methodologies were applied for the enantiomeric quality control of promethazine and trimeprazine enantiomers in commercially available pharmaceutical formulations. Resolution, accuracy, reproducibility, cost and sample throughput of the proposed methodologies make it suitable for quality control of the enantiomeric composition of promethazine and trimeprazine in pharmaceutical preparations.

  14. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions.

  15. Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

    PubMed

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J; Dahlberg, Leif E; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-07-25

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

  16. Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry*

    PubMed Central

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J.; Dahlberg, Leif E.; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-01-01

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided. PMID:24917676

  17. Simultaneous speciation of selenoproteins and selenometabolites in plasma and serum by dual size exclusion-affinity chromatography with online isotope dilution inductively coupled plasma mass spectrometry.

    PubMed

    García-Sevillano, M A; García-Barrera, T; Gómez-Ariza, J L

    2014-04-01

    A method for the simultaneous speciation of selenoproteins and selenometabolites in mouse plasma has been developed based on in series two-dimensional size exclusion and affinity high-performance liquid chromatography (2D/SE-AF-HPLC), using two columns of each type, and hyphenation to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-QMS). The method allows the quantitative determination of selenoprotein P (SeP), extracellular glutathione peroxidase (eGPx), selenoalbumin (SeAlb), and selenometabolites in mouse plasma using species-unspecific isotope dilution (SUID). The 2D chromatographic separation is proposed to remove typical spectral interferences in plasma from chloride and bromide on (77)Se ((40)Ar(37)Cl) and (82)Se ((81)Br(1)H). In addition, the approach increases chromatographic resolution allowing the separation of eGPx from Se metabolites of low molecular mass. The method is robust, reliable, and fast with a typical chromatographic runtime less than 20 min. Precision in terms of relative standard deviation (n = 5) is in the order of 4 %, and detection limits are in the range of 0.2 to 1.0 ng Se g(-1). Method accuracy for determination of total protein bound to Se was assessed by analyzing human serum reference material (BCR-637) certified for total Se content, and latterly applied to mouse plasma (Mus musculus). In summary, a reliable speciation method for the analysis of eGPx, selenometabolites, SeP, and SeAlb in plasma/serum samples is proposed for the first time and is applicable to the evaluation of Se status in human in clinical studies and other mammals for environmental or toxicological assessment.

  18. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    PubMed

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

  19. Synthesis of sulfonamide- and sulfonyl-phenylboronic acid-modified silica phases for boronate affinity chromatography at physiological pH.

    PubMed

    Li, Xiaobao; Pennington, Justin; Stobaugh, John F; Schöneich, Christian

    2008-01-15

    Two new types of boronate affinity solid phases were synthesized and characterized. The materials were prepared by silylation of porous silica gel with monochlorosilane derivatives containing synthetic sulfonyl- and sulfonamide-substituted phenylboronic acids. The new solid phases were evaluated for boronate affinity chromatography with aryl and alkyl cis-diol compounds and were found to be suitable for the retention of cis-diols under acidic conditions. Significant correlations between the retention factor (K) and the pH of the mobile phase demonstrate that the binding of cis-diols to the solid phases is best rationalized by chelation. Based on the lower pKa, caused by the electron-withdrawing effects of the sulfonyl and sulfonamide groups, these media display an enhanced affinity for cis-diols as compared with unsubstituted phenylboronic acid. Using isocratic elution, a mixture of various biologically relevant l-tyrosines, l-DOPA, and several catecholamines were resolved with a mobile phase composed of 0.05M phosphate buffer (pH 5.5). Mono-, di-, and triphosphates of adenosine were also separated at pH 6.0. Hence, the new boronate solid phase offers efficient affinity separation and purification of cis-diol-containing molecules under rather mild pH conditions.

  20. Chromatography

    MedlinePlus

    Chromatography is a way of separating two or more chemical compounds. Chemical compounds are chemicals that are ... of chemical compound. There are different kinds of chromatography. These include gas, high pressure liquid, or ion ...

  1. Affine Projection Algorithm with Improved Data-Selective Method Using the Condition Number

    NASA Astrophysics Data System (ADS)

    Ban, Sung Jun; Lee, Chang Woo; Kim, Sang Woo

    Recently, a data-selective method has been proposed to achieve low misalignment in affine projection algorithm (APA) by keeping the condition number of an input data matrix small. We present an improved method, and a complexity reduction algorithm for the APA with the data-selective method. Experimental results show that the proposed algorithm has lower misalignment and a lower condition number for an input data matrix than both the conventional APA and the APA with the previous data-selective method.

  2. Isolation of a Trypanosoma cruzi antigen by affinity chromatography with a monoclonal antibody. Preliminary evaluation of its possible applications in serological tests.

    PubMed Central

    Carbonetto, C H; Malchiodi, E L; Chiaramonte, M; Durante de Isola, E; Fossati, C A; Margni, R A

    1990-01-01

    By affinity chromatography with a monoclonal antibody (163B6), obtained in our laboratory, we have isolated a T. cruzi antigen which could be useful for differential diagnosis of Chagas' disease from leishmaniasis. This antigen, a 52-kD protein, reacted with all sera from Chagas' disease patients tested but not with sera from patients with leishmania, in ELISA. The 52-kD antigen is widely distributed in the Trypanosoma genus since the 163B6 monoclonal antibody reacts with T. rangeli and 8 strains and a clone of T. cruzi epimastigotes. Images Fig. 1 Fig. 2 PMID:2119921

  3. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis.

    PubMed

    Scopes, R K

    1984-02-01

    2-Keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) has been isolated from extracts of Zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. The one-step procedure gives an enzyme with specific activity 600 units mg-1. Only 1 out of 47 dyes, Procion Yellow MX-GR, bound the enzyme completely in 20 mM phosphate buffer, pH 6.5. A column of Navy HE-R adsorbent was used first to remove most of the potentially adsorbing proteins.

  4. Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.

    PubMed

    Stocker-Majd, Gisela; Hilbrig, Frank; Freitag, Ruth

    2008-06-13

    Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

  5. A new affine-invariant image matching method based on SIFT

    NASA Astrophysics Data System (ADS)

    Wang, Peng-cheng; Chen, Qian; Chen, Hai-xin; Cheng, Hong-chang; Gong, Zhen-fei

    2013-09-01

    Local invariant feature extraction, as one of the main problems in the field of computer vision, has been widely applied to image matching, splicing and target recognition etc. Lowe's scale invariant feature transform (known as SIFT) algorithm has attracted much attention due to its invariance to scale, rotation and illumination. However, SIFT is not robust to affine deformations, because it is based on the DoG detector which extracts keypoints in a circle region. Besides, the feature descriptor is represented by a 128-dimensional vector, which means that the algorithm complexity is extremely large especially when there is a great quantity of keypoints in the image. In this paper, a new feature descriptor, which is robust to affine deformations, is proposed. Considering that circles turn to be ellipses after affine deformations, some improvements have been made. Firstly, the Gaussian image pyramids are constructed by convoluting the source image and the elliptical Gaussian kernel with two volatile parameters, orientation and eccentricity. In addition, the two parameters are discretely selected in order to imitate the possibilities of the affine deformation, which can make sure that anisotropic regions are transformed into isotropic ones. Next, all extreme points can be extracted as the candidates for the affine-invariant keypoints in the image pyramids. After accurate keypoints localization is performed, the secondary moment of the keypoints' neighborhood is calculated to identify the elliptical region which is affineinvariant, the same as SIFT, the main orientation of the keypoints can be determined and the feature descriptor is generated based on the histogram constructed in this region. At last, the PCA method for the 128-dimensional descriptor's reduction is used to improve the computer calculating efficiency. The experiments show that this new algorithm inherits all SIFT's original advantages, and has a good resistance to affine deformations; what's more, it

  6. Predicting the relative binding affinity of mineralocorticoid receptor antagonists by density functional methods

    NASA Astrophysics Data System (ADS)

    Roos, Katarina; Hogner, Anders; Ogg, Derek; Packer, Martin J.; Hansson, Eva; Granberg, Kenneth L.; Evertsson, Emma; Nordqvist, Anneli

    2015-12-01

    In drug discovery, prediction of binding affinity ahead of synthesis to aid compound prioritization is still hampered by the low throughput of the more accurate methods and the lack of general pertinence of one method that fits all systems. Here we show the applicability of a method based on density functional theory using core fragments and a protein model with only the first shell residues surrounding the core, to predict relative binding affinity of a matched series of mineralocorticoid receptor (MR) antagonists. Antagonists of MR are used for treatment of chronic heart failure and hypertension. Marketed MR antagonists, spironolactone and eplerenone, are also believed to be highly efficacious in treatment of chronic kidney disease in diabetes patients, but is contra-indicated due to the increased risk for hyperkalemia. These findings and a significant unmet medical need among patients with chronic kidney disease continues to stimulate efforts in the discovery of new MR antagonist with maintained efficacy but low or no risk for hyperkalemia. Applied on a matched series of MR antagonists the quantum mechanical based method gave an R2 = 0.76 for the experimental lipophilic ligand efficiency versus relative predicted binding affinity calculated with the M06-2X functional in gas phase and an R2 = 0.64 for experimental binding affinity versus relative predicted binding affinity calculated with the M06-2X functional including an implicit solvation model. The quantum mechanical approach using core fragments was compared to free energy perturbation calculations using the full sized compound structures.

  7. Preparation and evaluation of a phenylboronate affinity monolith for selective capture of glycoproteins by capillary liquid chromatography.

    PubMed

    Lin, Zi An; Pang, Ji Lei; Lin, Yao; Huang, Hui; Cai, Zong Wei; Zhang, Lan; Chen, Guo Nan

    2011-08-21

    A phenylboronate affinity monolith was prepared and applied to the selective capture of glycoproteins from unfractionated protein mixtures. The monolith was synthesized in a 100 μm i.d capillary by an in situ polymerization procedure using a pre-polymerization mixture consisting of 4-vinylphenylboronic acid (VPBA) as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, diethylene glycol and ethylene glycol as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The prepared monolith was characterized in terms of the morphology, pore property, and recognition property. The selectivity and dynamic binding capacity were evaluated by using standard glycoproteins and nonglycoproteins as model proteins. The chromatographic results demonstrated that the phenylboronate affinity monolith had higher selectivity and binding capacity for glycoprotein than nonglycoprotein. The resulting phenylboronate affinity monolith was used as the sorbent for in-tube solid phase microextraction (in-tube SPME), and the extraction performance of the monolith was assessed by capture of ovalbumin from egg white sample.

  8. A Better Method for Filling Pasteur Pipet Chromatography Columns

    ERIC Educational Resources Information Center

    Ruekberg, Ben

    2006-01-01

    An alternative method for the preparation of Pasteur pipet chromatography columns is presented that allows the column to be filled with solvent without bubbles and allows greater control of fluid flow while the materials to be separated are added. Students are required to wear gloves and goggles and caution should be used while handling glass…

  9. Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins).

    PubMed Central

    Haddad, J G; Kowalski, M A; Sanger, J W

    1984-01-01

    The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein. Images Fig. 1. Fig. 3. Fig. 4. PMID:6547042

  10. Affinity membrane chromatography: relationship of dye-ligand type to surface polarity and their effect on lysozyme separation and purification.

    PubMed

    Arica, M Yakup; Yilmaz, Meltem; Yalçin, Emine; Bayramoğlu, Gülay

    2004-06-15

    Two different dye-ligands, i.e. Procion Brown MX-5BR (RB-10) and Procion Green H-4G (RG-5) were immobilised onto poly(2-hydroxyethylmethacrylate) (pHEMA) membranes. The polarities of the affinity membranes were determined by contact angle measurements. Separation and purification of lysozyme from solution and egg white were investigated. The adsorption data was analysed using two adsorption kinetic models the first order and the second order to determine the best-fit equation for the separation of lysozyme using affinity membranes. The second-order equation for the adsorption of lysozyme on the RB-10 and RG-5 immobilised membranes systems is the most appropriate equation to predict the adsorption capacity for the affinity membranes. The reversible lysozyme adsorption on the RB-10 and RG-5 did not follow the Langmuir model, but obeyed the Temkin and Freundlich isotherm model. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purities of the eluted lysozyme, as determined by HPLC, were 76 and 92% with recovery 63 and 77% for RB-10 and RG-5 membranes, respectively. For the separation and purification of lysozyme the RG-5 immobilised membrane provided the best results. The affinity membranes are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-elution cycles.

  11. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    PubMed

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug.

  12. Method for resurrecting negative electron affinity photocathodes after exposure to an oxidizing gas

    DOEpatents

    Mulhollan, Gregory A; Bierman, John C

    2012-10-30

    A method by which negative electron affinity photocathodes (201), single crystal, amorphous, or otherwise ordered, can be made to recover their quantum yield following exposure to an oxidizing gas has been discovered. Conventional recovery methods employ the use of cesium as a positive acting agent (104). In the improved recovery method, an electron beam (205), sufficiently energetic to generate a secondary electron cloud (207), is applied to the photocathode in need of recovery. The energetic beam, through the high secondary electron yield of the negative electron affinity surface (203), creates sufficient numbers of low energy electrons which act on the reduced-yield surface so as to negate the effects of absorbed oxidizing atoms thereby recovering the quantum yield to a pre-decay value.

  13. Protein purification-free method of binding affinity determination by microscale thermophoresis.

    PubMed

    Khavrutskii, Lyuba; Yeh, Joanna; Timofeeva, Olga; Tarasov, Sergey G; Pritt, Samuel; Stefanisko, Karen; Tarasova, Nadya

    2013-08-15

    Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

  14. A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity.

    PubMed

    Boger, D L; Fink, B E; Brunette, S R; Tse, W C; Hedrick, M P

    2001-06-27

    Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.

  15. A Lyapunov method for stability analysis of piecewise-affine systems over non-invariant domains

    NASA Astrophysics Data System (ADS)

    Rubagotti, Matteo; Zaccarian, Luca; Bemporad, Alberto

    2016-05-01

    This paper analyses stability of discrete-time piecewise-affine systems, defined on possibly non-invariant domains, taking into account the possible presence of multiple dynamics in each of the polytopic regions of the system. An algorithm based on linear programming is proposed, in order to prove exponential stability of the origin and to find a positively invariant estimate of its region of attraction. The results are based on the definition of a piecewise-affine Lyapunov function, which is in general discontinuous on the boundaries of the regions. The proposed method is proven to lead to feasible solutions in a broader range of cases as compared to a previously proposed approach. Two numerical examples are shown, among which a case where the proposed method is applied to a closed-loop system, to which model predictive control was applied without a-priori guarantee of stability.

  16. Virus-binding proteins recovered from bacterial culture derived from activated sludge by affinity chromatography assay using a viral capsid peptide.

    PubMed

    Sano, Daisuke; Matsuo, Takahiro; Omura, Tatsuo

    2004-06-01

    The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society. Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed. In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered. The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H(2)N-DNPASTTNKDKL-COOH) was immobilized as a ligand. VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay. The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography. On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively. As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force. Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa. Amino acid sequences of N termini of five VBPs were determined. Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins. These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments. Therefore, a virus removal technology

  17. Calculating protein-ligand binding affinities with MMPBSA: Method and error analysis.

    PubMed

    Wang, Changhao; Nguyen, Peter H; Pham, Kevin; Huynh, Danielle; Le, Thanh-Binh Nancy; Wang, Hongli; Ren, Pengyu; Luo, Ray

    2016-10-15

    Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) methods have become widely adopted in estimating protein-ligand binding affinities due to their efficiency and high correlation with experiment. Here different computational alternatives were investigated to assess their impact to the agreement of MMPBSA calculations with experiment. Seven receptor families with both high-quality crystal structures and binding affinities were selected. First the performance of nonpolar solvation models was studied and it was found that the modern approach that separately models hydrophobic and dispersion interactions dramatically reduces RMSD's of computed relative binding affinities. The numerical setup of the Poisson-Boltzmann methods was analyzed next. The data shows that the impact of grid spacing to the quality of MMPBSA calculations is small: the numerical error at the grid spacing of 0.5 Å is already small enough to be negligible. The impact of different atomic radius sets and different molecular surface definitions was further analyzed and weak influences were found on the agreement with experiment. The influence of solute dielectric constant was also analyzed: a higher dielectric constant generally improves the overall agreement with experiment, especially for highly charged binding pockets. The data also showed that the converged simulations caused slight reduction in the agreement with experiment. Finally the direction of estimating absolute binding free energies was briefly explored. Upon correction of the binding-induced rearrangement free energy and the binding entropy lost, the errors in absolute binding affinities were also reduced dramatically when the modern nonpolar solvent model was used, although further developments were apparently necessary to further improve the MMPBSA methods. © 2016 Wiley Periodicals, Inc.

  18. Ultra-fast liquid chromatography with tandem mass spectrometry determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up.

    PubMed

    Yang, Xihui; Hu, Yichen; Kong, Weijun; Chu, Xianfeng; Yang, Meihua; Zhao, Ming; Ouyang, Zhen

    2014-11-01

    A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r(2) ≥ 0.9993), low limit of detection (0.5-0.8 μg/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 μg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices.

  19. Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library.

    PubMed

    Smith, Richard G; Missailidis, Sotiris; Price, Michael R

    2002-01-05

    A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.

  20. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    PubMed

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  1. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

    PubMed

    Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U

    2016-02-15

    Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO.

  2. Affinity chromatography of proteins on non-porous copolymerized particles of styrene, methyl methacrylate and glycidyl methacrylate.

    PubMed

    Chen, C H; Lee, W C

    2001-06-29

    Non-porous particles having an average diameter of 2.1 microm were prepared by co-polymerization of styrene, methyl methacrylate and glycidyl methacrylate, which was abbreviated as P(S-MMA-GMA). The particles were mechanically stable due to the presence of benzene rings in the backbone of polymer chains, and could withstand high pressures when a column packed with these particles was operated in the HPLC mode. The polymer particles were advantaged by immobilization of ligands via the epoxy groups on the particle surface that were introduced by one of the monomers, glycidyl methacrylate. As a model system, Cibacron Blue 3G-A was covalently immobilized onto the non-porous copolymer beads. The dye-immobilized P(S-MMA-GMA) particles were slurry packed into a 1.0 cm x 0.46 cm I.D. column. This affinity column was effective for the separation of turkey egg white lysozyme from a protein mixture. The bound lysozyme could be eluted to yield a sharp peak by using a phosphate buffer containing 1 M NaCl. For a sample containing up to 8 microg of lysozyme, the retained portion of proteins could be completely eluted without any slit peak. Due to the use of a shorter column, the analysis time was shorter in comparison with other affinity systems reported in the literature. The retention time could be reduced significantly by increasing the flow-rate, while the capacity factor remained at the same level.

  3. Integrating computational methods and experimental data for understanding the recognition mechanism and binding affinity of protein-protein complexes.

    PubMed

    Gromiha, M Michael; Yugandhar, K

    2017-01-07

    Protein-protein interactions perform several functions inside the cell. Understanding the recognition mechanism and binding affinity of protein-protein complexes is a challenging problem in experimental and computational biology. In this review, we focus on two aspects (i) understanding the recognition mechanism and (ii) predicting the binding affinity. The first part deals with computational techniques for identifying the binding site residues and the contribution of important interactions for understanding the recognition mechanism of protein-protein complexes in comparison with experimental observations. The second part is devoted to the methods developed for discriminating high and low affinity complexes, and predicting the binding affinity of protein-protein complexes using three-dimensional structural information and just from the amino acid sequence. The overall view enhances our understanding of the integration of experimental data and computational methods, recognition mechanism of protein-protein complexes and the binding affinity.

  4. Potential of human serum albumin as chiral selector of basic drugs in affinity electrokinetic chromatography-partial filling technique.

    PubMed

    Martínez-Gómez, Maria A; Villanueva-Camañas, R M; Sagrado, Salvador; Medina-Hernández, Maria J

    2006-11-01

    The enantiomeric resolution of compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer (BGE), protein, and compound solutions), and plug length. In this paper, the possibility of using HSA as chiral selector for enantioseparation of 28 basic drugs using this methodology is studied. The effect of the physicochemical parameters, the structural properties of compounds, and compound-HSA protein binding percentages over their chiral resolution with HSA is outlined. Based on the results obtained, a decision tree is proposed for the "a priori" prediction of the capability of HSA for enantioseparation of basic drugs in AEKC. The results obtained indicated that enantioresolution of basic compounds with HSA depends on the hydrophobicity, polarity, and molar volume of compounds.

  5. Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

    PubMed

    Roy, Ipsita; Jain, Sulakshana; Teotia, Sunita; Gupta, Munishwar Nath

    2004-01-01

    Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed.

  6. Modified electrokinetic sample injection method in chromatography and electrophoresis analysis

    DOEpatents

    Davidson, J. Courtney; Balch, Joseph W.

    2001-01-01

    A sample injection method for horizontal configured multiple chromatography or electrophoresis units, each containing a number of separation/analysis channels, that enables efficient introduction of analyte samples. This method for loading when taken in conjunction with horizontal microchannels allows much reduced sample volumes and a means of sample stacking to greatly reduce the concentration of the sample. This reduction in the amount of sample can lead to great cost savings in sample preparation, particularly in massively parallel applications such as DNA sequencing. The essence of this method is in preparation of the input of the separation channel, the physical sample introduction, and subsequent removal of excess material. By this method, sample volumes of 100 nanoliter to 2 microliters have been used successfully, compared to the typical 5 microliters of sample required by the prior separation/analysis method.

  7. Multivariate optimization approach for chiral resolution of drugs using human serum albumin in affinity electrokinetic chromatography-partial filling technique.

    PubMed

    Martinez-Gomez, Maria A; Villanueva-Camañas, Rosa M; Sagrado, Salvador; Medina-Hernández, Maria J

    2005-11-01

    The enantiomeric resolution of chiral compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer, protein and compound solutions) and protein solution plug length. In this paper multivariate optimization approaches for chiral separation of four basic drugs (alprenolol, oxprenolol, promethazine and propranolol) using HSA as chiral selector in AEKC-partial filling technique are studied. The experimental conditions to achieve maximum resolution are optimized using the Box-Behnken experimental design. Partial least squares and pareto charts are used to analyse the main effects on the resolution. The experimental resolutions observed for all compounds studied in optimum conditions agree with the estimated values based on response surface models. The results obtained show that the range of experimental conditions that provided enantioresolution narrows as hydrophobicity of analytes decreases. This fact can be explained by assuming that hydrophobicity controls the interaction of basic compounds with HSA.

  8. Rapid column heating method for subcritical water chromatography.

    PubMed

    Fogwill, Michael O; Thurbide, Kevin B

    2007-01-19

    A novel resistive heating method is presented for subcritical water chromatography (SWC) that provides higher column heating rates than those conventionally obtained from temperature-programmed gas chromatography (GC) convection ovens. Since the polarity of water reduces dramatically with increasing temperature, SWC employs column heating to achieve gradient elution. As such, the rate at which the mobile phase is heated directly impacts the magnitude of such gradients applied in SWC. Data from the current study demonstrate that the maximum column heating rate attainable in a typical SWC apparatus (i.e. using a GC convection oven) is around 10 degrees C/min, even at instrument oven settings of over three times this value. Conversely, by wrapping the separation column with ceramic insulation and a resistively heated wire, the column heating rates are increased five-fold. As a result, elution times can be greatly decreased in SWC employing gradients. Separations of standard alcohol test mixtures demonstrate that the retention time of the latest eluting component decreases by 35 to 50% using the prototype method. Additionally, solute retention times in this mode deviate by less than 1% RSD over several trials, which compares very well to those obtained using a conventional GC convection oven. Results suggest that the developed method can be a useful alternative heating technique in SWC.

  9. Are analysts doing method validation in liquid chromatography?

    PubMed

    Ruiz-Angel, M J; García-Alvarez-Coque, M C; Berthod, A; Carda-Broch, S

    2014-08-01

    Method validation is being applied in the reported analytical methods for decades. Even before this protocol was defined, authors already somehow validated their methods without full awareness. They wished to assure the quality of their work. Validation is an applied approach to verify that a method is suitable and rugged enough to function as a quality control tool in different locations and times. The performance parameters and statistical protocols followed throughout a validation study vary with the source of guidelines. Before single laboratory validation, an analytical method should be fully developed and optimized. The purpose of the validation is to confirm performance parameters that are determined during method development, and it should provide information on how the method will perform under routine use. An unstable method may require re-validation. Further method development and optimization will be needed if validation results do not meet the accepted performance standards. When possible, the validation protocol should also be conducted as a collaborative study by multiple laboratories, on different instruments, reagents, and standards. At this point, it would be interesting to know how people are validating their methods. Are they evaluating all defined validation parameters? Are they indicating the followed guidelines? Is re-validation really currently used? Is validation performed by a single laboratory, or is it a collaborative work by several laboratories? Is it an evolving discipline? In this survey, we will try to answer these questions focused to the field of liquid chromatography.

  10. Lectin affinity chromatography of articular cartilage fibromodulin: Some molecules have keratan sulphate chains exclusively capped by α(2-3)-linked sialic acid.

    PubMed

    Lauder, Robert M; Huckerby, Thomas N; Nieduszynski, Ian A

    2011-10-01

    Fibromodulin from bovine articular cartilage has been subjected to lectin affinity chromatography by Sambucus nigra lectin which binds α(2-6)- linked N-acetylneuraminic acid, and the structure of the keratan sulphate in the binding and non-binding fractions examined by keratanase II digestion and subsequent high pH anion exchange chromatography. It has been confirmed that the keratan sulphate chains attached to fibromodulin isolated from bovine articular cartilage may have the chain terminating N-acetylneuraminic acid residue α(2-3)- or α(2-6)-linked to the adjacent galactose residue. Although the abundance of α(2-6)-linked N-acetylneuraminic acid (ca. 22%) is such that this could cap one of the four chains in almost all fibromodulin molecules, it was found that ca. 34% of the fibromodulin proteoglycan molecules from bovine articular cartilage were capped exclusively with α(2-3)-linked N-acetylneuraminic acid. The remainder of the fibromodulin proteoglycans, which bound to the lectin had a mixture of α(2-3)- and α(2-6)-linked N-acetylneuraminic acid capping structures. The keratan sulphates attached to fibromodulin molecules capped exclusively with α(2-3)- linked N-acetylneuraminic acid were found to have a higher level of galactose sulphation than those from fibromodulin with both α(2-3)- and α(2-6)-linked N-acetylneuraminic acid caps, which bound to the Sambucus nigra lectin. In addition, both pools contained chains of similar length (ca. 8-9 disaccharides). Both also contained α(1-3)-linked fucose, showing that this feature does not co-distribute with α(2-6)-linked N-acetylneuraminic acid, although these two features are present only in mature articular cartilage. These data show that there are discrete populations of fibromodulin within articular cartilage, which may have differing impacts upon tissue processes.

  11. New method for determining total dietary fiber by liquid chromatography.

    PubMed

    Ohkuma, K; Matsuda, I; Katta, Y; Tsuji, K; Ohkuma, K; Matsuda, I; Katta, Y; Tsuji, K

    2000-01-01

    The molecular weight limit of water-soluble dietary fiber (SDF) determined by the Prosky method was studied by liquid chromatography (LC). It was confirmed that only SDF with an average degree of polymerization of 12 or higher can be determined by the Prosky method. Total dietary fiber (TDF) was determined by 2 additional methods using LC. In the first method, the total quantity of insoluble dietary fiber (IDF) and high molecular weight SDF (HMSDF) was determined according to the modified Prosky method (MES-TRIS buffer-based). The quantitatively collected final filtrate was analyzed by LC for the quantity of low molecular weight SDF (LMSDF), and the 2 quantities were totaled to obtain TDF. TDF values thus determined for rice, polished or unpolished, soybean flour, and pressed barley were higher than those determined by the Prosky method by approximately 6, 3.5, and 3.5%, respectively. In the second method, direct determination by LC analysis was done on samples after enzymatic treatment according to the Prosky method. Results showed that the determination of LMSDF, in particular, was highly accurate and more effective. In both of these methods, the quantity of LMSDF was determined from its chromatographic peak area ratio to glucose as an internal standard, which was produced by hydrolysis.

  12. A new method for the evaluation of antibody affinity based on serial dilutions of studied antibody-antigen mixture.

    PubMed

    Bobrovnik, S A

    2002-01-01

    A new method for the evaluation of the affinity of bivalent antibodies were suggested. This method is based on the previously published by the author the idea of using so-called coordinates of dilutions. It was shown that the suggested method allows to evaluate the affinity of antibodies with high accuracy using this simple approach. It is supposed that at some conditions the suggested method could have substantial advantages in comparison to the traditional methods. This method allows to analyze situations when antibodies are already in a mixture with antigen, for example in the bloodstream in the case of infections or autoimmune diseases. The method provides useful approach for the evaluation not only antibody affinity, but also the concentration of circulated antigen.

  13. Serial lectin affinity chromatography with concavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures of prostatic acid phosphatase in human prostate carcinoma.

    PubMed

    Yoshida, K I; Honda, M; Arai, K; Hosoya, Y; Moriguchi, H; Sumi, S; Ueda, Y; Kitahara, S

    1997-08-01

    Differences between human prostate carcinoma (PCA, five cases) and benign prostatic hyperplasia (BPH, five cases) in asparagine-linked (Asn) sugar-chain structure of prostatic acid phosphatase (PAP) were investigated using lectin affinity chromatography with concanavalin A (Con A) and wheat germ agglutinin (WGA). PAP activities were significantly decreased in PCA-derived PAP, while no significant differences between the two PAP preparations were observed in the enzymatic properties (Michaelis-Menten value, optimal pH, thermal stability, and inhibition study). In these PAP preparations, all activities were found only in the fractions which bound strongly to the Con A column and were undetectable in the Con A unbound fractions and in the fractions which bound weakly to the Con A column. The relative amounts of PAP which bound strongly to the Con A column but passed through the WGA column, were significantly greater in BPH-derived PAP than in PCA-derived PAP. In contrast, the relative amounts of PAP which bound strongly to the Con A column and bound to the WGA column, were significantly greater in PCA-derived PAP than in BPH-derived PAP. The findings suggest that Asn-linked sugar-chain structures are altered during oncogenesis in human prostate and also suggest that studies of qualitative differences of sugar-chain structures of PAP might lead to a useful diagnostic tool for PCA.

  14. Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa.

    PubMed Central

    Yerima, A; Safi, A; Gastin, I; Michalski, J C; Saunier, M; Gueant, J L

    1996-01-01

    We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17,300, a yield of 63% and a cobalamin-binding activity of 11,260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor. PMID:8573109

  15. Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking.

    PubMed

    Savane, Tushar S; Kumar, Sanjit; Janakiraman, Vignesh Narasimhan; Kamalanathan, Agamudi S; Vijayalakshmi, Mookambeswaran A

    2016-05-15

    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.

  16. Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

    PubMed

    Li, Qian; Zhang, Tianlong; Bian, Liujiao

    2016-03-01

    Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties.

  17. Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts

    PubMed Central

    Ciesla, L.; Okine, M.; Rosenberg, A.; Dossou, K.S.S.; Toll, L.; Wainer, I.W.; Moaddel, R.

    2016-01-01

    The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs. PMID:26774122

  18. Separation and quantitation of hepatoma-associated gamma-glutamyltransferase by affinity chromatography with Affi-Gel blue and Con A-Sepharose.

    PubMed

    Izumi, M; Taketa, K

    1983-01-01

    Isozymes of serum gamma-glutamyltransferase (GGT) in patients with hepatocellular carcinoma (HCC) and other liver diseases were separated into two groups by double-affinity column chromatography with Affi-Gel blue and Con A-Sepharose, one recovered in the unbound fraction and the other in the bound fraction. Upon electrophoresis with polyacrylamide gradient gel slabs, the unbound fraction gave a GGTI1 band and a faint II1 band and the bound fraction gave a GGT I band and faint bands of GGT I", II' and X, when the original serum contained hepatoma-associated GGT (I1, I" and II') and high-molecular-weight lipid-protein complex, GGT(X). GGT I was present in all cases as a common isozyme. Other lipoprotein-associated GGT isozymes, III-IX, were removed by passing through Affi-Gel blue. GGT activities of unbound fraction in patients with HCC were generally higher than those in patients with non-HCC liver diseases, although the difference was not significant. When the percent of GGT activity of unbound (unbound + bound) was taken, 54% of patients with HCC had a ratio greater than 22%, whereas none of the healthy subjects or patients with other liver diseases gave values greater than this. The present technique may prove to be a useful clinical test for the diagnosis of HCC.

  19. Designed synthesis of Graphene @titania @mesoporous silica hybrid material as size-exclusive metal oxide affinity chromatography platform for selective enrichment of endogenous phosphopeptides.

    PubMed

    Yao, Jizong; Sun, Nianrong; Deng, Chunhui; Zhang, Xiangming

    2016-04-01

    In this work, a novel size-exclusive metal oxide affinity chromatography (SE-MOAC) platform was built for phosphoproteome research. The operation for preparing graphene @titania @mesoporous silica nanohybrids (denoted as G@TiO2@mSiO2) was facile and easy to conduct by grafting titania nanoparticles on polydopamine (PD)-covered graphene, following a layer of mesoporous silica was coated on the outermost layer. The G@TiO2@mSiO2 nanohybrids exhibited high sensitivity with a low detection limit of 5 amol/μL (a total amount of 1 fmol) and high selectivity for phosphopeptides at a mass ratio of phosphopeptides to non-phosphopeptides (1:1000). The size-exclusive capability of the nanohybrids were also demonstrated by enriching the phosphopeptides from the mixture of Bovine Serum Albumin (BSA), α-casein, and β-casein digests with a high mass ratio (β-casein digests: α-casein: BSA, 1:500:500), which was attributed to the large surface area and ordered mesoporous channels. In addition, the G@TiO2@mSiO2 nanohybrids were employed to capture the endogenous phosphopeptides from human serum successfully.

  20. Exploring the interplay between experimental methods and the performance of predictors of binding affinity change upon mutations in protein complexes.

    PubMed

    Geng, Cunliang; Vangone, Anna; Bonvin, Alexandre M J J

    2016-08-01

    Reliable prediction of binding affinity changes (ΔΔG) upon mutations in protein complexes relies not only on the performance of computational methods but also on the availability and quality of experimental data. Binding affinity changes can be measured by various experimental methods with different accuracies and limitations. To understand the impact of these on the prediction of binding affinity change, we present the Database of binding Affinity Change Upon Mutation (DACUM), a database of 1872 binding affinity changes upon single-point mutations, a subset of the SKEMPI database (Moal,I.H. and Fernández-Recio,J. Bioinformatics, 2012;28:2600-2607) extended with information on the experimental methods used for ΔΔG measurements. The ΔΔG data were classified into different data sets based on the experimental method used and the position of the mutation (interface and non-interface). We tested the prediction performance of the original HADDOCK score, a newly trained version of it and mutation Cutoff Scanning Matrix (Pires,D.E.V., Ascher,D.B. and Blundell,T.L. Bioinformatics 2014;30:335-342), one of the best reported ΔΔG predictors so far, on these various data sets. Our results demonstrate a strong impact of the experimental methods on the performance of binding affinity change predictors for protein complexes. This underscores the importance of properly considering and carefully choosing experimental methods in the development of novel binding affinity change predictors. The DACUM database is available online at https://github.com/haddocking/DACUM.

  1. Electron affinity of p-quinones. Improved method of electrochemical estimation

    NASA Astrophysics Data System (ADS)

    Jaworski, Jan S.

    1986-06-01

    Electron affinities of four p-quinones are estimated from enthalpy changes obtained on the basis of measured formal potentials and reaction entropies in the electroreduction process. A linear correlation between electron affinities of p-quinones and parent hydrocarbons is found.

  2. A novel affinity-based method for the isolation of highly purified extracellular vesicles

    PubMed Central

    Nakai, Wataru; Yoshida, Takeshi; Diez, Diego; Miyatake, Yuji; Nishibu, Takahiro; Imawaka, Naoko; Naruse, Ken; Sadamura, Yoshifusa; Hanayama, Rikinari

    2016-01-01

    Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies. PMID:27659060

  3. Method for performing site-specific affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    1999-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  4. Method for performing site-specific affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, A.D.; Lysov, Y.P.; Dubley, S.A.

    1999-05-18

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between the cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting the extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to the extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from the array. 14 figs.

  5. An affinity-based genome walking method to find transgene integration loci in transgenic genome.

    PubMed

    Thirulogachandar, V; Pandey, Prachi; Vaishnavi, C S; Reddy, Malireddy K

    2011-09-15

    Identifying a good transgenic event from the pool of putative transgenics is crucial for further characterization. In transgenic plants, the transgene can integrate in either single or multiple locations by disrupting the endogenes and/or in heterochromatin regions causing the positional effect. Apart from this, to protect the unauthorized use of transgenic plants, the signature of transgene integration for every commercial transgenic event needs to be characterized. Here we show an affinity-based genome walking method, named locus-finding (LF) PCR (polymerase chain reaction), to determine the transgene flanking sequences of rice plants transformed by Agrobacterium tumefaciens. LF PCR includes a primary PCR by a degenerated primer and transfer DNA (T-DNA)-specific primer, a nested PCR, and a method of enriching the desired amplicons by using a biotin-tagged primer that is complementary to the T-DNA. This enrichment technique separates the single strands of desired amplicons from the off-target amplicons, reducing the template complexity by several orders of magnitude. We analyzed eight transgenic rice plants and found the transgene integration loci in three different chromosomes. The characteristic illegitimate recombination of the Agrobacterium sp. was also observed from the sequenced integration loci. We believe that the LF PCR should be an indispensable technique in transgenic analysis.

  6. The synthesis and characterization of a nuclear membrane affinity chromatography column for the study of human breast cancer resistant protein (BCRP) using nuclear membranes obtained from the LN-229 cells.

    PubMed

    Habicht, K-L; Frazier, C; Singh, N; Shimmo, R; Wainer, I W; Moaddel, R

    2013-01-01

    BCRP expression has been reported in glioblastoma cell lines and clinical specimens and has been shown to be expressed both in purified nuclei and in the soluble cytoplasmic fraction. To date, the nuclear BCRP has not been characterized. Our laboratory has previously developed an online chromatographic approach for the study of binding interactions between ligands and protein, cellular membrane affinity chromatography. To this end, we have immobilized the nuclear membrane fragments onto an immobilized artificial membrane stationary phase (IAM), resulting in the nuclear membrane affinity chromatography (NMAC) column. Initial characterization was carried out on the radio flow detector, as well as the LC-MSD, using frontal displacement chromatography techniques. Etoposide, a substrate for BCRP, was initially tested, to determine the functional immobilization of BCRP. Frontal displacement experiments with multiple concentrations of etoposide were run and the binding affinity was determined to be 4.54 μM, which is in close agreement with literature. The BCRP was fully characterized on the NMAC column and this demonstrates that for the first time the nuclear membranes have been successfully immobilized.

  7. Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

    PubMed Central

    Jergil, Bengt; Guilford, Hugh; Mosbach, Klaus

    1974-01-01

    1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3′:5′-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N6-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP–Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50–100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N6-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP–Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4374933

  8. Liquid chromatography method to determine polyamines in thermosetting polymers.

    PubMed

    Dopico-García, M S; López-Vilariño, J M; Fernández-Martínez, G; González-Rodríguez, M V

    2010-05-14

    A simple, robust and sensitive analytical method to determine three polyamines commonly used as hardeners in epoxy resin systems and in the manufacture of polyurethane is reported. The studied polyamines are: one tetramine, TETA (triethylenetetramine), and two diamines, IPDA (Isophorone diamine) and TCD-diamine (4,7-methano-1H-indene-5,?-dimethanamine, octahydro-). The latter has an incompletely defined structure, and, as far as we know, has not been previously determined by other methods. All three polyamines contain primary amines; TETA also contains secondary amines. The analytical method involves derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, used for the first time for these compounds, followed by high performance liquid chromatography (HPLC) analysis with a fluorescence (FL) detector (lambda excitation 248nm, lambda emision 395nm). The HPLC-DAD-LTQ Orbitrap MS was used in order to provide structural information about the obtained derivatized compounds. The hybrid linear ion trap LTQ Orbitrap mass spectrometer has been introduced in recent years and provides a high mass accuracy. The structures of the derivatized analytes were identified from the protonated molecular ions [M+H](+) and corresponded to the fully labelled analytes. The following analytical parameters were determined for the method using the HPLC-FL: linearity, precision (2.5-10%), instrumental precision intraday (0.8-1.5%) and interday (2.9-6.3%), and detection limits (0.02-0.14mgL(-1)). The stability of stock solutions and derivatized compounds was also investigated. The method was applied to determine the amine free content in epoxy resin dust collected in workplaces.

  9. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity in a single chromatography step.

    PubMed

    Adem, Sevki; Ciftci, Mehmet

    2012-01-01

    The enzymes of glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) were purified from rat kidney in one chromatographic step consisting of the use of the 2',5'-ADP Sepharose 4B by using different elution buffers. This purification procedure was accomplished with the preparation of the homogenate and affinity chromatography on 2',5'-ADP Sepharose 4B. The purity and subunit molecular weights of the enzymes were checked on SDS-PAGE and purified enzymes showed a single band on the gel. The native molecular weights of the enzymes were found with Sephadex G-150 gel filtration chromatography. Using this procedure, G6PG, having the specific activity of 32 EU/mg protein, was purified 531-fold with a yield of 88%; 6PGD, having the specific activity of 25 EU/mg protein, was purified 494-fold with a yield of 73%; and GR, having the specific activity of 33 EU/mg protein, was purified 477-fold with a yield of 76%. Their native molecular masses were estimated to be 144 kDa for G6PD, 110 kDa for 6PGD, and 121 kDa for GR and the subunit molecular weights were found to be 68, 56, and 61 kDa, respectively. A new modified method to purify G6PD, 6PGD, and GR, namely one chromatographic step using the 2',5'-ADP Sepharose 4B, is described for the first time in this study. This procedure has several advantages for purification of enzymes, such as, rapid purification, produces high yield, and uses less chemical materials.

  10. Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

    SciTech Connect

    Zhao, Huaying Schuck, Peter

    2015-01-01

    Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design.

  11. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.

    PubMed

    Rudashevskaya, Elena L; Sacco, Roberto; Kratochwill, Klaus; Huber, Marie L; Gstaiger, Matthias; Superti-Furga, Giulio; Bennett, Keiryn L

    2013-01-01

    Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.

  12. A mathematical method for extracting cell secretion rate from affinity biosensors continuously monitoring cell activity

    PubMed Central

    Gao, Yandong; Zhou, Qing; Matharu, Zimple; Liu, Ying; Kwa, Timothy; Revzin, Alexander

    2014-01-01

    Our laboratory has previously developed miniature aptasensors that may be integrated at the site of a small group of cells for continuous detection of cell secreted molecules such as inflammatory cytokine interferon gamma (IFN-γ). In a system such as this, the signal measured at the sensor surfaces is a complex function of transport, reaction, as well as of cellular activity. Herein, we report on the development of a mathematical framework for extracting cell production rates from binding curves generated with affinity biosensors. This framework consisted of a diffusion-reaction model coupled to a root finding algorithm for determining cell production rates values causing convergence of a predetermined criterion. To experimentally validate model predictions, we deployed a microfluidic device with an integrated biosensor for measuring the IFN-γ release from CD4 T cells. We found close agreement between secretion rate observed theoretically and those observed experimentally. After taking into account the differences in sensor geometry and reaction kinetics, the method for cell secretion rate determination described in this paper may be broadly applied to any biosensor continuously measuring cellular activity. PMID:24803956

  13. Accurate Ionization Potentials and Electron Affinities of Acceptor Molecules: A Benchmark of GW Methods

    NASA Astrophysics Data System (ADS)

    Marom, Noa; Knight, Joseph; Wang, Xiaopeng; Gallandi, Lukas; Dolgounitcheva, Olga; Ren, Xinguo; Ortiz, Vincent; Rinke, Patrick; Korzdorfer, Thomas

    The performance of different GW methods is assessed for a set of 24 organic acceptors. Errors are evaluated with respect to coupled cluster singles, doubles, perturbative triples [CCSD(T)] reference data for the vertical ionization potentials (IPs) and electron affinities (EAs), extrapolated to the complete basis set limit. Additional comparisons are made to experimental data, where available. We consider fully self-consistent GW (scGW), partial self-consistency in the Green's function (scGW0) , non-self-consistent G0W0 based on several mean-field starting points, and a ``beyond GW'' second order screened exchange (SOSEX) correction to G0W0. The best performers overall are G0W0 + SOSEX and G0W0 based on an IP-tuned long range corrected hybrid functional with the former being more accurate for EAs and the latter for IPs. Both provide a balanced treatment of localized vs. delocalized states and valence spectra in good agreement with photoemission spectroscopy (PES) experiments.

  14. Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants.

    PubMed

    Rohila, Jai S; Chen, Mei; Cerny, Ronald; Fromm, Michael E

    2004-04-01

    A synthetic gene encoding the tandem affinity purification (TAP) tag has been constructed, and the TAP tag assayed for its effects on expression levels and subcellular localization by fusion to green fluorescent protein (GFP) as well as for its effects on steroid-dependent translocation to the nucleus and transcription when fused to a hybrid glucocorticoid receptor. A nuclear localization signal (NLS) was detected in the calmodulin-binding protein (CBP) domain and removed by mutation to improve the usefulness of the TAP tag. Additionally, purification improvements were made, including inhibition of a co-purifying protease, and adding a protein cross-linking step to increase the recovery of interacting proteins. The improved synthetic TAP tag gene and methods were used to isolate proteins interacting with the hybrid glucocorticoid receptor and to identify them by mass spectrometry. The two proteins identified, HSP70 and HSP90, are known to interact with the glucocorticoid receptor in vivo in mammalian cells and in vitro in plants.

  15. Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

    PubMed Central

    Leiker, Merced; Suzuki, Gen; Iyer, Vijay S.; Canty, John M.; Lee, Techung

    2010-01-01

    Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and β-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ∼2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte. PMID:19069634

  16. An improved method for analysis of biomass sugars and galacturonic acid by anion exchange chromatography

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While the most accurate method for analysis of sugars in biomass is based on gas chromatography of trimethylsilane or alditol acetate derivitives of sugars, the derivation method is time consuming and laborious. In comparison, sample preparation for sugar analysis using liquid chromatography is a si...

  17. Agonist high- and low-affinity states of dopamine D₂ receptors: methods of detection and clinical implications.

    PubMed

    van Wieringen, Jan-Peter; Booij, Jan; Shalgunov, Vladimir; Elsinga, Philip; Michel, Martin C

    2013-02-01

    Dopamine D(2) receptors, similar to other G-protein-coupled receptors, exist in a high- and low-affinity state for agonists. Based upon a review of the methods for detecting D(2) receptor agonist high-affinity states, we discuss alterations of such states in animal models of disease and the implications of such alterations for their labelling with positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers. The classic approach of detecting agonist high-affinity states compares agonist competition for antagonist radioligands, in most cases using [(3)H]-spiperone as the radioligand; alternative approaches and radioligands have been proposed, but their claimed advantages have not been substantiated by other investigators. In view of the advantages and disadvantages of various techniques, we critically have reviewed reported findings on the detection of D(2) receptor agonist high-affinity states in a variety of animal models. These data are compared to the less numerous findings from human in vivo studies based on PET and SPECT tracers; they are interpreted in light of the finding that D(2) receptor agonist high-affinity states under control conditions may differ between rodent and human brain. The potential advantages of agonist ligands in studies of pathophysiology and as diagnostics are being discussed.

  18. Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns.

    PubMed

    Tong, Zenghan; Schiel, John E; Papastavros, Efthimia; Ohnmacht, Corey M; Smith, Quentin R; Hage, David S

    2011-04-15

    Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins.

  19. Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity Purification Method

    PubMed Central

    Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein–protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein–protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  20. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  1. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  2. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    NASA Astrophysics Data System (ADS)

    Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

    2012-03-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  3. An electron paramagnetic resonance method for measuring the affinity of a spin-labeled analog of cholesterol for phospholipids.

    PubMed

    Williams, Justin A; Wassall, Cynthia D; Kemple, Marvin D; Wassall, Stephen R

    2013-09-01

    Cholesterol (chol)-lipid interactions are thought to play an intrinsic role in determining lateral organization within cellular membranes. Steric compatibility of the rigid steroid moiety for ordered saturated chains contributes to the high affinity that holds chol and sphingomyelin together in lipid rafts whereas, conversely, poor affinity of the sterol for highly disordered polyunsaturated fatty acids (PUFAs) is hypothesized to drive the formation of PUFA-containing phospholipid domains depleted in chol. Here, we describe a novel method using electron paramagnetic resonance (EPR) to measure the relative affinity of chol for different phospholipids. We monitor the partitioning of 3β-doxyl-5α-cholestane (chlstn), a spin-labeled analog of chol, between large unilamellar vesicles (LUVs) and cyclodextrin (mβCD) through analysis of EPR spectra. Because the shape of the EPR spectrum for chlstn is sensitive to the very different tumbling rates of the two environments, the ratio of the population of chlstn in LUVs and mβCD can be determined directly from spectra. Partition coefficients (K(B)(A)) between lipids derived from our results for chlstn agree with values obtained for chol and confirm that decreased affinity for the sterol accompanies increasing acyl chain unsaturation. The virtue of this EPR method is that it provides a measure of chol binding that is quick, employs a commercially available probe and avoids the necessity for physical separation of LUVs and mβCD.

  4. New methods and materials for solid phase extraction and high performance liquid chromatography

    SciTech Connect

    Dumont, Philip John

    1996-04-23

    This paper describes methods for solid phase extraction and high performance liquid chromatography (HPLC). The following are described: Effects of Resin Sulfonation on the Retention of Polar Organic Compounds in Solid Phase Extraction; Ion-Chromatographic Separation of Alkali Metals In Non-Aqueous Solvents; Cation-Exchange Chromatography in Non-Aqueous Solvents; and Silicalite As a Stationary Phase For HPLC.

  5. Investigation of calcium antagonist-L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method.

    PubMed

    Du, Hui; He, Jianyu; Wang, Sicen; He, Langchong

    2010-07-01

    The dissociation equilibrium constant (K(D)) is an important affinity parameter for studying drug-receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K(D) values for calcium antagonist-L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 degrees C. The K(D) values obtained using frontal analysis were 3.36 x 10(-6) M for nifedipine, 1.34 x 10(-6) M for nimodipine, 6.83 x 10(-7) M for nitrendipine, 1.23 x 10(-7) M for nicardipine, 1.09 x 10(-7) M for amlodipine, and 8.51 x 10(-8) M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug-receptor interactions.

  6. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  7. Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level.

    PubMed

    Bartlow, Patrick; Uechi, Guy T; Cardamone, John J; Sultana, Tamanna; Fruchtl, McKinzie; Beitle, Robert R; Ataai, Mohammad M

    2011-08-01

    Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.

  8. Comparison between high-performance liquid chromatography and gas chromatography methods for fatty acid identification and quantification in potato crisps.

    PubMed

    Sanches-Silva, A; Rodríguez-Bernaldo de Quirós, A; López-Hernández, J; Paseiro-Losada, P

    2004-04-02

    A reversed-phase high performance liquid chromatographic (RP-HLPC) method was compared with a gas chromatography-flame ionization detection (GC-FID) method for determining fatty acids in potato crisps. Different extraction procedures were used. Fatty acids were quantified by linear regression. Both methods presented good precision (R.S.D. < or = 5.88%) and recovery (> or = 82.31%). The precision using HPLC method was slightly better than for GC-FID method. There was good agreement between the fatty acid composition of potato crisps analysed by both methods. For most purposes the HPLC method would be better. However, when more fatty acids need to be analysed, GC is a more suitable method.

  9. Selective high-affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2013-09-17

    This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  10. A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles

    NASA Astrophysics Data System (ADS)

    Wang, Qi; Huang, Xi; Fu, Xuan; Deng, Huan; Ma, Meihu; Cai, Zhaoxia

    2016-06-01

    Avidin is a glycoprotein with antinutritional property, which should be limited in daily food. We developed an affinity biosensor system based on resonance Rayleigh scattering (RRS) and using affinity biotin labeling Au nanoparticles (AuNPs). This method was selective and sensitive for quick avidin detection due to the avidin-biotin affinitive interaction. Under optimal conditions, RRS intensity of biotin-AuNPs increase linearly with an increasing concentration of avidin from 5 to 160 ng/mL. The lower limit of detection was 0.59 ng/mL. This rapid and selective avidin detection method was used in synthetic samples and egg products with recoveries of between 102.97 and 107.92%, thereby demonstrating the feasible and practical application of this assay.

  11. A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles.

    PubMed

    Wang, Qi; Huang, Xi; Fu, Xuan; Deng, Huan; Ma, Meihu; Cai, Zhaoxia

    2016-06-05

    Avidin is a glycoprotein with antinutritional property, which should be limited in daily food. We developed an affinity biosensor system based on resonance Rayleigh scattering (RRS) and using affinity biotin labeling Au nanoparticles (AuNPs). This method was selective and sensitive for quick avidin detection due to the avidin-biotin affinitive interaction. Under optimal conditions, RRS intensity of biotin-AuNPs increase linearly with an increasing concentration of avidin from 5 to 160 ng/mL. The lower limit of detection was 0.59 ng/mL. This rapid and selective avidin detection method was used in synthetic samples and egg products with recoveries of between 102.97 and 107.92%, thereby demonstrating the feasible and practical application of this assay.

  12. Profiling of cis-Diol-containing Nucleosides and Ribosylated Metabolites by Boronate-affinity Organic-silica Hybrid Monolithic Capillary Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Jiang, Han-Peng; Qi, Chu-Bo; Chu, Jie-Mei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-01-01

    RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5′-deoxy-5′-methylthioadensine, N4-acetylcytidine, 1-ribosyl-N-propionylhistamine and N2,N2,7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers. PMID:25585609

  13. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  14. An online coupled peritoneal macrophage/cell membrane chromatography and high-performance liquid chromatography/mass spectrometry method to screen for anti-inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei.

    PubMed

    Li, Weifeng; Xing, Wei; Wang, Sicen; Fan, Ting; Huang, Huimin; Niu, Xiaofeng; He, Langchong

    2013-11-01

    Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti-inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor-α in lipopolysaccharide-stimulated mice and peritoneal macrophages in a dose-dependent manner. The results demonstrated that the PM/CMC-online-HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples.

  15. Evaluation between ultrahigh pressure liquid chromatography and high-performance liquid chromatography analytical methods for characterizing natural dyestuffs.

    PubMed

    Serrano, Ana; van Bommel, Maarten; Hallett, Jessica

    2013-11-29

    An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41-61% resolution and a decrease of 91-422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds' retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects.

  16. Development of analytical method for catechol compounds in mouse urine using hydrophilic interaction liquid chromatography with fluorescence detection.

    PubMed

    Kanamori, Takahiro; Isokawa, Muneki; Funatsu, Takashi; Tsunoda, Makoto

    2015-03-15

    An analytical method for catecholamines and related compounds using hydrophilic interaction liquid chromatography (HILIC) with native fluorescence detection has been developed. We found that ZIC-cHILIC with phosphorylcholine was suitable for the separation of catechol compounds with good peak shapes among six different HILIC columns (Inertsil SIL, Inertsil Amide, Inertsil Diol, TSKgel NH2-100, ZIC-HILIC, and ZIC-cHILIC). Using ZIC-cHILIC, eight catechol compounds (dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylglycol, 3,4-dihydroxymandelic acid, and internal standard 3,4-dihydroxybenzylamine) were separated within 15min. The limit of detection at a signal to noise ratio of 3 was 3-28nM. An improved sensitivity was obtained as compared to that of reversed-phase liquid chromatography. This was partly attributed to the increase in the fluorescence intensity of the catechol compounds in the acetonitrile-rich mobile phase. Solid phase extraction using a monolithic silica disk-packed spin column with phenylboronate moieties, which have affinity to catechol compounds, was performed for the selective extraction of catechol compounds from mouse urine. Dopamine, epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were successfully quantified in mouse urine.

  17. Species with negative electron affinity and standard DFT methods. Finding the valence anions.

    PubMed

    Puiatti, Marcelo; Vera, D Mariano A; Pierini, Adriana B

    2008-03-14

    Recently, we have shown that traditional bound-electron DFT models are reliable enough to reproduce negative electron affinities (EA) within a few meV, as long as the valence anion state is found, but they seem to fail in predicting the lowest EA when the ground anion state obtained is non-valence, which holds the extra electron in a diffuse orbital around the molecule; here we propose an alternative approach for finding the valence anion state, based on the stabilization exerted by a polar solvent; the methodology yields correct EA values (i.e. beyond the Koopman's theorem approximation) by gradually decreasing the dielectric constant of the medium.

  18. Path integral method for predicting relative binding affinities of protein-ligand complexes

    PubMed Central

    Mulakala, Chandrika; Kaznessis, Yiannis N.

    2009-01-01

    We present a novel approach for computing biomolecular interaction binding affinities based on a simple path integral solution of the Fokker-Planck equation. Computing the free energy of protein-ligand interactions can expedite structure-based drug design. Traditionally, the problem is seen through the lens of statistical thermodynamics. The computations can become, however, prohibitively long for the change in the free energy upon binding to be determined accurately. In this work we present a different approach based on a stochastic kinetic formalism. Inspired by Feynman's path integral formulation, we extend the theory to classical interacting systems. The ligand is modeled as a Brownian particle subjected to the effective non-bonding interaction potential of the receptor. This allows the calculation of the relative binding affinities of interacting biomolecules in water to be computed as a function of the ligand's diffusivity and the curvature of the potential surface in the vicinity of the binding minimum. The calculation is thus exceedingly rapid. In test cases, the correlation coefficient between actual and computed free energies is >0.93 for accurate data-sets. PMID:19275144

  19. Integration of gas chromatography mass spectrometry methods for differentiating ricin preparation methods.

    PubMed

    Wunschel, David S; Melville, Angela M; Ehrhardt, Christopher J; Colburn, Heather A; Victry, Kristin D; Antolick, Kathryn C; Wahl, Jon H; Wahl, Karen L

    2012-05-07

    The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.

  20. Advanced high-performance liquid chromatography method development. Discovering unexpected choices in chromatography.

    PubMed

    Rieger, H J; Molnar, I

    2002-03-01

    The influence of some important experimental parameters on the resolution of compounds as well as the validity of widely used rules of thumb and of common expectations about how to improve resolution is discussed. It will be shown, on the basis of selected examples, that the general expectations about how the experimental parameters have to be adjusted for better resolution does not cover all chances for resolution improvement. The tool for understanding the method and to discover all chances for increasing selectivity is the resolution map of a method.

  1. Analysis of biomolecular interactions using affinity microcolumns: A review

    PubMed Central

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L.; White, Christopher J.; Carter, NaTasha; Hage, David S.

    2014-01-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  2. Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

    PubMed

    Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

    2016-07-01

    An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice.

  3. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells.

    PubMed

    Bhatia, Prateek A; Moaddel, Ruin; Wainer, Irving W

    2010-06-15

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA, using a baculovirus expression system. The resulting CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [(3)H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9(MRP1)) column, etoposide and furosemide on the CMAC(Sf9(MRP2)) column and etoposide and fumitremorgin C on the CMAC(Sf9(BCPR)) column. The binding affinities (K(i) values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [(3)H]-etoposide on the CMAC(Sf9(MRP1)) column to a greater extent than (R)-verapamil and the relative IC(50) values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC(50) values were consistent with previously reported data. The results indicated that the CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system.

  4. Rapid contrast evaluation method based on affinity beads and backscattered electron imaging for the screening of electron stains.

    PubMed

    Kaku, Hiroki; Inoue, Kanako; Muranaka, Yoshinori; Park, Pyoyun; Ikeda, Kenichi

    2015-10-01

    Uranyl salts are toxic and radioactive; therefore, several studies have been conducted to screen for substitutes of electron stains. In this regard, the contrast evaluation process is time consuming and the results obtained are inconsistent. In this study, we developed a novel contrast evaluation method using affinity beads and a backscattered electron image (BSEI), obtained using scanning electron microscopy. The contrast ratios of BSEI in each electron stain treatment were correlated with those of transmission electron microscopic images. The affinity beads bound to cell components independently. Protein and DNA samples were enhanced by image contrast treated with electron stains; however, this was not observed for sugars. Protein-conjugated beads showed an additive effect of image contrast when double-stained with lead. However, additive effect of double staining was not observed in DNA-conjugated beads. The varying chemical properties of oligopeptides showed differences in image contrast when treated with each electron stain. This BSEI-based evaluation method not only enables screening for alternate electron stains, but also helps analyze the underlying mechanisms of electron staining of cellular structures.

  5. A Static Method as an Alternative to Gel Chromatography: An Experiment for the Undergraduate Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Burum, Alex D.; Splittgerber, Allan G.

    2008-01-01

    This article describes a static method as an alternative to gel chromatography, which may be used as an undergraduate laboratory experiment. In this method, a constant mass of Sephadex gel is swollen in a series of protein solutions. UV-vis spectrophotometry is used to find a partition coefficient, KD, that indicates the fraction of the interior…

  6. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method

    PubMed Central

    Nielsen, Morten; Lundegaard, Claus; Lund, Ole

    2007-01-01

    Background Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHC II) molecules. Identification of peptides that bind MHC II molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion The SMM-align method was

  7. Neuere Chromatographie

    NASA Astrophysics Data System (ADS)

    Hostettmann, K.

    1983-04-01

    Besides high-performance liquid chromatography (HPLC) which is now a well-established and currently used technique, several emerging methods for the isolation and separation of natural products are receiving considerable attention. Centrifugal thin-layer chromatography is a very rapid technique, but limited in resolution. Of special interest are the recently developed support-free liquid-liquid chromatography methods such as droplet counter-current chromatography (DCCC) and rotation locular counter-current chromatography (RLCC). This latter method was applied to the separation of the enantiomers of (±)-norephedrine.

  8. Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

    DOEpatents

    Cascao-Pereira, Luis G.; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D.

    2012-08-07

    The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

  9. Compositions and methods comprising cellulase variants with reduced affinity to non-cellulosic materials

    DOEpatents

    Cascao-Pereira, Luis G; Kaper, Thijs; Kelemen, Bradley R; Liu, Amy D

    2015-04-07

    The present disclosure relates to cellulase variants. In particular the present disclosure relates to cellulase variants having reduced binding to non-cellulosic materials. Also described are nucleic acids encoding the cellulase, compositions comprising said cellulase, methods of identifying cellulose variants and methods of using the compositions.

  10. A Simple Robust Orthogonal Background Correction Method for Two-Dimensional Liquid Chromatography

    PubMed Central

    Filgueira, Marcelo R.; Castells, Cecilia; Carr, Peter W.

    2012-01-01

    Background correction is a very important step that must be done before peak detection or any quantification procedure. When successful, this step greatly simplifies such procedures and enhances the accuracy of quantification. In the past, much effort has been invested to correct drifting baseline in one dimensional chromatography. In fast online comprehensive two-dimensional liquid chromatography (LC×LC) coupled with diode array detector (DAD), the change in the refractive index (RI) of the mobile phase in very fast gradients causes extremely serious baseline disturbances. The method reported here can be combined with many of existing baseline correction methods for one dimensional (1D) chromatography in two dimensional (2D) liquid chromatography and recreate the background structure for further correction. When such methods are applied orthogonally to the second dimension (2D), the background correction is dramatically improved. It results in an almost zero mean background level and it provides better background correction than does a simple subtraction of a blank. Indeed, the method proposed does not require running a blank sample. PMID:22702415

  11. Pre-staining paper chromatography method for quantification of gamma-aminobutyric acid.

    PubMed

    Li, Haixing; Qiu, Ting; Cao, Yusheng; Yang, Jiyan; Huang, Zhibing

    2009-06-19

    The routine method of paper chromatography includes five steps: spotting, separating, drying, spraying/immersing and color development. In this paper, a pre-staining paper chromatography which only consisted of spotting, separating and color development was developed for quantitative analysis of gamma-aminobutyric acid. Compared to the routine paper chromatography, the improved method is clean, rapid, inexpensive and reproducible. The effects of ninhydrin concentration, color temperature, color time and Cu(2+) concentration on the color yield in the ninhydrin reaction were optimized. And then the pre-staining paper chromatography coupled with vis spectrophotometry was applied to gamma-aminobutyric acid quantification. The results indicated that the limit of detection was 0.05 mg mL(-1) and the linear range was from 0.5 to 20.0 mg mL(-1). Furthermore, an excellent correlation coefficient was observed with an R(2)=0.998. The method is accurate (RSD<2.64%), and has good recoveries (102.7-103.9%). The validation of the modified technique was verified by a HPLC method.

  12. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  13. Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

    PubMed Central

    Kramberger, Petra; Urbas, Lidija; Štrancar, Aleš

    2015-01-01

    Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production. PMID:25751122

  14. Immobilized metal affinity chromatography in open-loop simulated moving bed technology: purification of a heat stable histidine tagged beta-glucosidase.

    PubMed

    Sahoo, Deepti; Andersson, Jonatan; Mattiasson, Bo

    2009-06-01

    Open-loop simulated moving bed (SMB) has been used for immobilized metal affinity chromatographic (IMAC) purification of his-tagged beta-glucosidase expressed in E. coli. A simplified approach based on an optimized single column protocol is used to design the open-loop SMB. A set of columns in the SMB represent one step in the chromatographic cycle i.e. there will be one set each of columns for load, wash, elution etc within the SMB. Only the wash and elution are operated with columns in sequence. The beta-glucosidase was purified to almost single band purity with a purification factor of 15 and a recovery of 91%. SMB-performance showed reduced buffer consumption, higher purification fold, a better yield and higher productivity.

  15. Fast Modeling of Binding Affinities by Means of Superposing Significant Interaction Rules (SSIR) Method

    PubMed Central

    Besalú, Emili

    2016-01-01

    The Superposing Significant Interaction Rules (SSIR) method is described. It is a general combinatorial and symbolic procedure able to rank compounds belonging to combinatorial analogue series. The procedure generates structure-activity relationship (SAR) models and also serves as an inverse SAR tool. The method is fast and can deal with large databases. SSIR operates from statistical significances calculated from the available library of compounds and according to the previously attached molecular labels of interest or non-interest. The required symbolic codification allows dealing with almost any combinatorial data set, even in a confidential manner, if desired. The application example categorizes molecules as binding or non-binding, and consensus ranking SAR models are generated from training and two distinct cross-validation methods: leave-one-out and balanced leave-two-out (BL2O), the latter being suited for the treatment of binary properties. PMID:27240346

  16. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  17. High-Throughput Melanin-Binding Affinity and In Silico Methods to Aid in the Prediction of Drug Exposure in Ocular Tissue.

    PubMed

    Reilly, John; Williams, Sarah L; Forster, Cornelia J; Kansara, Viral; End, Peter; Serrano-Wu, Michael H

    2015-12-01

    Drugs possessing the ability to bind to melanin-rich tissue, such as the eye, are linked with higher ocular exposure, and therefore have the potential to affect the efficacy and safety profiles of therapeutics. A high-throughput melanin chromatographic affinity assay has been developed and validated, which has allowed the rapid melanin affinity assessment for a large number of compounds. Melanin affinity of compounds can be quickly assigned as low, medium, or high melanin binders. A high-throughput chromatographic method has been developed and fully validated to assess melanin affinity of pharmaceuticals and has been useful in predicting ocular tissue distribution in vivo studies. The high-throughput experimental approach has also allowed for a specific training set of 263 molecules for a quantitative structure-affinity relationships (QSAR) method to be developed, which has also been shown to be a predictor of ocular tissue exposure. Previous studies have reported the development of in silico QSAR models based on training sets of relatively small and mostly similar compounds; this model covers a broader range of melanin-binding affinities than what has been previously published and identified several physiochemical descriptors to be considered in the design of compounds where melanin-binding modulation is desired.

  18. Chromatography of carbon nanotubes separated albumin from other serum proteins: a method for direct analysis of their interactions.

    PubMed

    Kuboki, Yoshinori; Koshikawa, Takamitu; Takita, Hiroko; Fujisawa, Ryuichi; Lee, Min-ho; Abe, Shigeaki; Akasaka, Tsukasa; Uo, Motohiro; Watari, Fumio; Sammons, Rachel

    2010-08-01

    Chromatography technology was employed to clarify the mechanism of interaction between multi-wall carbon nanotubes (MWCNT) and proteins. A column (16x100 mm) was packed with purified MWCNT, and various proteins were eluted with phosphate buffered saline (PBS) with and without gradient systems. It was found that albumin in bovine serum was eluted immediately from the column without any adsorption to MWCNT. Conversely, the non-albumin proteins, including a protein of 85 kDa molecular mass and a group of proteins with molecular masses higher than 115 kDa, exhibited considerably high affinity towards MWCNT. A sample of pure bovine serum albumin was also eluted immediately from the column, while lysozyme did not elute as a peak with PBS, but eluted with 0.6 M NaCl. Fundamentally, carbon nanotubes are devoid of any electrical charge. Therefore, other forces including the hydrogen bonds, hydrophilic interactions, and van der Waals forces were most probably responsible for the differential elution behaviors. In conclusion, this chromatographic method provided a simple and direct analysis of the interactions between carbon nanotubes and the various proteins.

  19. Retention modeling and method development in hydrophilic interaction chromatography.

    PubMed

    Tyteca, Eva; Périat, Aurélie; Rudaz, Serge; Desmet, Gert; Guillarme, Davy

    2014-04-11

    In the present study, the possibility of retention modeling in the HILIC mode was investigated, testing several different literature relationships over a wide range of different analytical conditions (column chemistries and mobile phase pH) and using analytes possessing diverse physico-chemical properties. Furthermore, it was investigated how the retention prediction depends on the number of isocratic or gradient trial or initial scouting runs. The most promising set of scouting runs seems to be a combination of three isocratic runs (95, 90 and 70%ACN) and one gradient run (95 to 65%ACN in 10min), as the average prediction errors were lower than using six equally spaced isocratic runs and because it is common in Method development (MD) to perform at least one scouting gradient run in the screening step to find out the best column, temperature and pH conditions. Overall, the retention predictions were much less accurate in HILIC than what is usually experienced in RPLC. This has severe implications for MD, as it restricts the use of commercial software packages that require the simulation of the retention of every peak in the chromatogram. To overcome this problem, the recently proposed predictive elution window shifting and stretching (PEWS(2)) approach can be used. In this computer-assisted MD strategy, only an (approximate) prediction of the retention of the first and the last peak in the chromatogram is required to conduct a well-targeted trial-and-error search, with suggested search conditions uniformly covering the entire possible search and elution space. This strategy was used to optimize the separation of three representative pharmaceutical mixtures possessing diverse physico-chemical properties (pteridins, saccharides and cocktail of drugs/metabolites). All problems could be successfully handled in less than 2.5h of instrument time (including equilibration).

  20. Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

    NASA Astrophysics Data System (ADS)

    Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

    2009-11-01

    The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants ( Ka) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02 × 10 7 and 2.07 × 10 4 L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S 0 → S 1 transition of esculin ( λexmax≈340 nm) appears, which is similar to the λemmax of BSA and HSA. The critical distance ( R0) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

  1. Comparison of different methods to estimate the uncertainty in composition measurement by chromatography.

    PubMed

    Ariza, Adriana Alexandra Aparicio; Ayala Blanco, Elizabeth; García Sánchez, Luis Eduardo; García Sánchez, Carlos Eduardo

    2015-06-01

    Natural gas is a mixture that contains hydrocarbons and other compounds, such as CO2 and N2. Natural gas composition is commonly measured by gas chromatography, and this measurement is important for the calculation of some thermodynamic properties that determine its commercial value. The estimation of uncertainty in chromatographic measurement is essential for an adequate presentation of the results and a necessary tool for supporting decision making. Various approaches have been proposed for the uncertainty estimation in chromatographic measurement. The present work is an evaluation of three approaches of uncertainty estimation, where two of them (guide to the expression of uncertainty in measurement method and prediction method) were compared with the Monte Carlo method, which has a wider scope of application. The aforementioned methods for uncertainty estimation were applied to gas chromatography assays of three different samples of natural gas. The results indicated that the prediction method and the guide to the expression of uncertainty in measurement method (in the simple version used) are not adequate to calculate the uncertainty in chromatography measurement, because uncertainty estimations obtained by those approaches are in general lower than those given by the Monte Carlo method.

  2. Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

    PubMed Central

    Zhao, Huaying; Schuck, Peter

    2015-01-01

    Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design. PMID:25615855

  3. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  4. Direct Delta-MBPT(2) method for ionization potentials, electron affinities, and excitation energies using fractional occupation numbers

    SciTech Connect

    Beste, Ariana; Vazquez-Mayagoitia, Alvaro; Ortiz, J. Vincent

    2013-01-01

    A direct method (D-Delta-MBPT(2)) to calculate second-order ionization potentials (IPs), electron affinities (EAs), and excitation energies is developed. The Delta-MBPT(2) method is defined as the correlated extension of the Delta-HF method. Energy differences are obtained by integrating the energy derivative with respect to occupation numbers over the appropriate parameter range. This is made possible by writing the second-order energy as a function of the occupation numbers. Relaxation effects are fully included at the SCF level. This is in contrast to linear response theory, which makes the D-Delta-MBPT(2) applicable not only to single excited but also higher excited states. We show the relationship of the D-Delta-MBPT(2) method for IPs and EAs to a second-order approximation of the effective Fock-space coupled-cluster Hamiltonian and a second-order electron propagator method. We also discuss the connection between the D-Delta-MBPT(2) method for excitation energies and the CIS-MP2 method. Finally, as a proof of principle, we apply our method to calculate ionization potentials and excitation energies of some small molecules. For IPs, the Delta-MBPT(2) results compare well to the second-order solution of the Dyson equation. For excitation energies, the deviation from EOM-CCSD increases when correlation becomes more important. When using the numerical integration technique, we encounter difficulties that prevented us from reaching the Delta-MBPT(2) values. Most importantly, relaxation beyond the Hartree Fock level is significant and needs to be included in future research.

  5. Fast and accurate numerical method for predicting gas chromatography retention time.

    PubMed

    Claumann, Carlos Alberto; Wüst Zibetti, André; Bolzan, Ariovaldo; Machado, Ricardo A F; Pinto, Leonel Teixeira

    2015-08-07

    Predictive modeling for gas chromatography compound retention depends on the retention factor (ki) and on the flow of the mobile phase. Thus, different approaches for determining an analyte ki in column chromatography have been developed. The main one is based on the thermodynamic properties of the component and on the characteristics of the stationary phase. These models can be used to estimate the parameters and to optimize the programming of temperatures, in gas chromatography, for the separation of compounds. Different authors have proposed the use of numerical methods for solving these models, but these methods demand greater computational time. Hence, a new method for solving the predictive modeling of analyte retention time is presented. This algorithm is an alternative to traditional methods because it transforms its attainments into root determination problems within defined intervals. The proposed approach allows for tr calculation, with accuracy determined by the user of the methods, and significant reductions in computational time; it can also be used to evaluate the performance of other prediction methods.

  6. Validation of a sensitive ion chromatography method for determination of monoethylsulfate in Indinavir sulfate drug substance.

    PubMed

    Prasanna, S John; Sharma, Hemant Kumar; Mukkanti, K; Sivakumaran, M; Pavan Kumar, K S R; Kumar, V Jagadeesh

    2009-12-05

    The present study relates to the optimization of an ion chromatography method to determine the content of monoethylsulfate at very low levels in Indinavir sulfate drug substance, and subsequent validation of the method to prove its suitability, reliability and sensitivity. Monoethylsulfate is a potential impurity of Indinavir sulfate, and may forms during the preparation as well as during storage. The ion chromatography method was developed in such a way that to enhance the detection level by introducing suppressor, and minimizing acquisition time by using suitable buffer of 3.2 mmole of sodium carbonate and 1 mmole of sodium hydrogen carbonate in water as eluent. The retention time of monoethylsulfate was about 9.5 min and the total acquisition time was 25 min. The optimized method was validated to prove its performance characteristics by demonstrating selectivity, sensitivity (limit of detection and quantification), linearity, precision and accuracy. The established limit of detection and quantification of monoethylsulfate in Indinavir sulfate by this method was found to be 24 ng/ml and 74 ng/ml respectively, and the overall percent accuracy (recovery) of samples evaluated at different concentration levels was found to be 97.1, indicating the sensitivity and accuracy of this optimized ion chromatography method.

  7. A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.

    PubMed

    Kaya, Mustafa Oguzhan; Arslan, Oktay; Guler, Ozen Ozensoy

    2015-01-01

    In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. L-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-L-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-L-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) values obtained from the Lineweaver-Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.

  8. Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

    PubMed

    Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

    2014-07-18

    This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins.

  9. A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

    2015-04-01

    A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

  10. High-throughput multiclass method for antibiotic residue analysis by liquid chromatography-tandem mass spectrometry.

    PubMed

    Chico, J; Rúbies, A; Centrich, F; Companyó, R; Prat, M D; Granados, M

    2008-12-12

    A simple and rapid method has been developed for the residue analysis of 39 antibiotics (tetracyclines, quinolones, penicillins, sulfonamides and macrolides) in foodstuffs of animal origin. The method combines an effective extraction technique, which uses water-methanol as extracting solvent, with ultra-high-pressure liquid chromatography-tandem mass spectrometry, allowing both confirmation and quantification in a single chromatographic run. The multiresidue method has been validated in chicken muscle matrix according to European Union Decision 2002/657/EC. It has been implemented as a routine method in a Public Health Laboratory, instead of the five plates test and LC methods previously used.

  11. An affinity selection-mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries: Discovery of a novel antagonist of E. coli dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Annis, D. Allen; Athanasopoulos, John; Curran, Patrick J.; Felsch, Jason S.; Kalghatgi, Krishna; Lee, William H.; Nash, Huw M.; Orminati, Jean-Paul A.; Rosner, Kristin E.; Shipps, Gerald W., Jr.; Thaddupathy, G. R. A.; Tyler, Andrew N.; Vilenchik, Lev; Wagner, Carston R.; Wintner, Edward A.

    2004-11-01

    The NeoGenesis Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry (AS-MS) process consisting of a rapid size-exclusion chromatography stage integrated with reverse-phase chromatography, electrospray mass spectrometry, and novel data searching algorithms, was used to screen mass-encoded, 2500-member combinatorial libraries, leading to the discovery of a novel, bioactive ligand for the anti-infective target Escherichia coli dihydrofolate reductase (DHFR). Synthesis of the mass-encoded, ligand-containing library, discussion of the deconvolution process for verifying the structure of the ligand through independent synthesis and screening in a small mixture (sub-library) format, and ALIS-MS/MS techniques to assign its regioisomeric connectivity are presented. ALIS-based competition experiments between the newly discovered ligand and other, known DHFR ligands, and biological activity assessments with stereo- and regioisomers of the hit compound confirm its DHFR-specific biological activity. The method described requires no foreknowledge of the structure or biochemistry of the protein target, consumes less than 1 [mu]g protein to screen >2500 compounds in a single experiment, and enables screening of >250,000 compounds per system per day. These advantages highlight the potential of the ALIS method for drug discovery against genomic targets with unknown biological function, as well as validated targets for which traditional discovery efforts have failed.

  12. Methods of analysis-Determination of pesticides in sediment using gas chromatography/mass spectrometry

    USGS Publications Warehouse

    Hladik, Michelle; McWayne, Megan M.

    2012-01-01

    A method for the determination of 119 pesticides in environmental sediment samples is described. The method was developed by the U.S. Geological Survey (USGS) in support of the National Water Quality Assessment (NAWQA) Program. The pesticides included in this method were chosen through prior prioritization. Herbicides, insecticides, and fungicides along with degradates are included in this method and span a variety of chemical classes including, but not limited to, chloroacetanilides, organochlorines, organophosphates, pyrethroids, triazines, and triazoles. Sediment samples are extracted by using an accelerated solvent extraction system (ASE®, and the compounds of interest are separated from co-extracted matrix interferences (including sulfur) by passing the extracts through high performance liquid chromatography (HPLC) with gel-permeation chromatography (GPC) along with the use of either stacked graphitized carbon and alumina solid-phase extraction (SPE) cartridges or packed Florisil®. Chromatographic separation, detection, and quantification of the pesticides from the sediment-sample extracts are done by using gas chromatography with mass spectrometry (GC/MS). Recoveries in test sediment samples fortified at 10 micrograms per kilogram (μg/kg) dry weight ranged from 75 to 102 percent; relative standard deviations ranged from 3 to 13 percent. Method detection limits (MDLs), calculated by using U.S. Environmental Protection Agency procedures (40 CFR 136, Appendix B), ranged from 0.6 to 3.4 μg/kg dry weight.

  13. Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

    PubMed

    Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

    2016-12-02

    Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

  14. Reagents and methods for the solid-phase synthesis of protein-EDTA (ethylenediaminetetraacetic acid) for use in affinity cleaving

    SciTech Connect

    Sluka, J.P.; Griffin, J.H.; Mack, D.P.; Dervan, P.B. )

    1990-08-15

    Synthetic procedures for the introduction of the metal chelator ethylenediaminetetraacetic acid (EDTA) at unique amino acid positions of proteins by solid-phase methods are described. Two protected derivatives of EDTA compatible with Merrifield solid-phase protein synthesis employing N-tert-butyloxycarbonyl- (Boc-) protected amino acids were developed. The first reagent is a dipeptide with three of four carboxyl groups of EDTA protected as benzyl esters and the fourth coupled to a {gamma}-aminobutanoic acid linker, referred to as tribenzyl-EDTA-GABA (BEG). A second reagent is the tricyclohexyl ester of EDTA, TCE. BEG and TCE allow the modification of the NH{sub 2} terminus and/or lysine side chains of resin-bound peptides and proteins. Upon deprotection and cleavage from the resin, a protein is produced with EDTA at a defined amino acid position. The availability of protein-EDTA conjugates extends the affinity cleaving method to the study of protein-DNA complexes in solution.

  15. Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    2000-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  16. Possibilities and limitations of the kinetic plot method in supercritical fluid chromatography.

    PubMed

    De Pauw, Ruben; Desmet, Gert; Broeckhoven, Ken

    2013-08-30

    Although supercritical fluid chromatography (SFC) is becoming a technique of increasing importance in the field of analytical chromatography, methods to compare the performance of SFC-columns and separations in an unbiased way are not fully developed. The present study uses mathematical models to investigate the possibilities and limitations of the kinetic plot method in SFC as this easily allows to investigate a wide range of operating pressures, retention and mobile phase conditions. The variable column length (L) kinetic plot method was further investigated in this work. Since the pressure history is identical for each measurement, this method gives the true kinetic performance limit in SFC. The deviations of the traditional way of measuring the performance as a function of flow rate (fixed back pressure and column length) and the isopycnic method with respect to this variable column length method were investigated under a wide range of operational conditions. It is found that using the variable L method, extrapolations towards other pressure drops are not valid in SFC (deviation of ∼15% for extrapolation from 50 to 200bar pressure drop). The isopycnic method provides the best prediction but its use is limited when operating closer towards critical point conditions. When an organic modifier is used, the predictions are improved for both methods with respect to the variable L method (e.g. deviations decreases from 20% to 2% when 20mol% of methanol is added).

  17. A quantitative method to identify microRNAs targeting a messenger RNA using a 3'UTR RNA affinity technique.

    PubMed

    Shi, Miao; Han, Weiguo; Spivack, Simon D

    2013-12-01

    The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3'UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitative polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA.

  18. Caprylic acid precipitation method for impurity reduction: an alternative to conventional chromatography for monoclonal antibody purification.

    PubMed

    Brodsky, Yan; Zhang, Cheng; Yigzaw, Yinges; Vedantham, Ganesh

    2012-10-01

    We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes.

  19. Development and validation of ultra-high performance supercritical fluid chromatography method for determination of illegal dyes and comparison to ultra-high performance liquid chromatography method.

    PubMed

    Khalikova, Maria A; Šatínský, Dalibor; Solich, Petr; Nováková, Lucie

    2015-05-18

    A novel simple, fast and efficient ultra-high performance supercritical fluid chromatography (UHPSFC) method was developed and validated for the separation and quantitative determination of eleven illegal dyes in chili-containing spices. The method involved a simple ultrasound-assisted liquid extraction of illegal compounds with tetrahydrofuran. The separation was performed using a supercritical fluid chromatography system and CSH Fluoro-Phenyl stationary phase at 70°C. The mobile phase was carbon dioxide and the mixture of methanol:acetonitrile (1:1, v/v) with 2.5% formic acid as an additive at the flow rate 2.0 mL min(-1). The UV-vis detection was accomplished at 500 nm for seven compounds and at 420 nm for Sudan Orange G, Butter Yellow, Fast Garnet GBC and Methyl Red due to their maximum of absorbance. All eleven compounds were separated in less than 5 min. The method was successfully validated and applied using three commercial samples of chili-containing spices - Chili sauce (Indonesia), Feferony sauce (Slovakia) and Mojo sauce (Spain). The linearity range of proposed method was 0.50-9.09 mg kg(-1) (r ≥ 0.995). The detection limits were determined as signal to noise ratio of 3 and were ranged from 0.15 mg kg(-1) to 0.60 mg kg(-1) (1.80 mg kg(-1) for Fast Garnet) for standard solution and from 0.25 mg kg(-1) to 1.00 mg kg(-1) (2.50 mg kg(-1) for Fast Garnet, 1.50 mg kg(-1) for Sudan Red 7B) for chili-containing samples. The recovery values were in the range of 73.5-107.2% and relative standard deviation ranging from 0.1% to 8.2% for within-day precision and from 0.5% to 8.8% for between-day precision. The method showed potential for being used to monitor forbidden dyes in food constituents. The developed UHPSFC method was compared to the UHPLC-UV method. The orthogonality of Sudan dyes separation by these two methods was demonstrated. Benefits and drawbacks were discussed showing the reliability of both methods for monitoring of studied illegal dyes in real

  20. Functionalized multi-walled carbon nanotubes as affinity ligands

    NASA Astrophysics Data System (ADS)

    Yu, L.; Li, C. M.; Zhou, Q.; Gan, Y.; Bao, Q. L.

    2007-03-01

    Functionalization of carbon nanotubes is very challenging for their applications. The paper here describes a new method to functionalize multi-walled carbon nanotubes (MWCNTs) as specific affinity adsorbents. MWCNTs were acid purified and pretreated with (3-aminopropyl)-triethoxysilane (APTES) in order to introduce abundant amino groups on the surface of MWCNTs. After the conversion of amino groups to carboxyl groups by succinic acid anhydride, MWCNTs were attached to protein A or aminodextran using 1-ethyl-3,3' (dimethylamion)-propylcarbodiimide as a biofunctional crosslinker. The incorporation of aminodextran as a spacer arm noticeably increased the binding capacity of the APTES-modified MWCNTs for protein A. The application of affinity MWCNTs for purification of immunoglobulin G was then evaluated. The affinity of MWCNTs with AMD spacer exhibited a high adsorption capacity of ~361 µg IgG/mg MWCNT (wet basis). About 75% of bound IgG was eluted from affinity MWCNTs (ANT-I and ANT-II) and ELISA confirmed that the biological activity of IgG was well preserved during the course of affinity separation. The functionalized MWCNTs could be potentially used in affinity chromatography.

  1. A Size-Exclusion Chromatography Method for Analysis of Clostridium difficile Vaccine Toxins.

    PubMed

    Lancaster, Catherine; Rustandi, Richard R; Pannizzo, Paola; Ha, Sha

    2016-01-01

    High-performance size-exclusion chromatography (HPSEC or SEC) is a method that can be applied to measure size distribution of proteins, including aggregates, monomers, and fragments. In the biopharmaceutical industry the quantitation of aggregates contained in biotherapeutics and protein-based vaccines is critical given the potential impact on safety, immunogenicity, and efficacy. Hence, aggregation analysis of therapeutic proteins or protein-based vaccine products is almost always a requirement of regulatory agencies. SEC, also referred to as gel-filtration chromatography, separates molecules by size through a porous resin stationary phase. Under isocratic flow small molecules are retained on the column longer than large molecules. Here we describe the use of this SEC technique to characterize aggregation levels for four different protein antigens for a Clostridium difficile vaccine.

  2. Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin-I for capturing sub-glycoproteomics from breast cancer and disease-free human sera.

    PubMed

    Selvaraju, Subhashini; El Rassi, Ziad

    2012-07-01

    In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed towards the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease-free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.

  3. An enzymatic method for assaying sulbactam in human serum: comparison with high performance liquid chromatography.

    PubMed

    Sotto, A; Peray, P; Geny, F; Brunschwig, C; Carrière, C; Galtier, M; Ramuz, M; Jourdan, J

    1995-03-01

    An enzymatic method using nitrocefin as substrate was developed to assay sulbactam in human serum. Serum containing sulbactam was incubated with purified titrated TEM-1 beta-lactamase and nitrocefin was then added to the mixture to determine the remaining beta-lactamase activity and consequently the concentration of sulbactam. Assays were carried out on five patients with pulmonary infections receiving sulbactam plus amoxycillin iv. The values for serum sulbactam concentrations determined by the enzymatic method were compared with those determined by high performance liquid chromatography (HPLC). The correlation coefficient was 0.990 for serum sulbactam concentrations below 15 mg/L.

  4. Comparison of photoacoustic radiometry to gas chromatography/mass spectrometry methods for monitoring chlorinated hydrocarbons

    SciTech Connect

    Sollid, J.E.; Trujillo, V.L.; Limback, S.P.; Woloshun, K.A.

    1996-03-01

    A comparison of two methods of gas chromatography mass spectrometry (GCMS) and a nondispersive infrared technique, photoacoustic radiometry (PAR), is presented in the context of field monitoring a disposal site. First is presented an historical account describing the site and early monitoring to provide an overview. The intent and nature of the monitoring program changed when it was proposed to expand the Radiological Waste Site close to the Hazardous Waste Site. Both the sampling methods and analysis techniques were refined in the course of this exercise.

  5. Development of Hydrophilic Interaction Liquid Chromatography Method for the Analysis of Moxonidine and Its Impurities

    PubMed Central

    2016-01-01

    Fast and simple hydrophilic interaction liquid chromatography (HILIC) method was developed and validated for the analysis of moxonidine and its four impurities (A, B, C, and D) in pharmaceutical dosage form. All experiments were performed on the Agilent Technologies 1200 high-performance liquid chromatography (HPLC) system using Zorbax RX-SIL, 250 mm × 4.6 mm, 5 μm column as stationary phase (T = 25°C, F = 1 mL/min, and λ = 255 nm), and mixture of acetonitrile and 40 mM ammonium formate buffer (pH 2.8) 80 : 20 (v/v) as mobile phase. Under the optimal chromatographic conditions, selected by central composite design, separation and analysis of moxonidine and its four impurities are enabled within 12 minutes. Validation of the method was conducted in accordance with ICH guidelines. Based on the obtained results selectivity, linearity (r ≥ 0.9976), accuracy (recovery: 93.66%–114.08%), precision (RSD: 0.56%–2.55%), and robustness of the method were confirmed. The obtained values of the limit of detection and quantification revealed that the method can be used for determination of impurities levels below 0.1%. Validated method was applied for determination of moxonidine and its impurities in commercially available tablet formulation. Obtained results confirmed that validated method is fast, simple, and reliable for analysis of moxonidine and its impurities in tablets. PMID:27847672

  6. Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high-performance countercurrent chromatography.

    PubMed

    Rho, Taewoong; Jung, Mila; Lee, Min Won; Chin, Young-Won; Yoon, Kee Dong

    2016-12-01

    Efficient high-performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n-Butanol-soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid (1), swertiamarin (2) and gentiopicroside (3), and a chromene derivative, macrophylloside D (4) which were successfully isolated by flow rate gradient (1.5 mL/min in 0-60 min, 5.0 mL/min in 60-120 min), and consecutive flow rate gradient HPCCC using n-butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1-4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high-performance countercurrent chromatography, and consecutive flow rate gradient high-performance countercurrent chromatography gave 1, 2, 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2-methoxyanofinic acid, was successfully separated by n-hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow-rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by (1) H, (13) C NMR and ESI-Q-TOF-MS spectroscopic data which were compared with previously reported values.

  7. Affinity electrophoresis as a method for determining substrate-binding specificity of carbohydrate-active enzymes for soluble polysaccharides.

    PubMed

    Moraïs, Sarah; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    Affinity electrophoresis is a simple and rapid tool for the analysis of protein-binding affinities to soluble polysaccharides. This approach is particularly suitable for the characterization of the carbohydrate-active enzymes that contain a carbohydrate-binding module and for their mutants and chimeras. Knowledge of the binding characteristics of these enzymes can be the first step to elucidate the enzymatic activity of a putative enzyme; moreover in some cases, enzymes are able to bind polysaccharides targets other than their specified substrate, and this knowledge can be essential to understand the basics of the intrinsic mechanism of these enzymes in their natural environment.

  8. Enhanced cell affinity of chitosan membranes mediated by superficial cross-linking: a straightforward method attainable by standard laboratory procedures.

    PubMed

    Rodríguez-Velázquez, Eustolia; Silva, Maite; Taboada, Pablo; Mano, João F; Suárez-Quintanilla, David; Alatorre-Meda, Manuel

    2014-01-13

    It is well accepted that the surface modification of biomaterials can improve their biocompatibility. In this context, techniques like ion etching, plasma-mediated chemical functionalization, electrospinning, and contact microprinting have successfully been employed to promote the cell adhesion and proliferation of chitosan (CH) substrates. However, they prove to be time-consuming, highly dependent on environmental conditions, and/or limited to the use of expensive materials and sophisticated instruments not accessible to standard laboratories, hindering to a high extent their straightforward application. Filling this gap, this paper proposes the superficial cross-linking of CH as a much simpler and accessible means to modify its superficial properties in order to enhance its cellular affinity. CH membranes were prepared by solvent casting followed by a cross-linking step mediated by the chemical vapor deposition (CVD) of glutaraldehyde (GA). The membranes were characterized against non- and solution cross-linked membranes in terms of their mechanical/surface properties and biological performance. Among others, the CVD membranes proved (i) to be more mechanically stable against cell culture and sterilization than membranes cross-linked in solution and (ii) to prompt the adherence and sustained proliferation of healthy cells to levels even superior to commercial tissue culture plates (TCPs). Accordingly, the CVD cross-linking approach was demonstrated to be a simple and cost-effective alternative to the aforementioned conventional methods. Interestingly, this concept can also be applied to other biomaterials as long as GA (or other volatile components alike) can be employed as a cross-linker, making possible the cross-linking reaction at mild experimental conditions, neither requiring sophisticated lab implements nor using any potentially harmful procedure.

  9. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

    PubMed

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A; de Greef, Tom F A; Abbaspourrad, Alireza; Weitz, David A; Chong, Shaorong

    2016-03-04

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (10(3)-10(6)). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

  10. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    PubMed Central

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  11. A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity.

    PubMed

    Huuk, Thiemo C; Briskot, Till; Hahn, Tobias; Hubbuch, Jürgen

    2016-05-01

    Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high-throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion-exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv-detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid-base titration. In contrast to acid-base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666-677, 2016.

  12. The Generally Useful Estimate of Solvent Systems (GUESS) method enables the rapid purification of methylpyridoxine regioisomers by countercurrent chromatography

    PubMed Central

    Liu, Yang; Friesen, J. Brent; Klein, Larry L.; McAlpine, James B.; Lankin, David C.; Pauli, Guido F.; Chen, Shao-Nong

    2016-01-01

    The TLC-based Generally Useful Estimate of Solvent Systems (GUESS) method was employed for countercurrent chromatography solvent system selection, in order to separate the three synthetic isomers: 3-O-methylpyridoxine, 4′-O-methylpyridoxine (ginkgotoxin) and 5′-O-methylpyridoxine. The Rf values of the three isomers indicated that ChMWat +2 (chloroformmethanol-water 10:5:5, v/v/v) was appropriate for the countercurrent chromatography. The isomer separation was highly selective and demonstrated that the TLC-based GUESS method can accelerate solvent system selection for countercurrent chromatography. Accordingly, the study re-emphasizes the practicality of TLC as a tool to facilitate the rapid development of new countercurrent and centrifugal partition chromatography methods for this solvent system. Purity and structure characterization of all samples was performed by quantitative 1H NMR. PMID:26680272

  13. Chemistry and liquid chromatography methods for the analyses of primary oxidation products of triacylglycerols.

    PubMed

    Zeb, A

    2015-05-01

    Triacylglycerols (TAGs) are one of the major components of the cells in higher biological systems, which can act as an energy reservoir in the living cells. The unsaturated fatty acid moiety is the key site of oxidation and formation of oxidation compounds. The TAG free radical generates several primary oxidation compounds. These include hydroperoxides, hydroxides, epidioxides, hydroperoxy epidioxides, hydroxyl epidioxides, and epoxides. The presence of these oxidized TAGs in the cell increases the chances of several detrimental processes. For this purpose, several liquid chromatography (LC) methods were reported in their analyses. This review is therefore focused on the chemistry, oxidation, extraction, and the LC methods reported in the analyses of oxidized TAGs. The studies on thin-layer chromatography were mostly focused on the total oxidized TAGs separation and employ hexane as major solvent. High-performance LC (HPLC) methods were discussed in details along with their merits and demerits. It was found that most of the HPLC methods employed isocratic elution with methanol and acetonitrile as major solvents with an ultraviolet detector. The coupling of HPLC with mass spectrometry (MS) highly increases the efficiency of analysis as well as enables reliable structural elucidation. The use of MS was found to be helpful in studying the oxidation chemistry of TAGs and needs to be extended to the complex biological systems.

  14. Method transfer from high-pressure liquid chromatography to ultra-high-pressure liquid chromatography. II. Temperature and pressure effects.

    PubMed

    Åsberg, Dennis; Samuelsson, Jörgen; Leśko, Marek; Cavazzini, Alberto; Kaczmarski, Krzysztof; Fornstedt, Torgny

    2015-07-03

    The importance of the generated temperature and pressure gradients in ultra-high-pressure liquid chromatography (UHPLC) are investigated and compared to high-pressure liquid chromatography (HPLC). The drug Omeprazole, together with three other model compounds (with different chemical characteristics, namely uncharged, positively and negatively charged) were used. Calculations of the complete temperature profile in the column at UHPLC conditions showed, in our experiments, a temperature difference between the inlet and outlet of 16 °C and a difference of 2 °C between the column center and the wall. Through van't Hoff plots, this information was used to single out the decrease in retention factor (k) solely due to the temperature gradient. The uncharged solute was least affected by temperature with a decrease in k of about 5% while for charged solutes the effect was more pronounced, with k decreases up to 14%. A pressure increase of 500 bar gave roughly 5% increase in k for the uncharged solute, while omeprazole and the other two charged solutes gave about 25, 20 and 15% increases in k, respectively. The stochastic model of chromatography was applied to estimate the dependence of the average number of adsorption/desorption events (n) and the average time spent by a molecule in the stationary phase (τs) on temperature and pressure on peak shape for the tailing, basic solute. Increasing the temperature yielded an increase in n and decrease in τs which resulted in less skew at high temperatures. With increasing pressure, the stochastic modeling gave interesting results for the basic solute showing that the skew of the peak increased with pressure. The conclusion is that pressure effects are more pronounced for both retention and peak shape than the temperature effects for the polar or charged compounds in our study.

  15. Rapid method for determination of carbonyl groups in lignin compounds by headspace gas chromatography.

    PubMed

    Li, Jing; Hu, Hui-Chao; Chai, Xin-Sheng

    2015-07-24

    The paper reports on a novel method for rapid determination of carbonyl in lignins by headspace gas chromatography (HS-GC). The method involves the quantitative carbonyl reduction for aldehydes in 2min at room temperature or for acetones in 30min at 80°C by sodium borohydride solution in a closed headspace sample vial. After the reaction, the solution was acidified by injecting sulfuric acid solution and the hydrogen released to the headspace was determined by GC using thermal-conductivity detector. The results showed that with the addition of SiO2 powder, the reduction reaction of carbonyl groups can be greatly facilitated. The method has a good measurement precision (RSD<7.74%) and accuracy (relative error <10% compared with a reference method) in the carbonyl quantification. It is suitable to be used for rapid determination of carbonyl content in lignin and related materials.

  16. Rapid quantitative method for total brominated vegetable oil in soft drinks using ion chromatography.

    PubMed

    Yousef, Ashraf A; Abbas, Alaa B; Badawi, Bassam Sh; Al-Jowhar, Wafaa Y; Zain, Esam A; El-Mufti, Seham A

    2012-08-01

    A simple, quantitative and rapid method for total brominated vegetable oil (BVO) using ion chromatography (IC) with suppressed conductivity detection was developed and successfully applied to soft drinks with results expressed as inorganic bromide anion. The procedure involves extraction of BVO with diethyl ether and treatment with zinc dust in a solution of acetic acid, giving recoveries ranging between 92.5 and 98.5%. The calibration curves obtained were linear with correlation coefficients (r²) of 0.998, a coefficient of variation (CV) of less than 5% and limit of detection (LOD) and limit of quantification (LOQ) of 250 and 750 µg l⁻¹, respectively. The method was successfully applied to the determination of BVO in several commercial soft drinks which were found to contain BVO in the range 1.8-14.510 mg l⁻¹. The method has less sources of error compared to previously published methods.

  17. First screening method for the simultaneous detection of seven allergens by liquid chromatography mass spectrometry.

    PubMed

    Heick, J; Fischer, M; Pöpping, B

    2011-02-18

    The development of a multi-method for the detection of seven allergens based on liquid chromatography and triple-quadrupole tandem mass spectrometry in multiple reaction mode is described. It is based on extraction of the allergenic proteins from a food matrix, followed by enzymatic digestion with trypsin. The chosen marker peptides were implemented into one method that is capable of the simultaneous detection of milk, egg, soy, hazelnut, peanut, walnut and almond. This method has been used to detect all seven allergenic commodities from incurred reference bread material, which was baked according to a standard recipe from the baking industry. Detected concentrations ranged from 10 to 1000 μg/g, demonstrating that the mass spectrometric based method is a useful tool for allergen screening.

  18. Development of a size exclusion chromatography method for the determination of molar mass for poloxamers.

    PubMed

    Erlandsson, Bengt; Wittgren, Bengt; Brinkmalm, Gunnar

    2003-04-01

    An aqueous size exclusion chromatography (SEC) method for determination of the molar mass of poloxamers 188 and 407 has been developed as an alternative to the pharmacopoeia methods. During the development work two different columns and several different eluent compositions were investigated. With a PL-aquagel-OH column, non-exclusion behaviour was obtained. A TSKgel column gave good separation of both poloxamers. The best separation was obtained with an eluent consisting of sodium chloride (0.01 M)-methanol (90:10, v/v) on the TSKgel column. The method was shown to be linear within the elution time of the two poloxamers and to have acceptable precision. The results from the SEC method was compared to results obtained using SEC with online multi angle light scattering detection (MALS) and to results obtained with matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS).

  19. Comparative Study of Three Methods for Affinity Measurements: Capillary Electrophoresis Coupled with UV Detection and Mass Spectrometry, and Direct Infusion Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Mironov, Gleb G.; Logie, Jennifer; Okhonin, Victor; Renaud, Justin B.; Mayer, Paul M.; Berezovski, Maxim V.

    2012-07-01

    We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and β-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

  20. Development and validation of a microemulsion electrokinetic chromatography method for patulin quantification in commercial apple juice.

    PubMed

    Murillo-Arbizu, M; González-Peñas, E; Hansen, S H; Amézqueta, S; Ostergaard, J

    2008-06-01

    A microemulsion electrokinetic chromatography (MEECK) method for patulin (PAT) quantification in apple juice samples has been developed. The effects of several important factors such as co-surfactant type, concentration of surfactant, acetonitrile percentage in the microemulsion, and running voltage and temperature were investigated to determine the optimum conditions. They resulted to be: a background electrolyte (BGE) composed of 25mM of sodium tetraborate, SDS (2.16%w/w), ethanol (6.49%w/w), n-octanol (0.82%w/w) and 2%v/v acetonitrile; applied voltage of +15kV; and a capillary temperature of 35 degrees C. PAT was detected at 276nm. Quantification and detection limits (LOQ and LOD) in apple juice samples were 8.0microgL(-1) and 3.2microgL(-1), respectively. Patulin was extracted from apple juice using ethyl acetate with a mean recovery value of 75.3% (RSD=4.5). This method was applied to the measurement of patulin in twenty commercial apple juice samples obtained from different Danish supermarkets. The PAT apple juice mean and median levels obtained were 35.9 and 10.9microgL(-1), respectively. The comparison with a previously validated micellar electrokinetic chromatography (MEKC) method for PAT analysis showed the suitability of using MEEKC for this mycotoxin analysis. However, the expectations of obtaining a higher efficiency and thus lower limits of detection and quantitation when using MEEKC were not met.

  1. Isotope dilution gas chromatography/mass spectrometry method for determination of pyrethroids in apple juice.

    PubMed

    Wong, Siu-kay; Yu, Kwok-chiu; Lam, Chi-ho

    2010-03-01

    This paper presents the development of a highly precise and accurate analytical method for the determination of three matrix-bound pyrethroids, namely, cypermethrin, permethrin, and bifenthrin, using an isotope dilution gas chromatography/mass spectrometry technique. Identification of the analytes was confirmed under selective ion monitoring mode by the presence of two dominant ion fragments within specific time windows and matching of relative ion intensities of the ions concerned in samples and calibration standards. Quantitation was based on the measurement of concentration ratios of the natural and isotope analogues in the sample and calibration blends. Intraday and interday repeatabilities of replicate analyses of the pyethroids in an apple juice sample were below 0.5%. The expanded relative uncertainty ranged from 3 to 6%, which was significantly lower than the range obtained using internal or external calibration methods. As a labeled analogue is not available for bifenthrin, bifenthrin was determined using labeled cis-permethrin as the internal standard. The results were counterchecked by a gas chromatography-electron capture detection technique using PCB 209 as the internal standard. The method developed was applied to a recent pilot study organized by CCQM and the results were consistent with those of other participants.

  2. Determination of vaporization enthalpies of the branched esters from correlation gas chromatography and transpiration methods

    SciTech Connect

    Verevkin, S.P.; Heintz, A.

    1999-12-01

    Vaporization enthalpies are indispensable for the assessment of the environmental fate and behavior of environmental contaminants. The temperature dependencies of retention indices of a set of 80 esters with branched molecular structures were measured on a nonpolar gas chromatographic column. The correlation gas chromatography method and reliable data set of 16 esters selected from the literature were used to derive a correlation for the prediction of the standard molar enthalpies of vaporization {Delta}{sub 1}{sup g}H{sub m}{sup {degree}} at the temperature T = 298.15 K. Experimental values of {Delta}{sub 1}{sup g}H{sub m}{sup {degree}} for 64 branched esters were obtained with the help of this correlation. The vaporization enthalpies of isopentyl acetate, ethyl hexanoate, and neopentyl pivalate were additionally obtained by the transpiration method from the temperature dependence of the vapor pressure measured in a flow system and used for checking the validity of the correlation gas chromatography method.

  3. Additive method for the prediction of protein-peptide binding affinity. Application to the MHC class I molecule HLA-A*0201.

    PubMed

    Doytchinova, Irini A; Blythe, Martin J; Flower, Darren R

    2002-01-01

    A method has been developed for prediction of binding affinities between proteins and peptides. We exemplify the method through its application to binding predictions of peptides with affinity to major histocompatibility complex class I molecule HLA-A*0201. The method is named "additive" because it is based on the assumption that the binding affinity of a peptide could be presented as a sum of the contributions of the amino acids at each position and the interactions between them. The amino acid contributions and the contributions of the interactions between adjacent side chains and every second side chain were derived using a partial least squares (PLS) statistical methodology using a training set of 420 experimental IC50 values. The predictive power of the method was assessed using rigorous cross-validation and using an independent test set of 89 peptides. The mean value of the residuals between the experimental and predicted pIC50 values was 0.508 for this test set. The additive method was implemented in a program for rapid T-cell epitope search. It is universal and can be applied to any peptide-protein interaction where binding data is known.

  4. New process for purifying high purity α1-antitrypsin from Cohn Fraction IV by chromatography: A promising method for the better utilization of plasma.

    PubMed

    Huangfu, Chaoji; Zhang, Jinchao; Ma, Yuyuan; Jia, Junting; Lv, Maomin; Zhao, Xiong; Zhang, Jingang

    2017-03-01

    α1-antitrypsin (AAT) is a 52kDa serine protease inhibitor that is abundant in plasma. It is synthesized mainly by hepatic cells, and widely used to treat patients with emphysema due to congenital deficiency of AAT. A new isolation method for the purification of AAT from Cohn Fraction IV (Cohn F IV) is described. Cohn F IV is usually discarded as a byproduct from Cohn process. Using Cohn F IV as starting material does not interfere with the production of other plasma proteins and the cost of purification could be reduced greatly. Parameters of each step during purification were optimized, 15% polyethyleneglycol (PEG) concentration and pH 5.2 for PEG precipitation, elution with 0.05M sodium acetate and pH 4.7 for ion-exchange chromatography, and two steps blue sepharose affinity chromatography were chosen for AAT purification. The final protein with purity of 98.17%, specific activity of 3893.29 IU/mg, and yield of 28.35%, was achieved. Western blotting was applied for qualitative identification of final product, which specifically reacted with goat anti-human AAT antibody. LC-ESI-MS/MS was also employed to confirm the final protein. High performance liquid chromatography was used to analyze the composition of purified protein suggesting that pure protein was achieved. The molecular weight of AAT is 51062.77Da which was identified by LC-MS-MS. The manufacturing process described here may make better use of human plasma with Cohn F IV as starting material. The simple process described in this study is simple and inexpensive, it has a potential value for large scale production.

  5. Novel orthogonal liquid chromatography methods to dose neurotransmitters involved in Parkinson's disease.

    PubMed

    Sardella, Roccaldo; Scorzoni, Stefania; Conte, Carmela; Lisanti, Antonella; Ianni, Federica; Natalini, Benedetto

    2014-09-01

    Parkinson's disease is a multifactorial neurodegenerative disorder, characterized by a reduction of dopamine (DA) levels. The molecular pathways involved in the pathogenesis of disease have not yet been fully disclosed. Therefore, developing new diagnostic methods and tools to evaluate the depletion of DA and of some of its metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine) is of outstanding importance for biochemical evaluations. Moreover, neurons responsible for DA release also produce the neurotransmitter gamma-aminobutyric acid (GABA), thus, quantitative measurements of GABA levels can have a relevant impact for a further understanding of the biochemical processes involved in the neurodegenerative event. In the present study, two HPLC methods based on the reversed-phase ion-pairing chromatography (RP-IPC) and the hydrophilic liquid interaction chromatography (HILIC) concepts were developed to allow the quantification of DA and its metabolites as well as GABA levels in mouse striatal and cortical tissue homogenates. The two fairly orthogonal HPLC methods were directly applied to the biological samples, without preliminary derivatization of the compounds of interest. A high level of selectivity was obtained for DA metabolites and GABA by running the gradient RP-IPC method with a volatile ion-pairing reagent, which makes it suitable for the quantitative assay of four out of five compounds. Matrix deriving interferences unabled the base-line separation of DA which was instead successfully achieved with the HILIC-based method. To avail of HPLC methods providing distinct selectivity profiles, makes possible the correct species quantification and allows to compensate the intrinsic limits characterizing all chromatographic methods.

  6. A validated high performance thin layer chromatography method for determination of yohimbine hydrochloride in pharmaceutical preparations

    PubMed Central

    Badr, Jihan M.

    2013-01-01

    Background: Yohimbine is an indole alkaloid used as a promising therapy for erectile dysfunction. A number of methods were reported for the analysis of yohimbine in the bark or in pharmaceutical preparations. Materials and Method: In the present work, a simple and sensitive high performance thin layer chromatographic method is developed for determination of yohimbine (occurring as yohimbine hydrochloride) in pharmaceutical preparations and validated according to International Conference of Harmonization (ICH) guidelines. The method employed thin layer chromatography aluminum sheets precoated with silica gel as the stationary phase and the mobile phase consisted of chloroform:methanol:ammonia (97:3:0.2), which gave compact bands of yohimbine hydrochloride. Results: Linear regression data for the calibration curves of standard yohimbine hydrochloride showed a good linear relationship over a concentration range of 80–1000 ng/spot with respect to the area and correlation coefficient (R2) was 0.9965. The method was evaluated regarding accuracy, precision, selectivity, and robustness. Limits of detection and quantitation were recorded as 5 and 40 ng/spot, respectively. The proposed method efficiently separated yohimbine hydrochloride from other components even in complex mixture containing powdered plants. The amount of yohimbine hydrochloride ranged from 2.3 to 5.2 mg/tablet or capsule in preparations containing the pure alkaloid, while it varied from zero (0) to 1.5–1.8 mg/capsule in dietary supplements containing powdered yohimbe bark. Conclusion: We concluded that this method employing high performance thin layer chromatography (HPTLC) in quantitative determination of yohimbine hydrochloride in pharmaceutical preparations is efficient, simple, accurate, and validated. PMID:23661986

  7. Generation of an affinity column for antibody purification by intein-mediated protein ligation.

    PubMed

    Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2003-11-01

    Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

  8. Development of high performance liquid chromatography method for miconazole analysis in powder sample

    NASA Astrophysics Data System (ADS)

    Hermawan, D.; Suwandri; Sulaeman, U.; Istiqomah, A.; Aboul-Enein, H. Y.

    2017-02-01

    A simple high performance liquid chromatography (HPLC) method has been developed in this study for the analysis of miconazole, an antifungal drug, in powder sample. The optimized HPLC system using C8 column was achieved using mobile phase composition containing methanol:water (85:15, v/v), a flow rate of 0.8 mL/min, and UV detection at 220 nm. The calibration graph was linear in the range from 10 to 50 mg/L with r 2 of 0.9983. The limit of detection (LOD) and limit of quantitation (LOQ) obtained were 2.24 mg/L and 7.47 mg/L, respectively. The present HPLC method is applicable for the determination of miconazole in the powder sample with a recovery of 101.28 % (RSD = 0.96%, n = 3). The developed HPLC method provides short analysis time, high reproducibility and high sensitivity.

  9. Practical method for the definition of chromatographic peak parameters in preparative liquid chromatography.

    PubMed

    Jin, Gaowa; Guo, Zhimou; Xiao, Yuansheng; Yan, Jingyu; Dong, Xuefang; Shen, Aijin; Wang, Chaoran; Liang, Xinmiao

    2016-10-01

    A practical method was established for the definition of chromatographic parameters in preparative liquid chromatography. The parameters contained both the peak broadening level under different amounts of sample loading and the concentration distribution of the target compound in the elution. The parameters of the peak broadening level were defined and expressed as a matrix, which consisted of sample loading, the forward broadening and the backward broadening levels. The concentration distribution of the target compound was described by the heat map of the elution profile. The most suitable stationary phase should exhibit the narrower peak broadening and it was best to broaden to both sides to compare to the peak under analytical conditions. Besides, the concentration distribution of the target compounds should be focused on the middle of the elution. The guiding principles were validated by purification of amitriptyline from the mixture of desipramine and amitriptyline. On the selected column, when the content of the impurity desipramine was lower than 0.1%, the recovery of target compound was much higher than the other columns even when the sample loading was as high as 8.03 mg/cm(3) . The parameters and methods could be used for the evaluation and selection of stationary phases in preparative chromatography.

  10. A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography.

    PubMed

    Zhao, Yan; Kim, Young Ho; Lee, Wonjae; Lee, Young Keun; Kim, Kyung Tae; Kang, Jong Seong

    2016-01-05

    An effective and rapid method was developed for the simultaneous identification of aloe, catechu and gambir by high performance liquid chromatography-diode array detector (HPLC-DAD). Identification of three maker compounds presented in three medicinal materials was performed on high performance liquid chromatography-mass spectrometry (HPLC-MS). Under the optimal HPLC chromatographic conditions, sixty-two samples were processed on an Optimapak C18 column using a solvent system of acetonitrile (from 10% to 35%) and 0.1% phosphoric acid solution (from 90% to 65%) at a total flow rate of 1.0 mL/min and detected at 270 nm. All calibration curves exhibited good linear relationship (r(2)>0.9992). The relative standard deviation values of intra-day and inter-day precision were less than 1% and 2%, respectively. The recoveries of three analytes ranged from 99.48 to 100.97% with low RSDs (<2%). For the first time, this study demonstrates that the processed aloe, catechu and gambir are sold in local material markets in China and Korea without their correct identification. It indicates the existent of high potential medicinal risk by misuse of three medicinal materials. The developed HPLC method can be applied to prevent unexpected biological activity due to misapplication of medicinal materials.

  11. On-line coupled high performance liquid chromatography-gas chromatography for the analysis of contamination by mineral oil. Part 1: method of analysis.

    PubMed

    Biedermann, Maurus; Grob, Koni

    2012-09-14

    For the analysis of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH), on-line coupled high performance liquid chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID) offers important advantages: it separates MOSH and MOAH in robust manner, enables direct injection of large aliquots of raw extracts (resulting in a low detection limit), avoids contamination of the sample during preparation and is fully automated. This review starts with an overview of the technology, particularly the fundamentals of introducing large volumes of solvent into GC, and their implementation into various transfer techniques. The main part deals with the concepts of MOSH and MOAH analysis, with a thorough discussion of the choices made. It is followed by a description of the method. Finally auxiliary tools are summarized to remove interfering components, enrich the sample in case of a high fat content and obtain additional information about the MOSH and MOAH composition.

  12. Quantification of PSP toxins in toxic shellfish matrices using post-column oxidation liquid chromatography and pre-column oxidation liquid chromatography methods suggests post-column oxidation liquid chromatography as a good monitoring method of choice.

    PubMed

    Rey, Verónica; Botana, Ana M; Botana, Luis M

    2017-04-01

    Different shellfish samples were analyzed by Pre- and Post-Column Oxidation Liquid Chromatography to compare the toxins profiles and get information about the degree of accomplishment of both methods. Comparison of the results obtained, the linear correlation coefficient (r(2) = 0.94) and the paired t test (two tails, α = 0.05), indicated that there were not significant differences between both sets of data. Nevertheless, important differences related to toxins profiles were found: it was remarkable the difference in results for both Gonyautoxins 1 and 4 and Decarbamoylgonyautoxins 2 and 3, depending on the method of choice, due to an overestimation in the Pre-Column method. It was necessary to modify the elution conditions in the Post-Column method to avoid the interference of matrix peaks at retention times closer to the retention times of the calibrants, mostly when working with oyster and scallop matrices, although it is a good method to use routinely.

  13. Gas chromatography of safranal as preferable method for the commercial grading of saffron (Crocus sativus L.).

    PubMed

    Bononi, Monica; Milella, Paola; Tateo, Fernando

    2015-06-01

    We present a new extraction protocol, using ethyl alcohol as a solvent, to evaluate safranal by gas chromatography (GC). A linear response was obtained with R(2)=0.995 and a reproducibility standard deviation of 4.7-6.0%. The limit of detection and limit of quantitation were 0.05 and 0.25gkg(-1), respectively. The GC data for several samples of powdered saffron from different origins were compared to specific absorbance values measured according to the ISO Normative 3632-1:2011 method. The aroma strength of saffron samples quantitated by GC and the specific absorbance values of safranal by the UV method did not correlate. Quantitative evaluation of safranal by GC appears to be more specific and useful for commercial comparisons of saffron quality.

  14. A high performance liquid chromatography method for determination of furfural in crude palm oil.

    PubMed

    Loi, Chia Chun; Boo, Huey Chern; Mohammed, Abdulkarim Sabo; Ariffin, Abdul Azis

    2011-09-01

    A modified steam distillation method was developed to extract furfural from crude palm oil (CPO). The collected distillates were analysed using high performance liquid chromatography (HPLC) coupled with an ultraviolet diode detector at 284nm. The HPLC method allowed identification and quantification of furfural in CPO. The unique thermal extraction of CPO whereby the fresh fruit bunches (FFB) are first subjected to steam treatment, distinguishes itself from other solvent-extracted or cold-pressed vegetable oils. The presence of furfural was also determined in the fresh palm oil from FFB (without undergoing the normal extraction process), palm olein, palm stearin, olive oil, coconut oil, sunflower oil, soya oil and corn oil. The chromatograms of the extracts were compared to that of standard furfural. Furfural was only detected in CPO. The CPO consignments obtained from four mills were shown to contain 7.54 to 20.60mg/kg furfural.

  15. A novel thin-layer chromatography method to screen 1,3-propanediol producers.

    PubMed

    Anand, Pinki; Saxena, Rajendra Kumar

    2012-11-01

    To date, there is no established protocol for the screening of 1,3-propanediol producers. The proposed method has a wide applicability to harness the commercial potential of microorganisms which produce 1,3-propanediol as the end product. Glycerol fermentation broth of 50 bacteria spotted on thin-layer chromatography plates and run by appropriate solvent systems followed by colour development using vanillin reagent gave different coloured spots with most of the compounds present in the fermentation broth. The appearance of a purple-coloured spot of 1,3-propanediol with a retention factor (R(f)) of 0.62 forms the basis for the selection of 1,3-propanediol producers. Apart from being a rapid detection system the proposed method is pH independent and its authenticity was reconfirmed by HPLC.

  16. [Methods of nanoparticles control in food and biological objects. Report 1. Use of microscopic and chromatography investigation methods].

    PubMed

    Raspopov, R V; Gmoshinskiĭ, I V; Popov, K I; Krasnoiarova, O V; Khotimchenko, S A

    2012-01-01

    One of the promising applications of modern nanotechnology are food productions, which includes the improvement of food packaging, creation of new forms of nutrients that are characterized by improved assimilation and technological characteristics, quality control through the creation of compact and cheap test kits. All these applications of nanomaterials related to the risks of the possibility of receipt of potentially toxic nanoparticles in the diet. The task of regulation and hygienic standardization requires developing of the methods, their qualitative and quantitative analysis for such complex, multicomponent systems, which are the agricultural commodities and food products. The best hope in this plan are assigned to a group of approaches related to the microscopic visualization of artificial nanoparticles in the biological objects. While the typical size of nanoparticles (<100 nm) are below the theoretical maximum-resolution light optical methods, transmission electron microscopy often allows not only to identify nanoparticles on their size and shape, but also a qualitative and quantitative analysis their chemical composition with the use of additional analytical options. Another group of elaborate methods used in solving the problems of qualitative and quantitative analysis of nanoparticles are chromatographic methods, in particular, the exclusion, hydrodynamic, high-performance liquid chromatography, and the flow-field fractionation. Limitation of chromatographic approaches related with the need of complex sample preparation, as well as specific difficulties in nanoparticles detecting in chromatographic fractions. Transmission electron microscopy and high-performance liquid chromatography methods are officially recommended in Russia for the analysis of artificial nanoparticles in natural biological systems, including food products.

  17. High-performance thin-layer chromatography method for inositol phosphate analysis.

    PubMed

    Hatzack, F; Rasmussen, S K

    1999-12-24

    A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol-25% ammonia solution-water (5:4:1) and substance quantities as low as 100-200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R(F) values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.

  18. Affinity-based target deconvolution of safranal

    PubMed Central

    2013-01-01

    Background and the purpose of the study Affinity-based target deconvolution is an emerging method for the identification of interactions between drugs/drug candidates and cellular proteins, and helps to predict potential activities and side effects of a given compound. In the present study, we hypothesized that a part of safranal pharmacological effects, one of the major constituent of Crocus sativus L., relies on its physical interaction with target proteins. Methods Affinity chromatography solid support was prepared by covalent attachment of safranal to agarose beads. After passing tissue lysate through the column, safranal-bound proteins were isolated and separated on SDS-PAGE or two-dimensional gel electrophoresis. Proteins were identified using MALDI-TOF/TOF mass spectrometry and Mascot software. Results and major conclusion Data showed that safranal physically binds to beta actin, cytochrome b-c1 complex sub-unit 1, trifunctional enzyme sub-unit beta and ATP synthase sub-unit alpha and beta. These interactions may explain part of safranal’s pharmacological effects. However, phenotypic and/or biological relevance of these interactions remains to be elucidated by future pharmacological studies. PMID:23514587

  19. Hydrophilic interaction liquid chromatography method development and validation for the assay of HEPES zwitterionic buffer.

    PubMed

    Xu, Xiaolong; Gevaert, Bert; Bracke, Nathalie; Yao, Han; Wynendaele, Evelien; De Spiegeleer, Bart

    2017-02-20

    HEPES is a zwitterionic buffer component used as a raw material in the GMP-manufacturing of advanced therapy medicinal products (ATMPs), hence requiring an adequate assay method with sufficient selectivity toward related impurities. Therefore, a hydrophilic interaction chromatography (HILIC) method was developed. Different factors were investigated towards the retention behavior of HEPES, its analogue EPPS and its starting material isethionate: pH, ion concentration and organic solvent ratio of the mobile phase, as well as column temperature. Moreover, stress testing resulted in the N-oxide degradant, identified by high resolution MS. The final method consisted of an isocratic system with an aqueous (pH 2.0 with H3PO4) acetonitrile (35:65, v/v) mobile phase on a zwitterionic HILIC (Obelisc N) column with a flow rate of 0.5mL/min and UV detection at 195nm. The assay method of HEPES was validated, obtaining adequate linearity (R(2)=0.999), precision (RSD of 0.5%) and accuracy (recovery of 100.08%). Finally, the applicability of the validated method was demonstrated by analysis of samples from different suppliers.

  20. Comprehensive measurement of total nondigestible carbohydrates in foods by enzymatic-gravimetric method and liquid chromatography.

    PubMed

    Nishibata, Toyohide; Tashiro, Kouichi; Kanahori, Sumiko; Hashizume, Chieko; Kitagawa, Machiko; Okuma, Kazuhiro; Gordon, Dennis T

    2009-09-09

    Total nondigestible carbohydrate (NDC) in foods was determined by combining, not modifications, AOAC Official Methods 991.43, 2001.03, and 2002.02. Total NDC included insoluble dietary fiber (IDF) + high-molecular-weight soluble dietary fiber (HMWSDF), nondigestible oligosaccharides (NDO) not precipitated in ethanol solution, and resistant starch (RS). Eight sources of NDC (cellulose, wheat bran, gum arabic, resistant maltodextrin, polydextrose, fructooligosaccharide, galactooligosaccharides, and RS) were incorporated in different combinations into standard formula bread samples. All of the NDC sources and bread samples were analyzed for their (1) IDF + HMWSDF content with corrections for residual RS amount using AOAC Official Method 991.43, (2) NDO by liquid chromatography (LC) in AOAC Official Method 2001.03, and (3) RS by AOAC Official Method 2002.02. The correlation coefficient (R(2)) comparing calculated amounts versus measured amounts of total NDC in 11 bread samples was 0.92. Analysis of commercial food samples was also well matched with the DF + NDO value on their nutritional label. Consequently, we confirmed a single measurement of LC can determine all NDO in foods, and total NDC in foods can be determined by unifying existing AOAC Official Methods.

  1. Ceftriaxone pharmacokinetics by new simple and sensitive ultra-high-performance liquid chromatography method.

    PubMed

    Campos, Michel Leandro; de Moura Alonso, Juliana; Dos Santos Martins, Evelin; Oliveira, Jonata Augusto; Hussni, Carlos Alberto; Peccinini, Rosângela Gonçalves

    2017-02-27

    Ceftriaxone is a cephalosporin antibiotic with a potent antimicrobial activity and excellent penetration in most body fluids such as pleural, peritoneal, spinal and brain. These facts contribute to the application of ceftriaxone in the treatment of bacterial peritonitis, an abdominal disorder in veterinary medicine, with potential risk of death. The determination of ceftriaxone levels in plasma and peritoneal fluid may be used to assess the pharmacokinetic profile at various instances of administration and allows observing if the concentrations needed are being achieved. Therefore a method was developed and validated for the determination of ceftriaxone in plasma and peritoneal fluid which after was applied in a pharmacokinetic profile study. The bioanalytical method validation was performed according to widely acceptable experiments. Two horses were used as a model of the method applicability; ceftriaxone was intraperitoneally administered to these animals as a single dose. The plasma and peritoneal fluid analysis were performed using an UHPLC system in reverse phase chromatography mode in fully validated conditions. The methods have shown linearity between 0.49 and 500μg/mL for plasma, and between 0.24 and 500μg/mL for peritoneal fluid. The quantitative analysis of ceftriaxone in these matrices allows monitoring of the therapy. This method showed improved sensitivity as well as the quantitation in peritoneal fluid.

  2. Determination of hormonal growth promoters in bovine hair: comparison of liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry methods for estradiol benzoate and nortestosterone decanoate.

    PubMed

    Duffy, Eleanor; Rambaud, Lauriane; Le Bizec, Bruno; O'Keeffe, Michael

    2009-04-01

    The detection of steroid residues in hair is a powerful strategy to demonstrate long-term administration of these growth promoters in meat production animals. Analysis of the ester form of administered steroids is an unambiguous approach to prove the illegal use of natural hormones. For detection, gas chromatography-mass spectrometry (GC-MS/MS) was generally used. However, recent advances in liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology have improved the robustness and potential sensitivity of this method. This paper describes development and validation, according to Commission Decision 2002/657/EC, of LC-MS/MS and GC-MS/MS methods, in two separate laboratories, for determination of steroid esters in bovine hair. Bovine hair samples taken from animals treated with estradiol-3-benzoate and nortestosterone decanoate, as well as from an untreated animal, were used to evaluate the comparability of the results of the two validated methods. The results of the inter-comparison demonstrate that both the LC-MS/MS and the GC-MS/MS methods are fit for purpose and capable of determining steroid esters in hair samples from treated bovine animals.

  3. Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

    SciTech Connect

    Politi, Regina; Rusyn, Ivan; Tropsha, Alexander

    2014-10-01

    The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R{sup 2} = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R{sup 2} = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables

  4. Assessment of the chemical changes during storage of phenol-formaldehyde resins pyrolysis gas chromatography mass spectrometry, inverse gas chromatography and Fourier transform infra red methods.

    PubMed

    Strzemiecka, B; Voelkel, A; Zięba-Palus, J; Lachowicz, T

    2014-09-12

    The chemical changes occurring in the phenol-formaldehyde resins (resol and novolac type) during their storage were investigated. In this paper the FT-IR, py-GCMS and inverse gas chromatography methods were applied for assessment of the changes occurring during storage of the phenolic resins. We have found that during storage some examined resins occurred partial curing. The results from all techniques applied are consistent. Py-GCMS is useful technique for screening the storage processes but IGC seems to be most sensitive one.

  5. Modern methods for vancomycin determination in biological fluids by methods based on high-performance liquid chromatography--A review.

    PubMed

    Javorska, Lenka; Krcmova, Lenka Kujovska; Solichova, Dagmar; Solich, Petr; Kaska, Milan

    2016-01-01

    Vancomycin is a glycopeptide antibiotic used in the therapy of severe bacterial infection. The monitoring of vancomycin levels is recommended because of its narrow therapeutic index and toxicity. This measurement is especially appropriate in patients with unstable renal functions, who receive high doses of vancomycin or present serious bacterial infections accompanied by important sequestration of liquids when it could be difficult to achieve the optimal therapeutic dose. Most of the methods for vancomycin determination in routine practice are immunoassays. However, chromatography-based techniques in combination with UV or mass spectrometry detection provide results with greater accuracy and precision also in complicated biological matrices. This review provides a detailed overview of modern approaches for the chromatographic separation of vancomycin in various biological samples and useful sample preparation procedures for vancomycin determination in various biological fluids.

  6. Integration of Gas Chromatography Mass Spectrometry Methods for Differentiating Ricin Preparation Methods

    SciTech Connect

    Wunschel, David S.; Melville, Angela M.; Ehrhardt, Christopher J.; Colburn, Heather A.; Victry, Kristin D.; Antolick, Kathryn C.; Wahl, Jon H.; Wahl, Karen L.

    2012-05-17

    The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of the castor plant Ricinus communis. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatographic - mass spectrometric (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method and independent of the seed source. In particular the abundance of mannose, arabinose, fucose, ricinoleic acid and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation.

  7. The Isotope-Coded Affinity Tag Method for Quantitative Protein Profile Comparison and Relative Quantitation of Cysteine Redox Modifications.

    PubMed

    Chan, James Chun Yip; Zhou, Lei; Chan, Eric Chun Yong

    2015-11-02

    The isotope-coded affinity tag (ICAT) technique has been applied to measure pairwise changes in protein expression through differential stable isotopic labeling of proteins or peptides followed by identification and quantification using a mass spectrometer. Changes in protein expression are observed when the identical peptide from each of two biological conditions is identified and a difference is detected in the measurements comparing the peptide labeled with the heavy isotope to the one with a normal isotopic distribution. This approach allows the simultaneous comparison of the expression of many proteins between two different biological states (e.g., yeast grown on galactose versus glucose, or normal versus cancer cells). Due to the cysteine-specificity of the ICAT reagents, the ICAT technique has also been applied to perform relative quantitation of cysteine redox modifications such as oxidation and nitrosylation. This unit describes both protein quantitation and profiling of cysteine redox modifications using the ICAT technique.

  8. A Novel User Classification Method for Femtocell Network by Using Affinity Propagation Algorithm and Artificial Neural Network

    PubMed Central

    Ahmed, Afaz Uddin; Tariqul Islam, Mohammad; Ismail, Mahamod; Kibria, Salehin; Arshad, Haslina

    2014-01-01

    An artificial neural network (ANN) and affinity propagation (AP) algorithm based user categorization technique is presented. The proposed algorithm is designed for closed access femtocell network. ANN is used for user classification process and AP algorithm is used to optimize the ANN training process. AP selects the best possible training samples for faster ANN training cycle. The users are distinguished by using the difference of received signal strength in a multielement femtocell device. A previously developed directive microstrip antenna is used to configure the femtocell device. Simulation results show that, for a particular house pattern, the categorization technique without AP algorithm takes 5 indoor users and 10 outdoor users to attain an error-free operation. While integrating AP algorithm with ANN, the system takes 60% less training samples reducing the training time up to 50%. This procedure makes the femtocell more effective for closed access operation. PMID:25133214

  9. A novel user classification method for femtocell network by using affinity propagation algorithm and artificial neural network.

    PubMed

    Ahmed, Afaz Uddin; Islam, Mohammad Tariqul; Ismail, Mahamod; Kibria, Salehin; Arshad, Haslina

    2014-01-01

    An artificial neural network (ANN) and affinity propagation (AP) algorithm based user categorization technique is presented. The proposed algorithm is designed for closed access femtocell network. ANN is used for user classification process and AP algorithm is used to optimize the ANN training process. AP selects the best possible training samples for faster ANN training cycle. The users are distinguished by using the difference of received signal strength in a multielement femtocell device. A previously developed directive microstrip antenna is used to configure the femtocell device. Simulation results show that, for a particular house pattern, the categorization technique without AP algorithm takes 5 indoor users and 10 outdoor users to attain an error-free operation. While integrating AP algorithm with ANN, the system takes 60% less training samples reducing the training time up to 50%. This procedure makes the femtocell more effective for closed access operation.

  10. Profiling methods to identify cold-regulated primary metabolites using gas chromatography coupled to mass spectrometry.

    PubMed

    Dethloff, Frederik; Erban, Alexander; Orf, Isabel; Alpers, Jessica; Fehrle, Ines; Beine-Golovchuk, Olga; Schmidt, Stefanie; Schwachtje, Jens; Kopka, Joachim

    2014-01-01

    This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.

  11. SILAC-iPAC: a quantitative method for distinguishing genuine from non-specific components of protein complexes by parallel affinity capture.

    PubMed

    Rees, Johanna S; Lilley, Kathryn S; Jackson, Antony P

    2015-02-06

    Pull-down assays can identify members of protein complexes but suffer from co-isolation of contaminants. The problem is particularly acute when the specifically interacting partners are of low-abundance and/or bind transiently with low affinity. To differentiate true interacting partners from contaminants, we have combined SILAC labelling with a proteomic method called "Interactomes by Parallel Affinity Capture" (iPAC). In our method, a cell-line stably expressing a doubly tagged target endogenous protein and its tag-less control cell-line are differentially SILAC labelled. Lysates from the two cell-lines are mixed and the tagged protein is independently purified for MS analysis using multiple affinity resins in parallel. This allows the quantitative identification of tagged proteins and their binding partners. SILAC-iPAC provides a rigorous and sensitive approach that can discriminate between genuine binding partners and contaminants, even when the contaminants in the pull-down are in large excess. We employed our method to examine the interacting partners of phosphatidyl inositol 5-phosphate 4-kinase 2β subunit (PI5P4K2β) and the Fanconi anaemia core complex in the chicken pre-B cell-line DT40. We confirmed known components of these two complexes, and we have identified new potential binding partners. Combining the iPAC approach with SILAC labelling provides a sensitive and fully quantitative method for the discrimination of specific interactions under conditions where low signal to noise ratios are unavoidable. In addition, our work provides the first characterisation of the most abundant proteins within the DT40 proteome and the non-specific DT40 'beadomes' (non-specific proteins binding to beads) for common epitope tags. Given the importance and widespread use of the DT40 cell-line, these will be important resources for the cell biology and immunology communities. Biological significance SILAC-iPAC provides an improved method for the analysis of low-affinity

  12. SILAC-iPAC: A quantitative method for distinguishing genuine from non-specific components of protein complexes by parallel affinity capture

    PubMed Central

    Rees, Johanna S.; Lilley, Kathryn S.; Jackson, Antony P.

    2015-01-01

    Pull-down assays can identify members of protein complexes but suffer from co-isolation of contaminants. The problem is particularly acute when the specifically interacting partners are of low-abundance and/or bind transiently with low affinity. To differentiate true interacting partners from contaminants, we have combined SILAC labelling with a proteomic method called “Interactomes by Parallel Affinity Capture” (iPAC). In our method, a cell-line stably expressing a doubly tagged target endogenous protein and its tag-less control cell-line are differentially SILAC labelled. Lysates from the two cell-lines are mixed and the tagged protein is independently purified for MS analysis using multiple affinity resins in parallel. This allows the quantitative identification of tagged proteins and their binding partners. SILAC–iPAC provides a rigorous and sensitive approach that can discriminate between genuine binding partners and contaminants, even when the contaminants in the pull-down are in large excess. We employed our method to examine the interacting partners of phosphatidyl inositol 5-phosphate 4-kinase 2β subunit (PI5P4K2β) and the Fanconi anaemia core complex in the chicken pre-B cell-line DT40. We confirmed known components of these two complexes, and we have identified new potential binding partners. Combining the iPAC approach with SILAC labelling provides a sensitive and fully quantitative method for the discrimination of specific interactions under conditions where low signal to noise ratios are unavoidable. In addition, our work provides the first characterisation of the most abundant proteins within the DT40 proteome and the non-specific DT40 ‘beadomes’ (non-specific proteins binding to beads) for common epitope tags. Given the importance and widespread use of the DT40 cell-line, these will be important resources for the cell biology and immunology communities. Biological significance SILAC–iPAC provides an improved method for the analysis of

  13. Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure based modeling methods

    PubMed Central

    Politi, Regina; Rusyn, Ivan; Tropsha, Alexander

    2016-01-01

    The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure-Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R2=0.55 and CCR=0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R2=0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. PMID:25058446

  14. Method of identity analyte-binding peptides

    DOEpatents

    Kauvar, L.M.

    1990-10-16

    A method for affinity chromatography or adsorption of a designated analyte utilizes a paralog as the affinity partner. The immobilized paralog can be used in purification or analysis of the analyte; the paralog can also be used as a substitute for antibody in an immunoassay. The paralog is identified by screening candidate peptide sequences of 4--20 amino acids for specific affinity to the analyte. 5 figs.

  15. Method of identity analyte-binding peptides

    DOEpatents

    Kauvar, Lawrence M.

    1990-01-01

    A method for affinity chromatography or adsorption of a designated analyte utilizes a paralog as the affinity partner. The immobilized paralog can be used in purification or analysis of the analyte; the paralog can also be used as a substitute for antibody in an immunoassay. The paralog is identified by screening candidate peptide sequences of 4-20 amino acids for specific affinity to the analyte.

  16. Histamine H1 receptor cell membrane chromatography online high-performance liquid chromatography with mass spectrometry method reveals houttuyfonate as an activator of the histamine H1 receptor.

    PubMed

    Guo, Ying; Han, Shengli; Cao, Jingjing; Zhang, Tao; He, Langchong

    2014-11-01

    Allergy is an abnormal reaction of the body to an allergen. Histamine is responsible for many of the acute symptoms of allergic diseases. Many of the allergic and inflammatory actions of histamine are mediated by the histamine H1 receptor. In the present study, we established a two-dimensional histamine H1 receptor/cell membrane chromatography with online high-performance liquid chromatography and mass spectrometry method for screening potential histamine-activating components in a traditional Chinese medicine injection. The specification of the method was validated by screening, separating, and identifying a mixed standard solution of diphenhydramine hydrochloride, gefitinib, tamsulosin, and nitrendipine. The Yujin injection, an example of traditional Chinese medicine injection, was screened and potential allergic components acting on the histamine H1 receptor were identified. A Ca(2+) flux assay showed that houttuyfonate and Yujin injection induced calcium release in a dose-dependent manner. This suggests that houttuyfonate is an activator of the histamine H1 receptor. The mechanism of houttuyfonate activation involves phosphorylation of the inositol-1,4,5-trisphosphate receptor. In conclusion, this two-dimensional method can rapidly detect and enrich target components isolated from the Yujin injection. This indicates that individuals with an overexpression of the histamine H1 receptor should be aware of possible allergic reactions when receiving the Yujin injection.

  17. Determination of nucleosides in Cordyceps sinensis and Ganoderma lucidum by high performance liquid chromatography method

    PubMed Central

    Khan, Masood Shah; Parveen, Rabea; Mishra, Kshipra; Tulsawani, Rajkumar; Ahmad, Sayeed

    2015-01-01

    Background: Nucleosides are supportive in the regulation and modulation of various physiological processes in body, they acts as precursors in nucleic acid synthesis, enhance immune response, help in absorption of iron and influence the metabolism of fatty acids. Cordyceps sinensis and Ganoderma lucidum are well-known for its use in traditional medicine of China, Nepal and India. They are rich in nucleosides such as adenine, adenosine, cordycepin, etc. Hence, a simple, economic and accurate high-performance liquid chromatography (HPLC) analytical method was proposed for determination of adenine and adenosine for the quality control of plants. Materials and Methods: Chromatographic experiments were conducted on YL9100 HPLC system (South Korea). Reversed-phase chromatography was performed on a C18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 mL/min. Detection was carried out at 254 nm, which gives a sharp peak of adenine and adenosine at a retention time of 6.53 ± 0.02 min and 12.41 ± 0.02, respectively. Results and Discussion: Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25–200 µg/mL for adenosine and 100–800 µg/mL for adenine with regression coefficient of 0.999 and 0.996, respectively. The adenine was found 0.16% and 0.71% w/w in G. lucidum and in C. sinensis, respectively, and adenosine was found to be 0.14% w/w in G. lucidum whereas absent in C. sinensis. Conclusion: The developed HPLC method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations, in which adenine and adenosine are present as major constituents. The wide linearity range, sensitivity, accuracy, and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with high precision and

  18. Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

    PubMed Central

    Kamal, Abid; Khan, Washim; Ahmad, Sayeed; Ahmad, F. J.; Saleem, Kishwar

    2015-01-01

    Objective: The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga. Materials and Methods: RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase. Results: The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin. Conclusions: The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient. PMID:26681890

  19. The analysis of carbohydrates in milk powder by a new "heart-cutting" two-dimensional liquid chromatography method.

    PubMed

    Ma, Jing; Hou, Xiaofang; Zhang, Bing; Wang, Yunan; He, Langchong

    2014-03-01

    In this study, a new"heart-cutting" two-dimensional liquid chromatography method for the simultaneous determination of carbohydrate contents in milk powder was presented. In this two dimensional liquid chromatography system, a Venusil XBP-C4 analysis column was used in the first dimension ((1)D) as a pre-separation column, a ZORBAX carbohydrates analysis column was used in the second dimension ((2)D) as a final-analysis column. The whole process was completed in less than 35min without a particular sample preparation procedure. The capability of the new two dimensional HPLC method was demonstrated in the determination of carbohydrates in various brands of milk powder samples. A conventional one dimensional chromatography method was also proposed. The two proposed methods were both validated in terms of linearity, limits of detection, accuracy and precision. The comparison between the results obtained with the two methods showed that the new and completely automated two dimensional liquid chromatography method is more suitable for milk powder sample because of its online cleanup effect involved.

  20. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  1. A novel fast gas chromatography method for higher time resolution measurements of speciated monoterpenes in air

    NASA Astrophysics Data System (ADS)

    Jones, C. E.; Kato, S.; Nakashima, Y.; Kajii, Y.

    2014-05-01

    Biogenic emissions supply the largest fraction of non-methane volatile organic compounds (VOC) from the biosphere to the atmospheric boundary layer, and typically comprise a complex mixture of reactive terpenes. Due to this chemical complexity, achieving comprehensive measurements of biogenic VOC (BVOC) in air within a satisfactory time resolution is analytically challenging. To address this, we have developed a novel, fully automated Fast Gas Chromatography (Fast-GC) based technique to provide higher time resolution monitoring of monoterpenes (and selected other C9-C15 terpenes) during plant emission studies and in ambient air. To our knowledge, this is the first study to apply a Fast-GC based separation technique to achieve quantification of terpenes in ambient air. Three chromatography methods have been developed for atmospheric terpene analysis under different sampling scenarios. Each method facilitates chromatographic separation of selected BVOC within a significantly reduced analysis time compared to conventional GC methods, whilst maintaining the ability to quantify individual monoterpene structural isomers. Using this approach, the C9-C15 BVOC composition of single plant emissions may be characterised within a 14.5 min analysis time. Moreover, in-situ quantification of 12 monoterpenes in unpolluted ambient air may be achieved within an 11.7 min chromatographic separation time (increasing to 19.7 min when simultaneous quantification of multiple oxygenated C9-C10 terpenoids is required, and/or when concentrations of anthropogenic VOC are significant). These analysis times potentially allow for a twofold to fivefold increase in measurement frequency compared to conventional GC methods. Here we outline the technical details and analytical capability of this chromatographic approach, and present the first in-situ Fast-GC observations of 6 monoterpenes and the oxygenated BVOC (OBVOC) linalool in ambient air. During this field deployment within a suburban forest

  2. Gas chromatography-mass spectrometric method for metabolic profiling of tobacco leaves.

    PubMed

    Li, Yong; Pang, Tao; Li, Yanli; Wang, Xiaolin; Li, Qinghua; Lu, Xin; Xu, Guowang

    2011-06-01

    A gas chromatography-mass spectrometric method was developed for profiling of tobacco leaves. The differentiation among tobacco leaves planted in two different regions was investigated. Prior to analysis, the extraction solvent formulation was optimized and a combination of water, methanol and acetonitrile with a volume ratio of 3:1:1 was found to be optimal. The reproducibility of the method was satisfactory. Kendall tau-b rank correlation coefficients were equal to 1 (p<0.05) for 82% of the resolved peaks (up to 95% of the overall peak areas), indicating the good response correlation. Forty-four compounds including 9 saccharides, 9 alcohols, 9 amino acids, 16 organic acids and phosphoric acid were identified based on standard compounds. The method was successfully applied for profiling of tobacco leaves from Zimbabwe and Yunnan of China. Our result revealed that levels of saccharides and their derivatives including xylose, ribose, fructose, glucose, turanose, xylitol and glyceric acid were more abundant while sucrose, glucitol and D-gluconic acid were less abundant in tobacco leaves from Yunnan as compared to those from Zimbabwe. Amino acids such as L-alanine, L-tyrosine and L-threonine were found to be richer in Zimbabwe tobacco than in Yunnan tobacco.

  3. Development of a new method for the determination of nitrosamines by micellar electrokinetic capillary chromatography.

    PubMed

    Filho, Pedro José Sanches; Rios, Angel; Valcárcel, Miguel; Caramao, Elina Bastos

    2003-09-01

    A new method was developed for separation and quantification of nitrosamines by micellar electrokinetic capillary chromatography (MEKC). The effects of composition of the buffer, concentration of sodium dodecyl sulfate (SDS), gamma-cyclodextrin (CD) and pH on the separation and migration times of the nitrosamines were investigated. The different instrumental variables affecting sensitivity and resolution, such as power supply, injection mode and wavelength for measurement, were carefully optimized. The direct application of this method to aqueous synthetic samples allowed the separation and determination of nitrosamine mixtures at the mg x L(-1) level. The calibration curves revealed a linear range between 0.25 and 10 mg x L(-1), with detection limit (DL) varying from 0.16 to 0.27 mg x L(-1), quantification limit (QL) varying from 0.52 to 0.90 mg x L(-1), and RSD relative standard deviation (RSD) from 4.5% to 8.5%. The proposed method was successfully applied to the determination of dimethylnitrosamine (DMN), diethylnitrosamine (DEN), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosopiperidine (NPIP).

  4. Integrated liquid chromatography method in enantioselective studies: Biodegradation of ofloxacin by an activated sludge consortium.

    PubMed

    Maia, Alexandra S; Castro, Paula M L; Tiritan, Maria Elizabeth

    2016-09-01

    Ofloxacin is a chiral fluoroquinolone commercialized as racemate and as its enantiomerically pure form levofloxacin. This work presents an integrated liquid chromatography (LC) method with fluorescence detection (FD) and exact mass spectrometry (EMS) developed to assess the enantiomeric biodegradation of ofloxacin and levofloxacin in laboratory-scale microcosms. The optimized enantioseparation conditions were achieved using a macrocyclic antibiotic ristocetin A-bonded CSP (150×2.1mm i.d.; particle size 5μm) under reversed-phase elution mode. The method was validated using a mineral salts medium as matrix and presented selectivity and linearity over a concentration range from 5μgL(-1) (quantification limit) to 350μgL(-1) for each enantiomer. The method was successfully applied to evaluate biodegradation of ofloxacin enantiomers at 250μgL(-1) by an activated sludge inoculum. Ofloxacin (racemic mixture) and (S)-enantiomer (levofloxacin) were degraded up to 58 and 52%, respectively. An additional degradable carbon source, acetate, enhanced biodegradation up to 23%. (S)-enantiomer presented the highest extent of degradation (66.8%) when ofloxacin was supplied along with acetate. Results indicated slightly higher biodegradation extents for the (S)-enantiomer when supplementation was done with ofloxacin. Degradation occurred faster in the first 3days and proceeded slowly until the end of the assays. The chromatographic results from LC-FD suggested the formation of the (R)-enantiomer during levofloxacin biodegradation which was confirmed by LC-MS with a LTQ Orbitrap XL.

  5. Capture chromatography for Mo-99 recovery from uranyl sulfate solutions: minimum-column-volume design method.

    PubMed

    Ling, Lei; Chung, Pei-Lun; Youker, Amanda; Stepinski, Dominique C; Vandegrift, George F; Wang, Nien-Hwa Linda

    2013-09-27

    Molybdenum-99 (Mo-99), generated from the fission of Uranium-235 (U-235), is the radioactive parent of the most widely used medical isotope, technetium-99m (Tc-99m). An efficient, robust, low-pressure process is developed for recovering Mo-99 from uranyl sulfate solutions. The minimum column volume and the maximum column length for required yield, pressure limit, and loading time are determined using a new graphical method. The method is based on dimensionless groups and intrinsic adsorption and diffusion parameters, which are estimated using a small number of experiments and simulations. The design is tested with bench-scale experiments with titania columns. The results show a high capture yield and a high stripping yield (95±5%). The design can be adapted to changes in design constraints or the variations in feed concentration, feed volume, or material properties. The graph shows clearly how the column utilization is affected by the required yield, loading time, and pressure limit. The cost effectiveness of various sorbent candidates can be evaluated based on the intrinsic parameters. This method can be used more generally for designing other capture chromatography processes.

  6. Method for (236)U Determination in Seawater Using Flow Injection Extraction Chromatography and Accelerator Mass Spectrometry.

    PubMed

    Qiao, Jixin; Hou, Xiaolin; Steier, Peter; Nielsen, Sven; Golser, Robin

    2015-07-21

    An automated analytical method implemented in a flow injection (FI) system was developed for rapid determination of (236)U in 10 L seawater samples. (238)U was used as a chemical yield tracer for the whole procedure, in which extraction chromatography (UTEVA) was exploited to purify uranium, after an effective iron hydroxide coprecipitation. Accelerator mass spectrometry (AMS) was applied for quantifying the (236)U/(238)U ratio, and inductively coupled plasma mass spectrometry (ICPMS) was used to determine the absolute concentration of (238)U; thus, the concentration of (236)U can be calculated. The key experimental parameters affecting the analytical effectiveness were investigated and optimized in order to achieve high chemical yields and simple and rapid analysis as well as low procedure background. Besides, the operational conditions for the target preparation prior to the AMS measurement were optimized, on the basis of studying the coprecipitation behavior of uranium with iron hydroxide. The analytical results indicate that the developed method is simple and robust, providing satisfactory chemical yields (80-100%) and high analysis speed (4 h/sample), which could be an appealing alternative to conventional manual methods for (236)U determination in its tracer application.

  7. Micellar electrokinetic chromatography method development for simultaneous determination of thiabendazole, carbendazim, and fuberidazole.

    PubMed

    Soliman, Laiel C; Donkor, Kingsley K

    2014-01-01

    Thiabendazole (TBz), carbendazim (CBz), and fuberidazole (FBz) are systemic benzimidazole-type fungicides used for pre- and post-harvest treatment to control various types of fungal diseases on a variety of crops. Significant levels of these fungicides could alter the composition or flavour of crops, and being possible carcinogens, they could also pose risks for humans and the environment. A mode of capillary electrophoresis called micellar electrokinetic chromatography (MEKC) was investigated for the determination of these three benzimidazole fungicides. The study involved two kinds of surfactants in which several experimental conditions were optimized, i.e., buffer concentration, pH, micelle concentration, and percent organic modifier (methanol). Using the optimum experimental conditions, the fungicides were successfully separated by MEKC. The limits of detection and quantification were in the range of 0.6-0.7 and 2.1-2.5 mg L(-1), respectively, and the calibration curves were linear over the range of 5-60 mg L(-1) for the three fungicides. The potential of the proposed MEKC method was demonstrated by analyzing water samples which were fortified with the fungicides. The proposed method enabled simultaneous determination of the three benzimidazole fungicides and method validation with spiked water samples yielded satisfactory quantitative recoveries for all the three fungicides.

  8. Method for analysis of polar volatile trace components in aqueous samples by gas chromatography.

    PubMed

    Pettersson, Johan; Roeraade, Johan

    2005-05-15

    A new method has been developed for direct analysis of volatile polar trace compounds in aqueous samples by gas chromatography. Water samples are injected onto a short packed precolumn containing anhydrous lithium chloride. A capillary column is coupled in series with the prefractionation column for final separation of the analytes. The enrichment principle of the salt precolumn is reverse to the principles employed in conventional methods such as SPE or SPME in which a sorbent or adsorbent is utilized to trap or concentrate the analytes. Such methods are not efficient for highly polar compounds. In the LiCl precolumn concept, the water matrix is strongly retained on the hygroscopic salt, whereas polar as well as nonpolar volatile organic compounds show very low retention and are eluted ahead of the water. After transfer of the analytes to the capillary column, the retained bulk water is removed by backflushing the precolumn at elevated temperature. For direct injections of 120 microL of aqueous samples, the combined time for injection and preseparation is only 3.5 min. With this procedure, direct repetitive automated analyses of highly volatile polar compounds such as methanol or tetrahydrofuran can be performed, and a limit of quantification in the low parts-per-billion region utilizing a flame ionization detector is demonstrated.

  9. AMS radiocarbon age for fossil bone by XAD-2 chromatography method

    NASA Astrophysics Data System (ADS)

    Minami, Masayo; Nakamura, Toshio

    2000-10-01

    The XAD-2 chromatography method was examined for its ability to efficiently eliminate exogenous organic matter from fossil bones and to improve the accuracy of radiocarbon ( 14C) dating and stable isotope determinations on bone proteins. The fossil bones used in the experiment were animal fossil bones collected from the Awazu submarine archaeological site, Shiga, Japan. For comparison, the gelatin-extraction method was also applied to the same samples. It was found that the gelatin-extraction method is sufficient for 14C dating on well-preserved bones, but insufficient on poorly preserved bones, containing less than 1% extractable gelatin. The XAD-2 resin is useful for the clean up of proteins especially from poorly preserved bones. The carbon stable isotope fractionation of around 1‰ by XAD-2 treatment on modern collagen standards was larger than reported previously. The isotopic variation by sequential extraction of bones probably originates from changes in the amino acid composition and seems to be less sensitive to the indication of the removal of organic contamination.

  10. Detection of fuel release in a nuclear accident: a method for preconcentration and isolation of reactor-borne (239)Np using ion-specific extraction chromatography.

    PubMed

    Rosenberg, Brett L; Shozugawa, Katsumi; Steinhauser, Georg

    2015-09-01

    Although actinides are the most informative elements with respect to the nature of a nuclear accident, plutonium analysis is complicated by the background created by fallout from atmospheric nuclear explosions. Therefore, we propose (239)Np, a short-lived actinide that emits several γ rays, as a preferred proxy. The aim of this study was to screen ion specific extraction chromatography resins (RE-, TEVA-, UTEVA-, TRU-, and Actinide-Resin) for the highest possible recovery and separation of trace amounts of (239)Np from samples with large activities of fission products such as radiocesium, radioiodine, and, most importantly, radiotellurium, the latter of which causes spectral interference in gamma spectrometry through overlapping peaks with (239)Np. The investigated environmental media for these separations were aqueous solutions simulating rainwater and soil. Spiked samples containing (239)Np and the aforementioned volatile radionuclides were separated through extraction chromatographic columns to ascertain the most effective means of separating (239)Np from other fission products for detection by gamma spectroscopy. We propose a method for nuclear accident preparedness based on the use of Eichrom's RE-Resin. The proposed method was found most effective for isolating (239)Np from interfering radionuclides in both aqueous solution and soil using 8 M HNO3 as the loading solution and H2O as the eluent. The RE-Resin outperforms the more commonly used TEVA-Resin because the TEVA-Resin showed a higher affinity for interfering radiotellurium and radioiodine.

  11. Chromatography as Method for Analytical Confirmation of Paracetamol in Postmortem Material Together with Psychoactive Substances

    PubMed Central

    Biscevic-Tokic, Jasmina; Tokic, Nedim; Ibrahimpasic, Elma

    2015-01-01

    Introduction: Paracetamol (Acetaminophen) in addition to aspirin is the most commonly used analgesic and antipyretic medication by millions of patients worldwide. It is an example that paracetamol as medicine that in the world is provided without a doctor’s prescription, can lead to death. Today paracetamol became an integral part of a heroin mixture and is very popular at the street market. The main reason for this is that it can be obtained without a prescription, it is cheap, and by most people well tolerated without side effects. It is probably used for “cutting” the pure heroin, as it says in the jargon, and in that manner from small amount of pure drug is obtained greater amount, which is then sold on the street. The goal is to identify presence of paracetamol, by analytical method of gas chromatography mass spectrometer (GC-MS) in postmortem material together with psychoactive substances. Material and methods: For chemical-toxicological analysis is used biological material collected trough autopsy of 20 deceased people, suspected to have died due to psychoactive substance overdose. All received samples are stored at -20 ° C until analysis at our laboratory. From processed 47 samples that were analyzed in the period from 2014 to 2015, 19 are blood samples, urine 19, 3 samples of stomach contents, and 6 samples of bile content. Deceased were middle-aged, of which only 7 were female. The tested samples were processed according to two methods of extraction. Extraction by XAD-2 resin, and the extraction by the method of salting out with sodium tungstate. Extracts of the samples were then dissolved in chloroform and continued analysis at the analytical instrument. Identification of the paracetamol presence, in the test biological samples is demonstrated by the technique of gas chromatography with mass spectometry (hereinafter referred to as GC-MS). The technique of GC-MS is a selective, sensitive and reliable, and is therefore considered a “gold standard

  12. Rapid, economical qualitative method for separation of aflatoxins B-1, B-2 & G-1, G-2 by dry column chromatography.

    PubMed

    Megalla, S E

    1983-12-01

    A good correlation of four components of aflatoxins was accomplished by using the dry column chromatography method. The decolorization process of interfering substances, by 0.01 N KOH and defatting the extract with petroleum ether yields a clean residue for DCC separation. It is clear that the dry column chromatography is a very simple and time-saving procedure for separation of aflatoxins. DCC columns are more economical than precoated 'thick layer' preparative plates and, in DCC, no large developing tanks need to be used. Hazards associated with the use of large volumes of flammable solvents are greatly reduced.

  13. Constructing a LabVIEW-Controlled High-Performance Liquid Chromatography (HPLC) System: An Undergraduate Instrumental Methods Exercise

    ERIC Educational Resources Information Center

    Smith, Eugene T.; Hill, Marc

    2011-01-01

    In this laboratory exercise, students develop a LabVIEW-controlled high-performance liquid chromatography system utilizing a data acquisition device, two pumps, a detector, and fraction collector. The programming experience involves a variety of methods for interface communication, including serial control, analog-to-digital conversion, and…

  14. CAPILLARY GAS CHROMATOGRAPHY-ATOMIC EMISSION DETECTION METHOD FOR THE DETERMINATION OF PENTYLATED ORGANOTIN COMPOUNDS: INTERLABORATORY STUDY

    EPA Science Inventory

    A capillary gas chromatography-atomic emission detection (GC-AED) method was developed for the U. S. Environmental Protection Agency's Environmental Monitoring Systems Laboratory in Las Vegas, NV, for determination of selected organotin compounds. Here we report on an interlabora...

  15. High-Affinity α-Conotoxin PnIA Analogs Designed on the Basis of the Protein Surface Topography Method

    PubMed Central

    Kasheverov, Igor E.; Chugunov, Anton O.; Kudryavtsev, Denis S.; Ivanov, Igor A.; Zhmak, Maxim N.; Shelukhina, Irina V.; Spirova, Ekaterina N.; Tabakmakher, Valentin M.; Zelepuga, Elena A.; Efremov, Roman G.; Tsetlin, Victor I.

    2016-01-01

    Despite some success for small molecules, elucidating structure–function relationships for biologically active peptides — the ligands for various targets in the organism — remains a great challenge and calls for the development of novel approaches. Some of us recently proposed the Protein Surface Topography (PST) approach, which benefits from a simplified representation of biomolecules’ surface as projection maps, which enables the exposure of the structure–function dependencies. Here, we use PST to uncover the “activity pattern” in α-conotoxins — neuroactive peptides that effectively target nicotinic acetylcholine receptors (nAChRs). PST was applied in order to design several variants of the α-conotoxin PnIA, which were synthesized and thoroughly studied. Among the best was PnIA[R9, L10], which exhibits nanomolar affinity for the α7 nAChR, selectivity and a slow wash-out from this target. Importantly, these mutations could hardly be delineated by “standard” structure-based drug design. The proposed combination of PST with a set of experiments proved very efficient for the rational construction of new bioactive molecules. PMID:27841338

  16. A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese.

    PubMed

    Mayer, H K; Fiechter, G; Fischer, E

    2010-05-07

    A fast and reliable ultra-performance liquid chromatography (UPLC) method for the determination of biogenic amines (ethanolamine, methylamine, agmatine, histamine, dimethylamine, ethylamine, octopamine, pyrrolidine, dopamine, isopropylamine, propylamine, tyramine, putrescine, butylamine, cadaverine, tryptamine, 2-phenylethylamine, 3-methylbutylamine, spermidine, spermine) in cheese was established. After pre-column derivatization with 6-aminoquinolyl-N-hydroxy-succinimidyl carbamate (AQC), 20 primary and secondary biogenic amines were separated on an Acquity UPLC column (BEH C(18), 1.7 microm; 2.1 mm x 50 mm) within 9 min. Limits of detection (mg/100g cheese) ranged from 0.04 (ethanolamine) to 1.62 (spermine), and limits of quantification were between 0.16 (ethanolamine) and 6.09 (spermine). The UPLC method was applied to the analysis of 58 cheese samples as retailed in Austria. About 13.8% of samples had a histamine content above 10mg/100g, and 22.4% had a tyramine content above 10mg/100g. Moreover, 8.6% of samples had a putrescine or cadaverine content higher than 10mg/100g. The total concentration of biogenic amines in two cheese samples was about 194 mg/100g. Thus, obligatory monitoring of biogenic amines should be considered to ensure quality of cheese in future.

  17. Analysis of Fluconazole in Human Urine Sample by High Performance Liquid Chromatography Method

    NASA Astrophysics Data System (ADS)

    Hermawan, D.; Ali, N. A. Md; Ibrahim, W. A. Wan; Sanagi, M. M.

    2013-04-01

    A method for determination of fluconazole, antifungal drug in human urine by using reversed-phased high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detector was developed. Optimization HPLC conditions were carried out by changing the flow rate and composition of mobile phase. The optimum separation conditions at a flow rate 0.85 mL/min with a composition of mobile phase containing methanol:water (70:30, v/v) with UV detection at a wavelength 254 nm was able to analyze fluconazole within 3 min. The excellent linearity was obtained in the range of concentration 1 to 10 μg/mL with r2 = 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.39 μg/mL and 1.28 μg/mL, respectively. Solid phase extraction (SPE) method using octadecylsilane (C18) as a sorbent was used to clean-up and pre-concentrated of the urine sample prior to HPLC analysis. The average recoveries of fluconazole in spiked urine sample was 72.4% with RSD of 3.21% (n=3).

  18. A novel method for the determination of guanfacine in urine by gas chromatography-mass spectrometry.

    PubMed

    Haglock, Carrie; Wolf, Carl; Poklis, Alphonse

    2008-10-01

    Guanfacine (Tenex), an antihypertensive available since 1975, has recently been indicated for the treatment of attention deficit hyperactivity disorder in children (Intuniv). Because of this new usage, a gas chromatography-mass spectrometry method was developed and validated for the determination of guanfacine in urine. Guanfacine and 100 ng of protriptyline (internal standard) were extracted from 1.0 mL urine with 0.5 mL of saturated carbonate/bicarbonate buffer and 2 mL of ethyl acetate. The solvent extract was evaporated and derivatized with heptaflurobutyric anhydride in n-butyl chloride. Chromatographic separation was achieved using a DB-5 capillary column (30 m x 0.32 mm, 0.25 microm). Ions monitored for guanfacine were m/z 86.1, 272.1, and 274.1, and ions monitored for protriptyline were m/z 191.1 and 189.1. Concentrations were determined using calibrators over the range of 0.1-2.0 mg/L. The linear regression for all calibration curves had r2 values > or = 0.99. The limit of detection was 0.05 mg/L; limit of quantitation was 0.1 mg/L; and upper limit of linearity was 10.0 mg/L. Percent recovery of guanfacine at 0.1 and 2.0 mg/L was 93% and 71%, respectively. The method was found acceptable for routine quantitative analysis of guanfacine in urine.

  19. Ultra performance liquid chromatography PDA method for determination of tigecycline in human plasma.

    PubMed

    D'Avolio, Antonio; Peila, Emanuela; Simiele, Marco; Pensi, Debora; Baietto, Lorena; Cusato, Jessica; Cinnirella, Giacoma; De Rosa, Francesco; Di Perri, Giovanni

    2013-12-01

    : A simple ultra performance liquid chromatography with photodiode array method for the quantification of human plasma concentrations of tigecycline was developed and validated. Quinaxoline, used as an internal standard, was added to 500 μL of plasma before adding 1 mL of protein precipitation solution. The extracts were dried in a vacuum centrifuge system at 60°C and reconstituted with 60 μL of water and acetonitrile (95:5, vol/vol), and 5 μL was injected onto an ACQUITY UPLC H-Class system. Chromatographic separation was performed on a C18 ACQUITY UPLC HSS T3 column using a gradient of potassium phosphate buffer (pH 3.2) and acetonitrile. Detection was performed using a photodiode array detector at 350 nm. Relative error at 3 quality control concentrations ranged from -2.49% to -8.74%. Intraday and interday (percent relative standard error) precision ranged from 3.93% to 12.27% and from 9.53% to 13.32%, respectively. Limit of quantification and limit of detection were 0.024 and 0.006 μg/mL, respectively. Mean recovery was 95%. The calibration curve was linear up to 6 μg/mL. This concentration range proved to be adequate to measure tigecycline concentrations in patients treated with the drug, therefore this method would be suitable for therapeutic drug monitoring.

  20. Multi-class method for biomonitoring of hair samples using gas chromatography-mass spectrometry.

    PubMed

    Martín, Julia; Möder, Monika; Gaudl, Alexander; Alonso, Esteban; Reemtsma, Thorsten

    2015-11-01

    Currently, non-invasive biomonitoring of human exposure to organic pollutants bases upon the analysis mainly of urine and human breast milk. While mostly persistent organic pollutants are the center of interest, the aim of our study was to develop a method for the determination of different chemical classes of emerging pollutants (organophosphorus flame retardants, plastic additives such as phthalates, bisphenol A, insecticides, antimicrobials, preservatives and musk fragrances) in hair by gas chromatography-mass spectrometry. The preferred sample preparation included hydrolysis of the hair with trifluoroacetic acid in methanol followed by a liquid-liquid extraction using hexane/ethyl acetate. The validated method is characterized by recoveries higher than 77 % for most analytes, relative standard deviations below 16 % and limits of detection between 2 pg mg(-1) (HHCB) and 292 pg mg(-1) (propylparaben) using 50 mg of dry hair. After respective blank corrections, bis-(2-ethylhexyl)phthalate (DEHP) and the musk fragrance HHCB were the predominant compounds determined in all hair samples at concentrations between 32 and 59 ng mg(-1) and 0.8-13 ng mg(-1), respectively. The bactericide triclosan and the insect repellent N,N-diethyl-3-methylbenzamide (DEET) were detected in selected hair samples at 2 and 0.8 ng mg(-1), respectively.

  1. Isotope-ratio-monitoring gas chromatography-mass spectrometry: methods for isotopic calibration

    NASA Technical Reports Server (NTRS)

    Merritt, D. A.; Brand, W. A.; Hayes, J. M.

    1994-01-01

    In trial analyses of a series of n-alkanes, precise determinations of 13C contents were based on isotopic standards introduced by five different techniques and results were compared. Specifically, organic-compound standards were coinjected with the analytes and carried through chromatography and combustion with them; or CO2 was supplied from a conventional inlet and mixed with the analyte in the ion source, or CO2 was supplied from an auxiliary mixing volume and transmitted to the source without interruption of the analyte stream. Additionally, two techniques were investigated in which the analyte stream was diverted and CO2 standards were placed on a near-zero background. All methods provided accurate results. Where applicable, methods not involving interruption of the analyte stream provided the highest performance (sigma = 0.00006 at.% 13C or 0.06% for 250 pmol C as CO2 reaching the ion source), but great care was required. Techniques involving diversion of the analyte stream were immune to interference from coeluting sample components and still provided high precision (0.0001 < or = sigma < or = 0.0002 at.% or 0.1 < or = sigma < or = 0.2%).

  2. System and method for chromatography and electrophoresis using circular optical scanning

    DOEpatents

    Balch, Joseph W.; Brewer, Laurence R.; Davidson, James C.; Kimbrough, Joseph R.

    2001-01-01

    A system and method is disclosed for chromatography and electrophoresis using circular optical scanning. One or more rectangular microchannel plates or radial microchannel plates has a set of analysis channels for insertion of molecular samples. One or more scanning devices repeatedly pass over the analysis channels in one direction at a predetermined rotational velocity and with a predetermined rotational radius. The rotational radius may be dynamically varied so as to monitor the molecular sample at various positions along a analysis channel. Sample loading robots may also be used to input molecular samples into the analysis channels. Radial microchannel plates are built from a substrate whose analysis channels are disposed at a non-parallel angle with respect to each other. A first step in the method accesses either a rectangular or radial microchannel plate, having a set of analysis channels, and second step passes a scanning device repeatedly in one direction over the analysis channels. As a third step, the scanning device is passed over the analysis channels at dynamically varying distances from a centerpoint of the scanning device. As a fourth step, molecular samples are loaded into the analysis channels with a robot.

  3. Quality Evaluation of Potentilla fruticosa L. by High Performance Liquid Chromatography Fingerprinting Associated with Chemometric Methods

    PubMed Central

    Liu, Wei; Wang, Dongmei; Liu, Jianjun; Li, Dengwu; Yin, Dongxue

    2016-01-01

    The present study was performed to assess the quality of Potentilla fruticosa L. sampled from distinct regions of China using high performance liquid chromatography (HPLC) fingerprinting coupled with a suite of chemometric methods. For this quantitative analysis, the main active phytochemical compositions and the antioxidant activity in P. fruticosa were also investigated. Considering the high percentages and antioxidant activities of phytochemicals, P. fruticosa samples from Kangding, Sichuan were selected as the most valuable raw materials. Similarity analysis (SA) of HPLC fingerprints, hierarchical cluster analysis (HCA), principle component analysis (PCA), and discriminant analysis (DA) were further employed to provide accurate classification and quality estimates of P. fruticosa. Two principal components (PCs) were collected by PCA. PC1 separated samples from Kangding, Sichuan, capturing 57.64% of the variance, whereas PC2 contributed to further separation, capturing 18.97% of the variance. Two kinds of discriminant functions with a 100% discrimination ratio were constructed. The results strongly supported the conclusion that the eight samples from different regions were clustered into three major groups, corresponding with their morphological classification, for which HPLC analysis confirmed the considerable variation in phytochemical compositions and that P. fruticosa samples from Kangding, Sichuan were of high quality. The results of SA, HCA, PCA, and DA were in agreement and performed well for the quality assessment of P. fruticosa. Consequently, HPLC fingerprinting coupled with chemometric techniques provides a highly flexible and reliable method for the quality evaluation of traditional Chinese medicines. PMID:26890416

  4. Improved liquid chromatography-tandem mass spectrometry method for the analysis of eptifibatide in human plasma.

    PubMed

    Zhou, Zhi-Ling; Yu, Xi-Yong; Yang, Min; Mai, Li-Ping; Lin, Qiu-Xiong; Deng, Chun-Yu; Shan, Zhi-Xin; Kuang, Su-Juan; Zhu, Ping; Huang, Xiao-Zhong; Li, Xiao-Hong; Chen, Tie-Feng; Lin, Shu-Guang

    2010-08-01

    A rapid, selective and highly sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C(18) column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6-->m/z 646.4 and m/z 931.6-->m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1-1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within +/-7.6% of nominal values. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 microg/kg bolus of eptifibatide to 8 healthy volunteers.

  5. A validated new method for nevirapine quantitation in human plasma via high-performance liquid chromatography.

    PubMed

    Silverthorn, Courtney F; Parsons, Teresa L

    2006-01-01

    A fully validated and clinically relevant assay was developed for the assessment of nevirapine concentrations in neonate blood plasma samples. Solid-phase extraction with an acid-base wash series was used to prepare subject samples for analysis. Samples were separated by high performance liquid chromatography and detected at 280 nm on a C8 reverse-phase column in an isocratic mobile phase. The retention times of nevirapine and its internal standard were 5.0 and 6.9 min, respectively. The method was validated by assessment of accuracy and precision (statistical values <15%), specificity, and stability. The assay was linear in the range 25-10,000 ng/mL (r2 > 0.996) and the average recovery was 93% (n = 18). The lower limit of quantification (relative standard deviation <20%) was determined to be 25 ng/mL for 50 microL of plasma, allowing detection of as little as 1.25 ng of nevirapine in a sample. This value represents an increase in sensitivity of up to 30-fold over previously published methods.

  6. A micellar liquid chromatography method for the quantification of abacavir, lamivudine and raltegravir in plasma.

    PubMed

    Peris-Vicente, Juan; Villareal-Traver, Mónica; Casas-Breva, Inmaculada; Carda-Broch, Samuel; Esteve-Romero, Josep

    2014-09-01

    An analytical methodology based on micellar liquid chromatography has been developed to quantify abacavir, lamivudine and raltegravir in plasma. These three antiretroviral drugs are prescribed as a set in highly active antiretroviral therapy to acquired immunodeficiency syndrome patients. The experimental procedure consists in the dilution of the sample in micellar media, followed by filtration and, without cleanup step. The analytes were resolved in less than 30min using a mobile phase of 0.05M sodium dodecyl sulphate at pH 7, running at 1mLmin(-1) under isocratic mode at room temperature through a C18 column (125×4.6mm, 5μm particle size). The UV detection wavelength was set at 260nm. The method was successfully validated following the requirements of ICH guidelines in terms of: linear range (0.25-2.5μgmL(-1)), linearity (r(2)>0.990), intra- and interday precision (<6.8%) and accuracy (92.3-104.2%) and robustness (<7.1%). To the extent of our knowledge, this is the first published method to quantify these three drugs in plasma. Several blood samples from AIDS patients taking this HAART set provided by a local hospital were analyzed with satisfactory results.

  7. Quality Evaluation of Potentilla fruticosa L. by High Performance Liquid Chromatography Fingerprinting Associated with Chemometric Methods.

    PubMed

    Liu, Wei; Wang, Dongmei; Liu, Jianjun; Li, Dengwu; Yin, Dongxue

    2016-01-01

    The present study was performed to assess the quality of Potentilla fruticosa L. sampled from distinct regions of China using high performance liquid chromatography (HPLC) fingerprinting coupled with a suite of chemometric methods. For this quantitative analysis, the main active phytochemical compositions and the antioxidant activity in P. fruticosa were also investigated. Considering the high percentages and antioxidant activities of phytochemicals, P. fruticosa samples from Kangding, Sichuan were selected as the most valuable raw materials. Similarity analysis (SA) of HPLC fingerprints, hierarchical cluster analysis (HCA), principle component analysis (PCA), and discriminant analysis (DA) were further employed to provide accurate classification and quality estimates of P. fruticosa. Two principal components (PCs) were collected by PCA. PC1 separated samples from Kangding, Sichuan, capturing 57.64% of the variance, whereas PC2 contributed to further separation, capturing 18.97% of the variance. Two kinds of discriminant functions with a 100% discrimination ratio were constructed. The results strongly supported the conclusion that the eight samples from different regions were clustered into three major groups, corresponding with their morphological classification, for which HPLC analysis confirmed the considerable variation in phytochemical compositions and that P. fruticosa samples from Kangding, Sichuan were of high quality. The results of SA, HCA, PCA, and DA were in agreement and performed well for the quality assessment of P. fruticosa. Consequently, HPLC fingerprinting coupled with chemometric techniques provides a highly flexible and reliable method for the quality evaluation of traditional Chinese medicines.

  8. Improved method for rapid detection of phthalates in bottled water by gas chromatography-mass spectrometry.

    PubMed

    Otero, Paz; Saha, Sushanta Kumar; Moane, Siobhan; Barron, John; Clancy, Gerard; Murray, Patrick

    2015-08-01

    An improved gas chromatography-mass spectrometry (GC-MS) method for simple, rapid and precise quantification of phthalates in drinking water is presented. This method was validated for bis (2-n-butoxyethyl) phthalate (DBEP), bis (2-n-ethylhexyl) phthalate (DEHP), butyl benzyl phthalate (BBP), di-butyl phthalate (DBP), diethyl phthalate (DEP), dihexyl phthalate (DHP), dimethyl phthalate (DMP), di-n-octyl phthalate (DNOP) and dinonyl phthalate (DINP). Linearity of 0.9984>r(2)>0.9975 in the range of 0.075-4.8μg/mL for the selected phthalates was obtained. Accuracy values were in the range of 93-114% and RSD% for the analysis of 1.2μg/mL of each phthalate was below 2.3% (n=9). This new method design has significantly improved the detection in terms of rapidity, specificity, repeatability and accuracy compared to available methods. The procedure has been applied to the analyses of three different brands of commercially available bottled mineral water and the corresponding plastic bottles. Phthalates were extracted with dichloromethane and re-constituted in cyclohexane prior to GC-MS analysis. When the validated GC-MS method was applied to the quantification of the selected phthalates in the samples, only DBP (up to 0.0675±0.0018μg/mL) and DEHP (up to 1.6848±0.1631μg/mL) were found. Furthermore, we provide specific data about the concentration of DBP and DEHP in bottled water attributable to migration of phthalates from respective plastic bottles.

  9. Ultra-high-performance liquid chromatography profiling method for chemical screening of proanthocyanidins in Czech hops.

    PubMed

    Olšovská, J; Kameník, Z; Čejka, P; Jurková, M; Mikyška, A

    2013-11-15

    Hops represent an important natural source of bioactive polyphenols, particularly proanthocyanidins, which can contribute to prevention of several civilization diseases, owing to their antioxidant and radical scavenging activity. We have developed a high-throughput ultra-high-performance liquid chromatography time-of-flight mass spectrometry profiling method, which can be used for monitoring of bioactive proanthocyanidins in hops. The method was applied for analysis of hops of four Czech varieties (Saaz, Sladek, Preminat and Agnus) from the 2011 crop (9 localities, 11 samples) and the 2012 crop (24 localities, 40 samples). Hop samples were extracted by acetone and the analytes were separated on the Acquity UPLC BEH Shield RP18 column. Partial validation of the method revealed a satisfactory intra-day repeatability of the method for retention times (relative standard deviation within 1.39%) as well as areas under the peaks (within 9.89%). Experimental data were evaluated using principal component analysis and cluster analysis. Significant amounts of di-, tri- and tetramer proanthocyanidins consisting of (epi)catechin and (epi)gallocatechin were found in the hop samples. The dependence of the proantocyanidin composition on both the variety and the growing locality was observed. Specifically, the traditional Saaz variety contained more frequently oligomers formed by (epi)catechin units only, whereas the varieties Premiant and Agnus produced oligomers consisting of (epi)catechin as well as (epi)gallocatechin units. The relative abundance of proanthocyanidins in studied hop varieties from the two crops, 2011 and 2012, did correspond to each other. In the further perspective, the method may also be used for prediction of qualitative marks or authenticity verification of hops.

  10. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

  11. Characterization and classification of pseudo-stationary phases in micellar electrokinetic chromatography using chemometric methods.

    PubMed

    Fu, Cexiong; Khaledi, Morteza G

    2014-03-04

    Two types of chemometric methods, principal component analysis (PCA) and cluster analysis, are employed to characterize and classify a total of 70 pseudostationary phases (54 distinct systems and 16 decoy systems) in micellar electrokinetic chromatography (MEKC). PCA excels at removing redundant information for micellar phase characterization and retaining principal determinants for phase classification. While PCA is useful in the characterization of micelle selectivities, it is ineffective in defining the grouping of micellar phases. Hierarchical clustering yields a complete dendrogram of cluster structures but provides only limited cluster characterizations. The combination of these two chemometric methods leads to a comprehensive interpretation of the micellar phase classification. Moreover, the k-means analysis can further discern subtle differences among those closely located micellar phases. All three chemometric methods result in similar classifications with respect to the similarities and differences of the 70 micelle systems investigated. These systems are categorized into 3 major clusters: fluoro-surfactants represent cluster I, identified as strong hydrogen bond donors and dipolar but weak hydrogen bond acceptors. Cluster II includes sulfonated acrylamide/acrylate copolymers and surfactants with trimethylammonium head groups, characterized by strong hydrophobicity (v) and weak hydrogen bond acidity (b). The last cluster consists of two subclusters: clusters III and IV. Cluster III includes siloxane-based polymeric micelles, exhibiting weak hydrophobicity and medium hydrogen bond acidity and basicity (a), and the cluster IV micellar systems are characterized by their strong hydrophobicity and medium hydrogen bond acidity and basicity but rather weak dipolarity. Cluster III differs from cluster IV by its slightly weaker hydrophobicity and hydrogen bond donating capability. The classification by chemometric methods is in good agreement with the

  12. Rapid liquid chromatography for paralytic shellfish toxin analysis using superficially porous chromatography with AOAC Official Method 2005.06.

    PubMed

    Hatfield, Robert G; Turner, Andrew D

    2012-01-01

    The bioaccumulation of paralytic shellfish toxins in mussels, oysters, cockles, hard clams, razors, and king scallops is monitored in England, Scotland, and Wales by AOAC Official Method 2005.06 LC-with fluorescence detection (FLD). One of the commonly perceived disadvantages of using this method is the long turnaround time and low throughput in a busy laboratory environment. The chromatographic analysis of each sample typically utilizes a 15 min cycle time to achieve toxin oxidation product separation and column equilibration prior to subsequent analysis. A standard RP C18 analytical column, used successfully in recent years, achieves good separation with a long column lifetime. The analysis of a 40 sample qualitative screening batch takes approximately 18 h, including blanks, standards, and other QC samples. The availability of superficially porous column technology has offered the potential to reduce analysis time while retaining column performance on existing hardware. In this study, AOAC Official Method 2005.06 with LC-FLD was transferred to two different commercially available superficially porous columns, and the method performance characteristics were evaluated. Both columns separated all toxins adequately with cycle times less than half that of the existing method. Linearity for each toxin was acceptable up to two times the European maximum permitted limit of 800 microg di-HCl saxitoxin equivalent/kg flesh. LOD and LOQ values were substantially improved for the majority of toxins, with gonyautoxin 1&4 and neosaxitoxin showing up to a two- and fourfold improvement, respectively, depending on the column used. Quantification results obtained from parallel analysis of contaminated samples were acceptable on both columns. Comparative screen results gave a slight increase in the occurrence of contaminated samples, which was attributed to the improved detection limit for most toxins. Issues with rapidly increasing back pressure, however, were identified with both

  13. New methods for determination of cinnarizine in mixture with piracetam by spectrodensitometry, spectrophotometry, and liquid chromatography.

    PubMed

    Metwally, Fadia H; Elzeany, B A; Darwish, H W

    2005-01-01

    Four new methods were developed and validated for the determination of cinnarizine HCl in its binary mixture with piracetam in pure and pharmaceutical preparations. The first one was a densitometric analysis that provides a simple and rapid method for the separation and quantification of cinnarizine HCI. The method depends on the quantitative densitometric evaluation of thin-layer chromatograms of cinnarizine HCI at 252 nm over concentration range of 1-6 microg/spot, with a mean accuracy of 100.05 +/- 0.91%. The second method was determination of the drug using a colorimetric method that utilizes the reaction of 3-methyl-benzothiazolin-2-one in the presence of FeCl3 as an oxidant. The green color of the resulting product was measured at 630 nm over concentration range 10-40 microg/mL, with a mean accuracy of 100.10 +/- 1.13%. The third method was a direct spectrophotometric determination of cinnarizine HCI at 252 nm over the concentration range 7-20 microg/mL, while piracetam was determined by derivative ratio spectrophotometry at 221.6 nm over concentration range 5-30 microg/mL, with a mean accuracy of 100.14 +/- 0.79 and 100.26 +/- 1.24% for cinnarizine HCI and piracetam, respectively. The last method was a liquid chromatography analysis of both cinnarizine HCI and piracetam, depending on quantitative evaluation of chromatograms of cinnarizine HCI and piracetam at 252 and 212 nm, respectively, over the concentration range 10-200 microg/mL for cinnarizine HCI and 20-500 microg/mL for piracetam, with a mean accuracy of 100.03 +/- 0.89 and 100.40 +/- 0.94% for cinnarizine HCI and piracetam, respectively. The proposed procedures were checked using laboratory-prepared mixtures and successfully applied for the analysis of their pharmaceutical preparations. The validity of the proposed procedures was further assessed by applying the standard addition technique. Recoveries were quantitative, and the results obtained agreed with those obtained by other reported methods.

  14. Method transfer from high-pressure liquid chromatography to ultra-high-pressure liquid chromatography. I. A thermodynamic perspective.

    PubMed

    Åsberg, Dennis; Leśko, Marek; Samuelsson, Jörgen; Kaczmarski, Krzysztof; Fornstedt, Torgny

    2014-10-03

    This is the first investigation in a series that aims to enhance the scientific knowledge needed for reliable analytical method transfer between HPLC and UHPLC using the quality by design (QbD) framework. Here, we investigated the differences and similarities from a thermodynamic point of view between RP-LC separations conducted with 3.5μm (HPLC) and 1.7μm (UHPLC) C18 particles. Three different model solutes and one pharmaceutical compound were used: the uncharged cycloheptanone, the cationic benzyltriethylammonium chloride, the anionic sodium 2-naphatlene sulfonate and the pharmaceutical compound omeprazole, which was anionic at the studied pH. Adsorption data were determined for the four solutes at varying fractions of organic modifier and in gradient elution in both the HPLC and UHPLC system, respectively. From the adsorption data, the adsorption energy distribution of each compound was calculated and the adsorption isotherm model was estimated. We found that the adsorption energy distribution was similar, with only minor differences in degree of homogeneity, for HPLC and UHPLC stationary phases. The adsorption isotherm model did not change between HPLC and UHPLC, but the parameter values changed considerably especially for the ionic compounds. The dependence of the organic modifier followed the same trend in HPLC as in UHPLC. These results indicates that the adsorption mechanism of a solute is the same on HPLC and UHPLC stationary phases which simplifies design of a single analytical method applicable to both HPLC and UHPLC conditions within the QbD framework.

  15. Liquid chromatography/tandem mass spectrometry method for quantitation of cremophor el and its applications.

    PubMed

    Vijaya Bhaskar, V; Middha, Anil

    2013-01-01

    A rapid sensitive and selective MRM based method for the determination of Cremophor EL (CrEL) in rat plasma was developed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). CrEL and polypropylene glycol (internal standard) were extracted from rat plasma with acetonitrile and analysed on C18 column (XBridge, 50 × 4.6 mm, 3.5  μ m). The most abundant molecular ions corresponding to PEG oligomers at m/z 828, 872, 916 and 960 with daughter ion at m/z 89 were selected for multiple reaction monitoring (MRM) in electrospray mode of ionisation. Plasma concentrations of CrEL were quantified after administration through oral and intravenous routes in male sprague dawley rats at a dose of 0.26 g/kg. The standard curve was linear (0.9972) over the concentration range of 1.00 to 200  μ g/mL. The lower limit of quantitation for CrEL was 1.00  μ g/mL using 50  μ L plasma. The coefficient of variation and relative error for inter and intra assay at three QC levels were 0.69 to 9.21 and -7.60 to 4.74 respectively. A novel proposal was conveyed to the scientific community, where formulation excipient can be analysed as qualifier in the analysis of NCEs to address the spiky plasma concentration profiles.

  16. A gas chromatography-mass spectrometry method to monitor detergents removal from a membrane protein sample.

    PubMed

    Shi, Chaowei; Han, Fang; Xiong, Ying; Tian, Changlin

    2009-12-01

    In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography-mass spectrometry selected ion monitoring (GC-MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC-MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.

  17. Simple micellar electrokinetic chromatography method for the determination of hydrogen sulfide in hen tissues.

    PubMed

    Kubalczyk, Paweł; Borowczyk, Kamila; Chwatko, Grażyna; Głowacki, Rafał

    2015-04-01

    A new method for the determination of hydrogen sulfide in hen tissues has been developed and validated. For estimation of hydrogen sulfide content, a sample (0.1 g) of hen tissue was treated according to the procedure consisted of some essential steps: simultaneous homogenization of a tissue and derivatization of hydrogen sulfide to its S-quinolinium derivative with 2-chloro-1-methylquinolinium tetrafluoroborate, separation of so-formed derivative by micellar electrokinetic chromatography with sweeping, and detection and quantitation with the use of UV detector set to measure analytical signals at 375 nm. Effective electrophoretic separation was achieved using fused silica capillary (effective length 41.5 cm, 75 μm id) and 0.05 mol/L, pH 8 phosphate buffer with the addition of 0.04 mol/L SDS and 26% ACN. The lower limit of quantification was 0.12 μmol hydrogen sulfide in 1 g of tissue. The calibration curve prepared in tissue homogenate for hydrogen sulfide showed linearity in the range from 0.15 to 2.0 μmol/g, with the coefficient of correlation 0.9978. The relative standard deviation of the points of the calibration curve varied from 8.3 to 3.2% RSD.

  18. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

    PubMed

    Wang, Dongdong; Hincapie, Marina; Rejtar, Tomas; Karger, Barry L

    2011-03-15

    Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace

  19. Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography-tandem mass spectrometry method.

    PubMed

    Liu, Jia; Duan, Xiaotao; Chen, Xiaoyan; Zhong, Dafang

    2009-02-15

    A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine the concentration of eptifibatide in human plasma. Following protein precipitation, the analyte was separated on a reversed-phase C(18) column. Acetonitrile:5mM ammonium acetate:acetic acid (30:70:0.1, v/v/v) was used at a flow-rate of 0.5mL/min with the isocratic mobile phase. An API 4000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. "Truncated" multiple reaction monitoring using the transition of m/z 832.6-->m/z 832.6 and m/z 931.3-->m/z 931.3 was performed to quantify eptifibatide and the internal standard (EPM-05), respectively. The method had a lower limit of quantification of 4.61ng/mL for eptifibatide. The calibration curve was demonstrated to be linear over the concentration range of 4.61-2770ng/mL. The intra- and inter-day precisions were less than 10.5% for each QC level, and the inter-day relative errors were 2.0%, 5.6%, and 2.8% for 9.22, 184, and 2490ng/mL, respectively. The validated method was successfully applied to the quantification of eptifibatide concentration in human plasma after intravenous (i.v.) administration of a 270-microg/kg bolus of eptifibatide and i.v. administration of eptifibatide at a constant rate of infusion of 2microg/(kgmin) for 18h in order to evaluate the pharmacokinetics.

  20. Evaluation of injection methods for fast, high peak capacity separations with low thermal mass gas chromatography.

    PubMed

    Fitz, Brian D; Mannion, Brandyn C; To, Khang; Hoac, Trinh; Synovec, Robert E

    2015-05-01

    Low thermal mass gas chromatography (LTM-GC) was evaluated for rapid, high peak capacity separations with three injection methods: liquid, headspace solid phase micro-extraction (HS-SPME), and direct vapor. An Agilent LTM equipped with a short microbore capillary column was operated at a column heating rate of 250 °C/min to produce a 60s separation. Two sets of experiments were conducted in parallel to characterize the instrumental platform. First, the three injection methods were performed in conjunction with in-house built high-speed cryo-focusing injection (HSCFI) to cryogenically trap and re-inject the analytes onto the LTM-GC column in a narrower band. Next, the three injection methods were performed natively with LTM-GC. Using HSCFI, the peak capacity of a separation of 50 nl of a 73 component liquid test mixture was 270, which was 23% higher than without HSCFI. Similar peak capacity gains were obtained when using the HSCFI with HS-SPME (25%), and even greater with vapor injection (56%). For the 100 μl vapor sample injected without HSCFI, the preconcentration factor, defined as the ratio of the maximum concentration of the detected analyte peak relative to the analyte concentration injected with the syringe, was determined to be 11 for the earliest eluting peak (most volatile analyte). In contrast, the preconcentration factor for the earliest eluting peak using HSCFI was 103. Therefore, LTM-GC is demonstrated to natively provide in situ analyte trapping, although not to as great an extent as with HSCFI. We also report the use of LTM-GC applied with time-of-flight mass spectrometry (TOFMS) detection for rapid, high peak capacity separations from SPME sampled banana peel headspace.

  1. Determination of Alternaria mycotoxins in wine and juice using ionic liquid modified countercurrent chromatography as a pretreatment method followed by high-performance liquid chromatography.

    PubMed

    Fan, Chen; Cao, Xueli; Liu, Man; Wang, Wei

    2016-03-04

    Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are some of the main Alternaria mycotoxins that can be found as contaminants in food materials. The objective of this study was to develop a pretreatment method with countercurrent chromatography (CCC) for enrichment and cleanup of trace Alternaria mycotoxins in food samples prior to high-performance liquid chromatography (HPLC) analysis. An Analytical CCC instrument with a column volume 22.5mL was used, and a two-phase solvent system composed of ethyl acetate and water modified with 6% [HOOMIM][Cl] in mass to volume ratio was selected. Under the optimized CCC operation conditions, trace amounts of AOH, AME, and TeA in large volume of liquid sample were efficiently extracted and enriched in the stationary phase, and then eluted out just by reversing the stationary phase as mobile phase in the opposite flowing direction tail-to-head. The enrichment and elution strategies are unique and can be fulfilled online with high enrichment factors (87-114) and high recoveries (81.14-110.94%). The method has been successively applied to the determination of Alternaria mycotoxins in real apple juice and wine samples with the limits of detection (LOD) in the range of 0.03-0.14μgL(-1). Totally 12 wine samples and 15 apple juice samples from the local market were analyzed. The detection rate of AOH and AME in both kinds of the samples were more than 50%, while TeA was found in relatively high level of 1.75-49.61μgL(-1) in some of the apple juice samples. The proposed method is simple, rapid, and sensitive and could also be used for the analysis and monitoring of Alternaria mycotoxin in other food samples.

  2. Ultrafast gas chromatography method with direct injection for the quantitative determination of benzene, toluene, ethylbenzene, and xylenes in commercial gasoline.

    PubMed

    Miranda, Nahieh Toscano; Sequinel, Rodrigo; Hatanaka, Rafael Rodrigues; de Oliveira, José Eduardo; Flumignan, Danilo Luiz

    2017-04-01

    Benzene, toluene, ethylbenzene, and xylenes are some of the most hazardous constituents found in commercial gasoline samples; therefore, these components must be monitored to avoid toxicological problems. We propose a new routine method of ultrafast gas chromatography coupled to flame ionization detection for the direct determination of benzene, toluene, ethylbenzene, and xylenes in commercial gasoline. This method is based on external standard calibration to quantify each compound, including the validation step of the study of linearity, detection and quantification limits, precision, and accuracy. The time of analysis was less than 3.2 min, with quantitative statements regarding the separation and quantification of all compounds in commercial gasoline samples. Ultrafast gas chromatography is a promising alternative method to official analytical techniques. Government laboratories could consider using this method for quality control.

  3. [Measurement of free urinary cortisol and cortisone using liquid chromatography associated with tandem mass spectrometry method].

    PubMed

    Vieira, José Gilberto H; Nakamura, Odete H; Carvalho, Valdemir M

    2005-04-01

    Free urinary cortisol (UFF) measurement is one of the most useful screening tests for Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a solution containing a known quantity of deuterated cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the steroids tested presented cross reactivity of <1% for cortisol and cortisone. Functional sensitivity is <1 microg/L for both steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF cortisone allows the study of the activity of the enzyme 11beta-HSD2 and the diagnosis of the apparent mineralocorticoid excess syndrome. The method represents the first steroid assay of a new generation

  4. Ultra-high-pressure liquid chromatography-tandem mass spectrometry method for the determination of alkylphenols in soil.

    PubMed

    Wang, Jing; Pan, Hefang; Liu, Zhengzheng; Ge, Fei

    2009-03-20

    A novel method has been developed for the determination of alkylphenols in soil by ultra-high-pressure liquid chromatography employing small particle sizes, combined with tandem mass spectrometry. Soil samples were extracted with pressurized liquid extraction (PLE) and then cleaned with solid-phase extraction (SPE). The extracts were separated on C18 column (1.7 microm, 50 mm x 2.1mm) with a gradient elution and a mobile phase consisting of water and acetonitrile, and then detected by an electrospray ionization tandem mass spectrometry in negative ion mode with multiple reaction monitoring (MRM). Compared with traditional liquid chromatography, it took ultra-high-pressure liquid chromatography much less time to analyze alkylphenols. Additionally, the ultra-high-pressure liquid chromatography/tandem mass spectrometry method produces satisfactory reliability, sensitivity, and accuracy. The average recoveries of the three target analytes were 74.0-103.4%, with the RSD<15%. The calibration curves for alkylphenols were linear within the range of 0.01-0.4 microg/ml, with the correlation coefficients greater than 0.99. When 10 g soil sample was used for analysis, the limits of quantification (LOQs) of the three alkylphenols were all 1.0 microg/kg.

  5. Novel method for rapid copper chelation assessment confirmed low affinity of D-penicillamine for copper in comparison with trientine and 8-hydroxyquinolines.

    PubMed

    Říha, Michal; Karlíčková, Jana; Filipský, Tomáš; Macáková, Kateřina; Hrdina, Radomír; Mladěnka, Přemysl

    2013-06-01

    Copper is an essential trace element involved in many physiological processes. Since disorder of copper homeostasis is observed in various pathologies, copper chelators may represent a promising therapeutic tool. This study was aimed at: 1) formation of an in vitro methodology for screening of copper chelators, and 2) detailed analysis of the interaction of copper with clinically used D-penicillamine (D-PEN), triethylenetetramine (trientine), experimentally tested 8-hydroxyquinolines, and the disodium salt of EDTA as a standard chelator. Methodology based on bathocuproinedisulfonic acid disodium salt (BCS), usable at (patho)physiologically relevant pHs (4.5-7.5), enabled assessment of both cuprous and cupric ions chelation and comparison of the relative affinities of the tested compounds for copper. In the case of potent chelators, the stoichiometry could be estimated too. Clioquinol, chloroxine and EDTA formed very stable complexes with Cu(+)/Cu(2+) at all tested pHs, while copper complexes with trientine were stable only under neutral or slightly acidic conditions. Non-substituted 8-hydroxyquinoline was a less efficient copper chelator, but still unequivocally more potent than D-PEN. Both 8-hydroxyquinoline and D-PEN chelation potencies, similarly to that of trientine, were pH-dependent and decreased with pH. Moreover, only D-PEN was able to reduce cupric ions. Conclusively, BCS assay represents a rapid, simple and precise method for copper chelation measurement. In addition, lower binding affinity of D-PEN compared with 8-hydroxyquinolines and trientine was demonstrated.

  6. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    PubMed

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

  7. Influence of drying method on the surface energy of cellulose nanofibrils determined by inverse gas chromatography.

    PubMed

    Peng, Yucheng; Gardner, Douglas J; Han, Yousoo; Cai, Zhiyong; Tshabalala, Mandla A

    2013-09-01

    Research and development of the renewable nanomaterial cellulose nanofibrils (CNFs) has received considerable attention. The effect of drying on the surface energy of CNFs was investigated. Samples of nanofibrillated cellulose (NFC) and cellulose nanocrystals (CNC) were each subjected to four separate drying methods: air-drying, freeze-drying, spray-drying, and supercritical-drying. The surface morphology of the dried CNFs was examined using a scanning electron microscope. The surface energy of the dried CNFs was determined using inverse gas chromatography at infinite dilution and column temperatures: 30, 40, 50, 55, and 60 °C. Surface energy measurements of supercritical-dried NFCs were performed also at column temperatures: 70, 75, and 80 °C. Different drying methods produced CNFs with different morphologies which in turn significantly influenced their surface energy. Supercritical-drying resulted in NFCs having a dispersion component of surface energy of 98.3±5.8 mJ/m(2) at 30 °C. The dispersion component of surface energy of freeze-dried NFCs (44.3±0.4 mJ/m(2) at 30 °C) and CNCs (46.5±0.9 mJ/m(2) at 30 °C) were the lowest among all the CNFs. The pre-freezing treatment during the freeze-drying process is hypothesized to have a major impact on the dispersion component of surface energy of the CNFs. The acid and base parameters of all the dried CNFs were amphoteric (acidic and basic) although predominantly basic in nature.

  8. The Generally Useful Estimate of Solvent Systems (GUESS) method enables the rapid purification of methylpyridoxine regioisomers by countercurrent chromatography.

    PubMed

    Liu, Yang; Friesen, J Brent; Klein, Larry L; McAlpine, James B; Lankin, David C; Pauli, Guido F; Chen, Shao-Nong

    2015-12-24

    The TLC-based Generally Useful Estimate of Solvent Systems (GUESS) method was employed for countercurrent chromatography solvent system selection, in order to separate the three synthetic isomers: 3-O-methylpyridoxine, 4'-O-methylpyridoxine (ginkgotoxin), and 5'-O-methylpyridoxine. The Rf values of the three isomers indicated that ChMWat+2 (chloroform-methanol-water 10:5:5, v/v/v) was appropriate for the countercurrent separation. The isomer separation was highly selective and demonstrated that the TLC-based GUESS method can accelerate solvent system selection for countercurrent separation. Accordingly, the study re-emphasizes the practicality of TLC as a tool to facilitate the rapid development of new countercurrent and centrifugal partition chromatography methods for this solvent system. Purity and structure characterization of all samples was performed by quantitative (1)H NMR.

  9. Method developments approaches in supercritical fluid chromatography applied to the analysis of cosmetics.

    PubMed

    Lesellier, E; Mith, D; Dubrulle, I

    2015-12-04

    Analyses of complex samples of cosmetics, such as creams or lotions, are generally achieved by HPLC. These analyses are often multistep gradients, due to the presence of compounds with a large range of polarity. For instance, the bioactive compounds may be polar, while the matrix contains lipid components that are rather non-polar, thus cosmetic formulations are usually oil-water emulsions. Supercritical fluid chromatography (SFC) uses mobile phases composed of carbon dioxide and organic co-solvents, allowing for good solubility of both the active compounds and the matrix excipients. Moreover, the classical and well-known properties of these mobile phases yield fast analyses and ensure rapid method development. However, due to the large number of stationary phases available for SFC and to the varied additional parameters acting both on retention and separation factors (co-solvent nature and percentage, temperature, backpressure, flow rate, column dimensions and particle size), a simplified approach can be followed to ensure a fast method development. First, suited stationary phases should be carefully selected for an initial screening, and then the other operating parameters can be limited to the co-solvent nature and percentage, maintaining the oven temperature and back-pressure constant. To describe simple method development guidelines in SFC, three sample applications are discussed in this paper: UV-filters (sunscreens) in sunscreen cream, glyceryl caprylate in eye liner and caffeine in eye serum. Firstly, five stationary phases (ACQUITY UPC(2)) are screened with isocratic elution conditions (10% methanol in carbon dioxide). Complementary of the stationary phases is assessed based on our spider diagram classification which compares a large number of stationary phases based on five molecular interactions. Secondly, the one or two best stationary phases are retained for further optimization of mobile phase composition, with isocratic elution conditions or, when

  10. How reliable are gas-phase proton affinity values of small carbanions? A comparison of experimental data with values calculated using Gaussian-3 and CBS compound methods

    NASA Astrophysics Data System (ADS)

    Danikiewicz, Witold

    2009-08-01

    Gas-phase proton affinities (PA) of a series of 25 small, aliphatic carbanions were computed using different Gaussian-3 methods: G3, G3(B3LYP), G3(MP2) and G3(MP2, B3LYP) and Complete Basis Set Extrapolation methods: CBS-4M, CBS-Q, CBS-QB3, and CBS-APNO. The results were compared with critically selected experimental data. The analysis of the results shows that for the majority of the studied molecules all compound methods (Gaussian-3 and CBS), except for CBS-4M, give comparable results, which differ no more than +/-2 kcal mol-1 from the experimental data. Taking into account the calculation time, G3(MP2) and G3(MP2, B3LYP) methods offer the best compromise between accuracy and computational cost. As an additional proof, the results obtained by these two methods were compared with the values obtained using CCSD(T) ab initio method with large basis set. It was found also that some of the published experimental data are erroneous and should be corrected. The results described in this work show that for the majority of the studied compounds PA values calculated using compound methods can be used with the same or even higher confidence as the experimental ones because even the largest differences between Gaussian-3 and CBS methods listed above are still comparable with the accuracy of the typical PA measurements.

  11. Ion chromatography to detect salts in stone structures and to assess salt removal methods

    NASA Astrophysics Data System (ADS)

    Alvarez de Buergo, M.; Lopez-Arce, P.; Fort, R.

    2012-04-01

    thorough previous inspection, we can select the most representative points by a drilling net - as minimum as possible- and make some profiles of the inner salt content of a structure. Moreover, this procedure is not only reliable for determining the nature and extent of salts damage, but to assess the efficacy of salts removal methods in cultural heritage. Here we present two case studies from relevant buildings of the Spanish cultural heritage in which this procedure was performed with successful and useful results, in both terms of understanding what types of salts were decaying the stones structures, and also whether the salts removal methods that were planned in the restoration project were efficient or not. It should be remarked that even ion chromatography is not a non destructive technique (can be considered as a minimally destructive one due to the few quantity it is needed for the analysis), the information it can provide is so useful that should not be ruled out from the beginning, depending of each specific case.

  12. EPA Method 525.3 - Determination of Semivolatile Organic Chemicals in Drinking Water by Solid Phase Extraction and Capillary Column Gas Chromatography/Mass Spectrometry (GC/MS)

    EPA Science Inventory

    Method 525.3 is an analytical method that uses solid phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS) for the identification and quantitation of 125 selected semi-volatile organic chemicals in drinking water.

  13. Affinity separation of human plasma gelsolin on Affi-Gel Blue.

    PubMed

    Yamamoto, H; Terabayashi, M; Egawa, T; Hayashi, E; Nakamura, H; Kishimoto, S

    1989-05-01

    Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.

  14. Ion Chromatography.

    ERIC Educational Resources Information Center

    Mulik, James D.; Sawicki, Eugene

    1979-01-01

    Accurate for the analysis of ions in solution, this form of analysis enables the analyst to directly assay many compounds that previously were difficult or impossible to analyze. The method is a combination of the methodologies of ion exchange, liquid chromatography, and conductimetric determination with eluant suppression. (Author/RE)

  15. Sensitive and rapid high-performance liquid chromatography tandem mass spectrometry method for estimation of fulvestrant in rabbit plasma.

    PubMed

    Balaram, Varanasi Murali; Parmar, Dharmesh; Teja, Bulusu B; Rathnam, Shivprakash; Rao, Jangala Venkateswara; Dasandi, Bhavesh

    2010-08-01

    A rapid and sensitive high-performance liquid chromatography and electrospray tandem mass spectrometry method was developed and validated for estimation of fulvestrant in rabbit plasma using liquid-liquid extraction. The separation and quantification of fulvestrant were achieved by reverse-phase chromatography on a Sunfire C18 column (50 x 2.1. i.d., 3.5 microm) with isocratic elution at a flow rate of 300 microL/min using norethistrone as an internal standard from 500 microL plasma sample. The method was validated over the concentration range from 0.092 to 16.937 ng/mL with a lower limit of detection of 0.023 ng/mL. The intra-day and inter-day accuracy and precision were within 10%. The recovery was 85 and 90% for fulvestrant and norethistrone respectively. The chromatographic run time was only 2.5 min.

  16. Identification of atmospheric organic sources using the carbon hollow tube-gas chromatography method and factor analysis

    SciTech Connect

    Cobb, G.P.; Braman, R.S.; Gilbert, R.A. )

    1989-04-15

    Atmospheric organics were sampled and analyzed by using the carbon hollow tube-gas chromatography method. Chromatograms from spice mixtures, cigarettes, and ambient air were analyzed. Principal factor analysis of row order chromatographic data produces factors which are eigenchromatograms of the components in the samples. Component sources are identified from the eigenchromatograms in all experiments and the individual eigenchromatogram corresponding to a particular source is determined in most cases. Organic sources in ambient air and in cigaretts are identified with 87% certainty. Analysis of clove cigarettes allows the determination of the relative amount of clove in different cigarettes. A new nondestructive quality control method using the hollow tube-gas chromatography analysis is discussed.

  17. Liquid chromatography-tandem mass spectrometry method for the determination of anthelmintics in alfalfa plants.

    PubMed

    Islam, M Dabalus; Haberhauer, G; Gerzabek, M; Cannavan, A

    2012-01-01

    A simple and inexpensive liquid chromatography-tandem mass spectrometric method for the determination of anthelmintics in alfalfa plants (Medicago sativa L.) was developed and validated. Anthelmintics in plant leaves and stems (green chops) were extracted with methanol/acetonitrile (7:3, v/v) followed by a concentration and clean-up step using solid-phase extraction (Strata-X, 500 mg, 6 ml cartridge). After drying with nitrogen gas, the adsorbed analytes were eluted with methanol/acetonitrile (50:50, v/v) mixture followed by 100% acetonitrile. Chromatographic separation was achieved on an Atlantis T-3 (2.1 × 100 mm × 3 µm) analytical column with a Phenomenex guard cartridge (C8, 4 × 3 mm) attached to a Waters triple quadrupole mass spectrometer operated in positive electrospray ionisation mode with selected reaction monitoring. Samples were analysed using gradient elution at a flow rate of 0.35 ml min⁻¹. The mobile phase consisted of a 10 mM ammonium formate solution in (A) water/acetonitrile (90:10, v/v) and (B) methanol/acetonitrile (50:50, v/v). The method was validated for levamisole, fenbendazole, fenbendazole sulphoxide and fenbendazole sulphone at 10, 20 and 40 µg kg⁻¹ and for eprinomectin at 20, 40 and 80 µg kg⁻¹. Limits of quantification (LOQ) were 10 µg kg⁻¹ for all analytes except eprinomectin, which had an LOQ of 20 µg kg⁻¹. The overall mean recovery in green plants was between 74.2% and 81.4% with repeatabilities ranging from 2.2% to 19.1% and reproducibilities in the range 3.8-8.7%. The validated method was applied to plant samples in a study on the behaviour of anthelmintic drugs in a soil, plant and water system.

  18. Use of thermal desorption/gas chromatography as a performance-based screening method for petroleum hydrocarbons

    SciTech Connect

    Slavin, P.J. |; Crandall, K.; Dawson, L.; Kottenstette, R.; Wade, M. |

    1996-08-01

    Thermal desorption/gas chromatography (TD/GC) was used to screen soil samples on site for total petroleum hydrocarbon (TPH) content during a RCRA Facility Investigation (RFI). It proved to be a rapid, cost- effective tool for detecting non-aromatic mineral oil in soil. The on- site TD/GC results correlated well with those generated at an off- site laboratory for samples analyzed in accordance with EPA Method 418.1.

  19. Comparison of standard methods and gas chromatography method in determination of formaldehyde emission from MDF bonded with formaldehyde-based resins.

    PubMed

    Kim, Sumin; Kim, Hyun-Joong

    2005-09-01

    Formaldehyde emissions from MDF bonded with urea-formaldehyde resin (UF), melamine-formaldehyde resin (MF) and the co-polycondensation resin of urea-melamine-formaldehyde (UMF) and melamine-formaldehyde, measured by the Japanese standard method of determining formaldehyde emission with a desiccator (JIS A 5908) and the DIN EN 120 (European Committee For Standardization, 1991) method using the perforator value, were used as the typical standard methods. While the UF resin showed a desiccator value of 7.05 ppm and a perforator value of 12.1 mg/100 g panel, the MF resin exhibited a desiccator value of 0.6 ppm and a perforator value of 2.88 mg/100 g panel. According to the Japanese industrial standard and the European standard, the formaldehyde emission level of the MDF panels made with UF resin in this study was E(2) grade. The formaldehyde emission level was dramatically reduced by the addition of MF resin. This is because the addition of formaldehyde to melamine occurs more easily and completely than its addition to urea, even though the condensation reaction of melamine with formaldehyde is similar to that between urea and formaldehyde. These two methods, the desiccator method and the perforator method, produced proportionally equivalent results. Gas chromatography, a more sensitive and advanced method, was also used. The samples used for gas chromatography were gathered during the experiment involving the perforator method. The formaldehyde emission levels obtained from gas chromatography were similar to those obtained from the perforator method. The formaldehyde contents measured by gas chromatography were directly proportional to the perforator values.

  20. High-throughput fragment screening by affinity LC-MS.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in <4 h (corresponding to >3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  1. Green chromatography.

    PubMed

    Płotka, Justyna; Tobiszewski, Marek; Sulej, Anna Maria; Kupska, Magdalena; Górecki, Tadeusz; Namieśnik, Jacek

    2013-09-13

    Analysis of organic compounds in samples characterized by different composition of the matrix is very important in many areas. A vast majority of organic compound determinations are performed using gas or liquid chromatographic methods. It is thus very important that these methods have negligible environmental impact. Chromatographic techniques have the potential to be greener at all steps of the analysis, from sample collection and preparation to separation and final determination. The paper summarizes the approaches used to accomplish the goals of green chromatography. While complete elimination of sample preparation would be an ideal approach, it is not always practical. Solventless extraction techniques offer a very good alternative. Where solvents must be used, the focus should be on the minimization of their consumption. The approaches used to make chromatographic separations greener differ depending on the type of chromatography. In gas chromatography it is advisable to move away from using helium as the carrier gas because it is a non-renewable resource. GC separations using low thermal mass technology can be greener because of energy savings offered by this technology. In liquid chromatography the focus should be on the reduction of solvent consumption and replacement of toxic and environmentally hazardous solvents with more benign alternatives. Multidimensional separation techniques have the potential to make the analysis greener in both GC and LC. The environmental impact of the method is often determined by the location of the instrument with respect to the sample collection point.

  2. Development of a rapid diagnostic method for identification of Staphylococcus aureus and antimicrobial resistance in positive blood culture bottles using a PCR-DNA-chromatography method.

    PubMed

    Ohshiro, Takeya; Miyagi, Chihiro; Tamaki, Yoshikazu; Mizuno, Takuya; Ezaki, Takayuki

    2016-06-01

    Blood culturing and the rapid reporting of results are essential for infectious disease clinics to obtain bacterial information that can affect patient prognosis. When gram-positive coccoid cells are observed in blood culture bottles, it is important to determine whether the strain is Staphylococcus aureus and whether the strain has resistance genes, such as mecA and blaZ, for proper antibiotic selection. Previous work led to the development of a PCR method that is useful for rapid identification of bacterial species and antimicrobial susceptibility. However, that method has not yet been adopted in community hospitals due to the high cost and methodological complexity. We report here the development of a quick PCR and DNA-chromatography test, based on single-tag hybridization chromatography, that permits detection of S. aureus and the mecA and blaZ genes; results can be obtained within 1 h for positive blood culture bottles. We evaluated this method using 42 clinical isolates. Detection of S. aureus and the resistance genes by the PCR-DNA-chromatography method was compared with that obtained via the conventional identification method and actual antimicrobial susceptibility testing. Our method had a sensitivity of 97.0% and a specificity of 100% for the identification of the bacterial species. For the detection of the mecA gene of S. aureus, the sensitivity was 100% and the specificity was 95.2%. For the detection of the blaZ gene of S. aureus, the sensitivity was 100% and the specificity was 88.9%. The speed and simplicity of this PCR-DNA-chromatography method suggest that our method will facilitate rapid diagnoses.

  3. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  4. Advances in analytical techniques for neutron capture therapy: thin layer chromatography matrix and track etch thin layer chromatography methods for boron-10 analysis

    SciTech Connect

    Schremmer, J.M.; Noonan, D.J.

    1987-09-01

    A new track etch autoradiographic technique for quantitating boron-10 containing compounds used for neutron capture therapy is described. Instead of applying solutions of Cs2B12H11SH and its oxidation products directly to solid-state nuclear track detectors, diethylaminoethyl cellulose thin layer chromatography (TLC) plates are utilized as sample matrices. The plates are juxtaposed with Lexan polycarbonate detectors and irradiated in a beam of thermal neutrons. The detectors are then chemically etched, and the resultant tracks counted with an optoelectronic image analyzer. Sensitivity to boron-10 in solution reaches the 1 pg/microliter level, or 1 ppb. In heparinized blood samples, 100 pg boron-10/microliter are detected. This TLC matrix method has the advantage that sample plates can be reanalyzed under different reactor conditions to optimize detector response to the boron-10 carrier material. Track etch/TLC allows quantitation of the purity of boron neutron capture therapy compounds by utilizing the above method with TLC plates developed in solvent systems that resolve Cs2B12H11SH and its oxidative analogs. Detectors irradiated in juxtaposition to the thin layer chromatograms are chemically etched, and the tracks are counted in the sample lane from the origin of the plate to the solvent front. A graphic depiction of the number of tracks per field yields a quantitative analysis of compound purity.

  5. [THE DEVELOPMENT AND VALIDATION OF THE GAS CHROMATOGRAPHY METHOD AND VALIDATION DEVELOPMENT FOR THE ORGANIC CYANIDE (CYANOETHYLENE) DETERMINATION IN EXPIRED AIR].

    PubMed

    Zaĭtseva, N V; Ulanova, T S; Nurislamova, T V; Popova, N A; Mal'tseva, O A

    2015-01-01

    There are presented results of experimental studies on the development of gas chromatography method for the cyan ethylene determination in expired air During the process of the study there was chosen and proved the capillary gas chromatography method; there were investigated and elaborated optimal parameters of the gas chromatography separation of cyanoethylene with associated hydrocarbons together with the sample preparation and quantitative measurement methods. There was achieved the optimal level of gas chromatography quantification method for the cyan ethylene determination at 0, 00012 mg/m3, with the method uncertainty not more than 25%. The method was tried during the medical and biological examination of groups of 6-8 years old children, living in the territory of exposition from the moment of the birth and in the control territory.

  6. In silico prediction of estrogen receptor subtype binding affinity and selectivity using statistical methods and molecular docking with 2-arylnaphthalenes and 2-arylquinolines.

    PubMed

    Wang, Zhizhong; Li, Yan; Ai, Chunzhi; Wang, Yonghua

    2010-09-20

    Over the years development of selective estrogen receptor (ER) ligands has been of great concern to researchers involved in the chemistry and pharmacology of anticancer drugs, resulting in numerous synthesized selective ER subtype inhibitors. In this work, a data set of 82 ER ligands with ERα and ERβ inhibitory activities was built, and quantitative structure-activity relationship (QSAR) methods based on the two linear (multiple linear regression, MLR, partial least squares regression, PLSR) and a nonlinear statistical method (Bayesian regularized neural network, BRNN) were applied to investigate the potential relationship of molecular structural features related to the activity and selectivity of these ligands. For ERα and ERβ, the performances of the MLR and PLSR models are superior to the BRNN model, giving more reasonable statistical properties (ERα: for MLR, R(tr) (2) = 0.72, Q(te) (2) = 0.63; for PLSR, R(tr) (2) = 0.92, Q(te) (2) = 0.84. ERβ: for MLR, R(tr) (2) = 0.75, Q(te) (2) = 0.75; for PLSR, R(tr) (2) = 0.98, Q(te) (2) = 0.80). The MLR method is also more powerful than other two methods for generating the subtype selectivity models, resulting in R(tr) (2) = 0.74 and Q(te) (2) = 0.80. In addition, the molecular docking method was also used to explore the possible binding modes of the ligands and a relationship between the 3D-binding modes and the 2D-molecular structural features of ligands was further explored. The results show that the binding affinity strength for both ERα and ERβ is more correlated with the atom fragment type, polarity, electronegativites and hydrophobicity. The substitutent in position 8 of the naphthalene or the quinoline plane and the space orientation of these two planes contribute the most to the subtype selectivity on the basis of similar hydrogen bond interactions between binding ligands and both ER subtypes. The QSAR models built together with the docking procedure should be of great advantage for screening and

  7. The use of multivariate curve resolution methods to improve the analysis of muramic acid as bacterial marker using gas chromatography-mass spectrometry: an alternative method to gas chromatography-tandem mass spectrometry.

    PubMed

    Moazeni-Pourasil, Roudabeh Sadat; Piri, Farhad; Ghassempour, Alireza; Jalali-Heravi, Mehdi

    2014-02-15

    In analysis of muramic acid (MA) as bacterial marker, two dominant disturbing factors lead the researchers to use gas chromatography-tandem mass spectrometry (GC-MS/MS) technique instead of gas chromatography-mass spectrometry (GC-MS). These factors are the trace concentration of MA and fundamental disturbance of base line mass channels in GC-MS technique. This study aimed to utilize multivariate curve resolution (MCR) methods combined with GC-MS to improve the analysis of MA. First, the background and noise in GC-MS analysis were corrected and reduced using MCR methods. In addition, the MA overlapped peaks were resolved to its pure chromatographic and mass spectral profiles. Then the two-way response of each component was reconstructed by the outer product of the pure chromatographic and mass spectral profiles. The overall volume integration (OVI) method was used for quantitative determination. The MA peak area was decreased dramatically after the background correction and noise reduction. The findings severely ratify the appropriateness of using MCR techniques combined with GC-MS analysis as a simple, fast and inexpensive method for the analysis of MA in complex mixtures. The proposed method may be considered as an alternative method to GC-MS/MS for thorough analysis of the bacterial marker.

  8. Basic Principles of Chromatography

    NASA Astrophysics Data System (ADS)

    Ismail, Baraem; Nielsen, S. Suzanne

    Chromatography has a great impact on all areas of analysis and, therefore, on the progress of science in general. Chromatography differs from other methods of separation in that a wide variety of materials, equipment, and techniques can be used. [Readers are referred to references (1-19) for general and specific information on chromatography.]. This chapter will focus on the principles of chromatography, mainly liquid chromatography (LC). Detailed principles and applications of gas chromatography (GC) will be discussed in Chap. 29. In view of its widespread use and applications, high-performance liquid chromatography (HPLC) will be discussed in a separate chapter (Chap. 28). The general principles of extraction are first described as a basis for understanding chromatography.

  9. Development of a liquid chromatography-tandem mass spectrometry method for the determination of sulfite in food.

    PubMed

    Robbins, Katherine S; Shah, Romina; MacMahon, Shaun; de Jager, Lowri S

    2015-06-03

    Sulfites are widely used food preservatives that can cause severe reactions in sensitive individuals. As a result, the U.S. FDA requires that sulfites be listed on the label of any food product containing >10 mg/kg (ppm) sulfite (measured as sulfur dioxide). Currently, the optimized Monier-Williams (MW) method (AOAC Official Method 990.28) is the most common approach for determining sulfite concentrations in food samples. However, this method is time-consuming and lacks specificity in certain matrices. An improved rapid, sensitive, and selective method has been developed using electrospray ionization (ESI) high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of sulfite in various food matrices. A total of 12 different types of foods were evaluated. These included dried fruits and vegetables, frozen seafood, sweeteners, and juices. The matrix is extracted with a buffered formaldehyde solution, converting free and reversibly bound sulfite to the stable formaldehyde adduct, hydroxymethylsulfonate (HMS). Extracts are prepared for injection using a C18 SPE cartridge to remove any lipophilic compounds. HMS is then separated from other matrix components using hydrophilic interaction chromatography (HILIC) and detected using multiple reaction monitoring (MRM). The method was validated at 5 concentrations in 12 food matrices. Accuracy data showed spiked recoveries ranging from 84 to 115% in representative foods. Six commercially available sulfited products were analyzed using the LC-MS/MS method, as well as the MW method, to determine if differences exist.

  10. Evidence of multi-affinity in the Japanese stock market

    NASA Astrophysics Data System (ADS)

    Katsuragi, Hiroaki

    2000-04-01

    Fluctuations of the Japanese stock market (Tokyo Stock Price Index: TOPIX) are analyzed using a multi-affine analysis method. In the research to date, only some simulated self-affine models have shown multi-affinity. In most experiments using observations of self-affine fractal profiles, multi-affinity has not been found. However, we find evidence of multi-affinity in fluctuations of the Japanese stock market (TOPIX). The qth-order Hurst exponent Hq varies with changes in q. This multi-affinity indicates that there are plural mechanisms that affect the same time scale as stock market price fluctuation dynamics.

  11. Evaluation of a liquid chromatography method for quality control of methylated cyclodextrins.

    PubMed

    Fougère, L; Elfakir, C; Lafosse, M

    2013-02-15

    Halo C18 column (fused core particles) and Chromolith RP18 column (monolith) were evaluated in liquid chromatography in order to analyze methylated-β-cyclodextrins (Me-β-CD) with various degrees of substitution, DS such as the number of methyl groups per cyclodextrin ring. Chromolith RP18 enables a performing analysis of Me-β-CD with low DS but is not suitable for dimethyl-β-cyclodextrins (DM-β-CD). On the other hand, Halo C18 column allows an improved fingerprint of CDs having a DS from 4.9 up to a value major than 14 and avoiding the use of various chromatographic systems. Thus, liquid chromatography performed with this column and an evaporative light scattering detector can be used as a generic system for methylated CD analysis. Moreover, fused core particles of Halo C18 column enables a rapid analysis and liquid chromatography coupled with electrospray-mass spectrometry appears as a powerful tool to determine co-elution and to characterize various isomers of complex methylated-β-cyclodextrin mixtures.

  12. A reverse-phase liquid chromatography/mass spectrometry method for the analysis of high-molecular-weight fructooligosaccharides.

    PubMed

    Harrison, Scott J; Fraser, Karl; Lane, Geoffrey A; Villas-Boas, Silas; Rasmussen, Susanne

    2009-12-01

    Many important crop and forage plants accumulate polymeric water-soluble carbohydrates as fructooligosaccharides (or fructans). We have developed an improved method for the analysis of the full fructan complement in plant extracts based on porous graphitized carbon chromatography coupled to negative electrospray ionization mass spectrometry. By the use of profile data collection and multiple charge state ions, the effective mass range of the ion trap was extended to allow for the analysis of very high-molecular-weight oligosaccharides. This method allows the separation and quantification of isomeric fructan oligomers ranging from degree of polymerization (DP) 3 to DP 49.

  13. Rapid method for hydrocarbon-type analysis of heavy oils and synthetic fuels by pyrolysis thin layer chromatography

    SciTech Connect

    Poirier, M.A.; George, A.E.

    1982-09-01

    This work describes a rapid method for hydrocargon-type analysis applying thin layer chromatography (TLC) to the pentane-soluble fraction *malthenes) of the petroleum and synthetic fuels boiling above 200/sup 0/C. The principal component types encountered in this paper are saturates (SA), aromatics (AR), (mono and di together) polynuclear aromatics (PNA) and polar material (PO). The method uses a Iatroscan TLC pyrolyzer which combines the resolution capabilities of TLC with the possibility of quantification by using a flame-ionization detector (FID). Comparison of the results with those obtained by the API-60 procedure is presented.

  14. Development of a liquid chromatography-mass spectrometry method for the high-accuracy determination of creatinine in serum.

    PubMed

    Stokes, Peter; O'Connor, Gavin

    2003-08-25

    An LC-MS method for the high-accuracy determination of creatinine in serum has been developed and used to provide results for an international measurement evaluation programme (IMEP) and the Comité Consultatif pour la Quantité de Matière (CCQM) international inter-laboratory studies. An assessment of different sample preparation methods, including ion-exchange chromatography, solid-phase extraction, plasma ultrafiltration and ethanol protein precipitation, revealed that no bias or reduced precision was associated with the quicker less extensive clean-up methods, when using liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) for quantitation. A number of different calibration regimes were also investigated. External calibration was shown to provide adequate calibration for most routine analysis with a relative associated expanded uncertainty (k=2) of 6% at the 95% confidence level. The use of a non-isotopically labelled internal standard was shown to improve the relative expanded uncertainty (k=2) to 4%. However, the difference in retention time between the internal standard and the creatinine was such that a matrix interferent produced an observed bias of over 16%. The use of an isotopically labelled internal standard was shown to reduce any bias to less than 0.2% with an expanded uncertainty (k=2) of less than 0.3%. The developed method was then used, in a blind trial organised jointly by IMEP and CCQM, to determine the amount of creatinine in human serum. The method performed well against the established reference method of ion-exchange chromatography followed by derivatisation gas chromatography (GC)-IDMS. The observed difference between the values determined by LC-IDMS and the key comparison reference value (average of all the submitted results) was less than 0.3%. The biggest advantage of the described method is in the speed of analysis. With a chromatographic run time of less than 10 min and sample preparation consisting of a simple protein

  15. Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

    PubMed Central

    Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

    2016-01-01

    Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance. PMID:27846293

  16. Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

    PubMed

    Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan

    2014-07-22

    Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV.

  17. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    PubMed

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  18. METHOD 332.0: DETERMINATION OF PERCHLORATE IN DRINKING WATER BY ION CHROMATOGRAPHY WITH SUPPRESSED CONDUCTIVITY AND ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    This method is applicable to the identification and quantitation of perchlorate in raw and finished drinking waters. The approach used is ion chromatography with suppressed conductivity and electrospray ionization mass spectrometry (IC-ESI/MS)

  19. EPA Method 507: Determination of Nitrogen- and Phosphorus-Containing Pesticides in Water by Gas Chromatography with a Nitrogen-Phosphorus Detector

    EPA Pesticide Factsheets

    Method 507 describes procedures for sample preparation and analysis using solvent extraction of organophosphate pesticides in drinking water samples which are analyzed using a gas chromatography –nitrogen-phosphorus detector (GC-NPD).

  20. EPA Method 8321B (SW-846): Solvent-Extractable Nonvolatile Compounds by High Performance Liquid Chromatography-Thermospray-Mass Spectrometry (HPLC-TS-MS) or Ultraviolet (UV) Detection

    EPA Pesticide Factsheets

    Method 8321B describes procedures for preparation and analysis of solid, aqueous liquid, drinking water and wipe samples using high performance liquid chromatography and mass spectrometry for extractable non-volatile compounds.

  1. Thermal desorption-gas chromatography-mass spectrometry method to determine phthalate and organophosphate esters from air samples.

    PubMed

    Aragón, M; Borrull, F; Marcé, R M

    2013-08-16

    A method based on thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) has been developed to determine four organophosphate esters, seven phthalate esters, and bis(2-ethylhexyl) adipate in the gas phase from harbour and urban air samples. The method involves the sampling of 1.5L of air in a Tenax TA sorbent tube followed by thermal desorption (using a Tenax TA cryogenic trap) coupled to gas chromatography-mass spectrometry. The repeatability of the method expressed as %RSD (n=3) is less than 15% and the MQLs are between 0.007μgm(-3) (DMP, TBP, BBP, TPP and DnOP) and 6.7μgm(-3) (DEHP). The method was successfully applied in two areas (urban and harbour) testing two and three points in each one, respectively. Some of these compounds were found in both urban and harbour samples. Di-(2-ethylhexyl)phthalate was the most abundant compound found in both areas at concentration levels between 6.7μgm(-3) and 136.4μgm(-3). This study demonstrates that thermal desorption is an efficient method for the determination of these semi-volatile compounds in the gas phase fraction of air samples.

  2. A Simple and Sensitive Method for Quantitative Measurement of Methylmalonic Acid by Turbulent Flow Chromatography and Tandem Mass Spectrometry

    PubMed Central

    Tecleab, AG; Schofield, RC; Ramanathan, LV; Carlow, Dean C

    2016-01-01

    A simple and sensitive method for the detection of methylmalonic acid in serum without derivatization has been developed. This method implements protein precipitation using methanol followed by additional sample clean up by turbulent flow liquid chromatography (TFLC). The sample was directly injected into the turbulent flow liquid chromatography tandem mass spectrometry system (TFLC-MS/MS) for online extraction followed by HPLC separation. The eluent was transferred to the mass spectrometer and ionized by heated electrospray negative ionization (HESI) and the analyte was quantified using a six-point calibration curve. The validated analytical measurement range (AMR) is 30–1,000 nMol/L. Dilutions of 10 and 200-fold were validated giving a clinical reportable range (CRR) of 30–200,000 nMol/L. The between-day and within-day imprecision values at concentrations spanning the AMR were less than 15%. This method was compared to an established LC-MS/MS method at a CLIA certified national reference laboratory and shows an excellent correlation with our TFLC-MS/MS method. PMID:27833786

  3. Application of chromatography technology in the separation of active components from nature derived drugs.

    PubMed

    Zhao, H-Y; Jiang, J-G

    2010-11-01

    Chromatography technology has been widely applied in various aspects of the pharmacy research on traditional Chinese medicine (TCM). This paper reviews literatures, published in the past decades, on the separation of active component from TCM using chromatography technology. Ultra-performance liquid chromatography (UPLC), high-speed counter-current chromatography (HSCCC), rapid resolution liquid chromatography (RRLC), supercritical fluid chromatography (SFC), affinity chromatography (AC), and bio-chromatography (BC) are introduced in detail. Compared to high performance of high-performance liquid chromatography (HPLC), analysis time and solvent loss are significantly reduced by UPLC with increase in resolution and sensitivity. Some ingredients from nature derived drugs can be separated more completely by HSCCC, which has remarkable characteristics such as low cost, simple operation and no pollution. Trace components from complex systems can be selectively and efficiently separated and purified by AC, This feature makes it effective in isolation and identification of active components of Chinese herbs. Interference of some impurities could be excluded by BC. Active ingredients that are difficult to be separated by normal method can be acquired by SFC. Currently, application of novel chromatography techniques in TCM is still in the exploratory stage and many problems, such as preparation of stationary phase and detection, need to be solved.

  4. Insulin and epidermal growth factor-urogastrone: Affinity crosslinking to specific binding sites in rat liver membranes

    PubMed Central

    Sahyoun, N.; Hock, R. A.; Hollenberg, M. D.

    1978-01-01

    Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with 125I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with 125I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization. PMID:205865

  5. Determination of (R)-timolol in (S)-timolol maleate active pharmaceutical ingredient: validation of a new supercritical fluid chromatography method with an established normal phase liquid chromatography method.

    PubMed

    Marley, Adrian; Connolly, Damian

    2014-01-17

    An enantioselective supercritical fluid chromatography (SFC) method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The developed method is presented as an alternative to the current normal phase high performance liquid chromatography (NP-HPLC) method described in the European Pharmacopoeia (Timolol Maleate Monograph). Using a 4.6mm×250mm Chiralcel OD-H (dp: 5μm) column and a mobile phase of (93:7) CO2/0.1% (v/v) TEA in MeOH delivered at 4.0mLmin(-1) resolution of 2.0 was achieved within 5min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the NP-HPLC method. Method robustness was examined by the variation of flow rate (±0.5mLmin(-1)), column temperature (±5°C) and column back-pressure (±10bar) and resolution was maintained at ≥1.9 in all cases. R-timolol was resolved from all potential impurities and the limit of detection was improved by increasing the sample concentration threefold compared to the NP-HPLC method such that the method could detect the R-timolol enantiomer at 0.5% (w/w) with respect to S-timolol maleate. Additional validation parameters demonstrated that the potential of the method to be used for routine release testing of timolol maleate raw material for drug product manufacturing in which the quantitation of R-timolol impurity in S-timolol maleate drug substance would be a requirement.

  6. Stability-indicating High-performance Liquid Chromatography Method for Simultaneous Determination of Aminophylline and Chlorpheniramine Maleate in Pharmaceutical Formulations

    PubMed Central

    Ali, A.; Ahmed, M.; Mahmud, T.; Qadir, M. A.; Nadeem, K.; Saleem, A.

    2015-01-01

    The present work deals with the development and validation of method for simultaneous determination of antihistaminic drugs in pharmaceutical formulations. A precise, specific and accurate reverse phase-high-performance liquid chromatography method for the simultaneous measurement of aminophylline and chlorpheniramine maleate was developed. The separation of drugs was achieved on C-18 (5 μm, 250×4.6 mm) high-performance liquid chromatography column. The runtime for analysis was 10 min. Mobile phase is mixture containing dilute H2SO4:methanol (60:40% v/v) with flow rate adjusted at 1.5 ml/min. The detection of components was performed at a wavelength of 264 nm. Retention times of aminophylline and chlorphinramine maleate were found to be 2.00 and 3.25 min, respectively. Linearity was found in the range of 16-24 μg/ml for chlorpheniramine maleate and 102.4-153.6 μg/ml for aminophylline with a correlation coefficient of 0.9998 and 0.9996, respectively. High peak purity index of 99.99% indicated the complete separation of analytes in the presence of degradation products is justification of method stability. Linearity, accuracy, specificity, precision and robustness studies were performed for method validation. PMID:26798164

  7. Ion chromatography as highly suitable method for rapid and accurate determination of antibiotic fosfomycin in pharmaceutical wastewater.

    PubMed

    Zeng, Ping; Xie, Xiaolin; Song, Yonghui; Liu, Ruixia; Zhu, Chaowei; Galarneau, Anne; Pic, Jean-Stéphane

    2014-01-01

    A rapid and accurate ion chromatography (IC) method (limit of detection as low as 0.06 mg L(-1)) for fosfomycin concentration determination in pharmaceutical industrial wastewater was developed. This method was compared with the performance of high performance liquid chromatography determination (with a high detection limit of 96.0 mg L(-1)) and ultraviolet spectrometry after reacting with alizarin (difficult to perform in colored solutions). The accuracy of the IC method was established in the linear range of 1.0-15.0 mg L(-1) and a linear correlation was found with a correlation coefficient of 0.9998. The recoveries of fosfomycin from industrial pharmaceutical wastewater at spiking concentrations of 2.0, 5.0 and 8.0 mg L(-1) ranged from 81.91 to 94.74%, with a relative standard deviation (RSD) from 1 to 4%. The recoveries of effluent from a sequencing batch reactor treated fosfomycin with activated sludge at spiking concentrations of 5.0, 8.0, 10.0 mg L(-1) ranging from 98.25 to 99.91%, with a RSD from 1 to 2%. The developed IC procedure provided a rapid, reliable and sensitive method for the determination of fosfomycin concentration in industrial pharmaceutical wastewater and samples containing complex components.

  8. Stability-indicating High-performance Liquid Chromatography Method for Simultaneous Determination of Aminophylline and Chlorpheniramine Maleate in Pharmaceutical Formulations.

    PubMed

    Ali, A; Ahmed, M; Mahmud, T; Qadir, M A; Nadeem, K; Saleem, A

    2015-01-01

    The present work deals with the development and validation of method for simultaneous determination of antihistaminic drugs in pharmaceutical formulations. A precise, specific and accurate reverse phase-high-performance liquid chromatography method for the simultaneous measurement of aminophylline and chlorpheniramine maleate was developed. The separation of drugs was achieved on C-18 (5 μm, 250×4.6 mm) high-performance liquid chromatography column. The runtime for analysis was 10 min. Mobile phase is mixture containing dilute H2SO4:methanol (60:40% v/v) with flow rate adjusted at 1.5 ml/min. The detection of components was performed at a wavelength of 264 nm. Retention times of aminophylline and chlorphinramine maleate were found to be 2.00 and 3.25 min, respectively. Linearity was found in the range of 16-24 μg/ml for chlorpheniramine maleate and 102.4-153.6 μg/ml for aminophylline with a correlation coefficient of 0.9998 and 0.9996, respectively. High peak purity index of 99.99% indicated the complete separation of analytes in the presence of degradation products is justification of method stability. Linearity, accuracy, specificity, precision and robustness studies were performed for method validation.

  9. Chromatographic fingerprint analysis of metabolites in natural and artificial agarwood using gas chromatography-mass spectrometry combined with chemometric methods.

    PubMed

    Gao, Xiaoxia; Xie, Mingrong; Liu, Shaofeng; Guo, Xiaoling; Chen, Xiaoying; Zhong, Zhaojian; Wang, Lei; Zhang, Weimin

    2014-09-15

    Agarwood is a resinous material formed in wounded Aquilaria sinensis in China, which is widely used as an effective traditional Chinese medicine (TCM). This study is aimed to use gas chromatography-mass spectrometry combined with chemometric methods to create reliable criteria for accurate identification of natural agarwood and artificial agarwood, as well as for quality evaluation of artificial agarwood. Natural agarwood and artificial agarwood (stimulated by formic acid or formic acid plus fungal inoculation) were used as standards and controls for the gas chromatography-mass spectrometry (GC-MS) and multivariate analysis. The identification criteria developed were applied to commercial agarwood. A reliable criteria including correlation coefficient of GC-MS fingerprint of natural agarwood and 22 markers of metabolism in natural and artificial agarwood was constructed. Compared with chemically stimulated agarwood (formic acid) and in terms of the 22 markers, artificial agarwood obtained by formic acid stimulation and fungal inoculation were much closer to natural agarwood. The study demonstrates that the chemical components of artificial agarwood obtained by comprehensive stimulated method (formic acid plus fungal inoculation) are much closer to the natural agarwood than those obtained by chemically stimulated method (formic acid), as times goes by. A reliable criteria containing correlation coefficient of GC-MS fingerprint of natural agarwood and 22 metabolism markers can be used to evaluate the quality of the agarwood. As an application case, three samples were identified as natural agarwood from the 25 commercial agarwood by using the evaluation method.

  10. A novel derivatization-free method of formaldehyde and propylene glycol determination in hydrogels by liquid chromatography with refractometric detection.

    PubMed

    Isakau, Henadz; Robert, Marielle; Shingel, Kirill I

    2009-04-05

    The paper describes the development and validation of a new derivatization-free liquid chromatography method for simultaneous determination of propylene glycol and formaldehyde in the formulations containing formaldehyde-releasing preservative. Highly swollen hydrogel made of poly(ethylene glycol)-protein conjugates was taken as a model formulation for integration of the propylene glycol and the diazolydinyl urea as formaldehyde releaser. The method is shown to be simple and selective and, more importantly, allows determining an existing level of formaldehyde at the moment of analysis instead of all available formaldehyde that might be released during chemical derivatization. After liquid extraction the propylene glycol (PG) and formaldehyde (FA) amounts are determined chromatographically on a Shodex SH 1011 ligand-exchange column using 0.01 M sulfuric acid mobile phase, a flow rate of 1.0 ml/min and RI detection. The assay is validated showing good linearity, precision, and accuracy. The limits of detection of formaldehyde and propylene glycol in the analyzed solutions were estimated to be 25 ng and 87 ng, respectively. This analytical assay is considered useful for product stability studies and in developing new formaldehyde releaser-containing formulations where the concentration of formaldehyde is a presumable subject of labeling requirements. This method can also provide a rapid and convenient alternative to gas chromatography method of propylene glycol quantification.

  11. A quantitative method to identify microRNAs targeting a messenger RNA using a 3′UTR RNA affinity technique

    PubMed Central

    Shi, Miao; Han, Weiguo; Spivack, Simon D.

    2014-01-01

    The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3′UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitatvie polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA. PMID:23938772

  12. [Isolation and purification of enhanced green fluorescent protein using chromatography].

    PubMed

    Hou, Qinghua; Song, Shuliang; Liang, Hao; Wang, Weili; Ji, Aiguo

    2013-02-01

    Enhanced green fluorescent protein (EGFP) is a common biological marker. In this research, on the foundation of successful clone and expression of EGFP, a two-step chromatographic method was established to separate and purify EGFP, which includes the use of HisTrap HP immobilized metal affinity chromatography (IMAC) and Sephadex G-10 HR size exclusion chromatography in sequence. Sephacryl S-300 HR size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to check out the purity of EGFP. At last, it was found that EGFP still had fluorescent activity using fluorescence spectrophotometric detection and Native-PAGE detection. This method can effectively separate the active EGFP. The purity of the obtained EGFP was more than 98%.

  13. Methods of Analysis - Determination of Pyrethroid Insecticides in Water and Sediment Using Gas Chromatography/Mass Spectrometry

    USGS Publications Warehouse

    Hladik, Michelle; Smalling, Kelly L.; Kuivila, Kathryn

    2009-01-01

    A method for the determination of 14 pyrethroid insecticides in environmental water and sediment samples is described. The method was developed by the U.S. Geological Survey in response to increasing concern over the effects of pyrethroids on aquatic organisms. The pyrethroids included in this method are ones that are applied to many agricultural and urban areas. Filtered water samples are extracted for pyrethroids using solid-phase extraction (SPE) with no additional cleanup steps. Sediment and soil samples are extracted using a microwave-assisted extraction system, and the pyrethroids of interest are separated from co-extracted matrix interferences by passing the extracts through stacked graphitized carbon and alumina SPE cartridges, along with the use of high-performance liquid chromatography and gel-permeation chromatography (HPLC/GPC). Quantification of the pyrethroids from the extracted water and sediment samples is done using gas chromatography with mass spectrometry (GC/MS) or gas chromatography with tandem mass spectrometry (GC/MS/MS). Recoveries in test water samples fortified at 10 ng/L ranged from 83 to 107 percent, and recoveries in test sediment samples fortified at 10 ug/kg ranged from 82 to 101 percent; relative standard deviations ranged from 5 to 9 percent in the water samples and 3 to 9 percent in the sediment samples. Method detection limits (MDLs), calculated using U.S. Environmental Protection Agency procedures (40 CFR 136, Appendix B), in water ranged from 2.0 to 6.0 ng/L using GC/MS and 0.5 to 1.0 ng/L using GC/MS/MS. For sediment, the MDLs ranged from 1.0 to 2.6 ug/kg dry weight using GC/MS and 0.2 to 0.5 ug/kg dry weight using GC/MS/MS. The matrix-spike recoveries for each compound, when averaged for 12 environmental water samples, ranged from 84 to 96 percent, and when averaged for 27 environmental sediment samples, ranged from 88 to 100 percent.

  14. Centrifugal precipitation chromatography.

    PubMed

    Ito, Yoichiro; Qi, Lin

    2010-01-15

    Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.

  15. Portable system and method combining chromatography and array of electrochemical sensors

    DOEpatents

    Zaromb, Solomon; Stetter, Joseph R.

    1989-01-01

    A portable system for analyzing a fluid sample includes a small, portable, low-pressure and low-power chromatographic analyzer and a chemical parameter spectrometry monitor including an array of sensors for detecting, identifying and measuring the concentrations of a variety of components in the eluent from the chromatographic analyzer. The monitor includes one or more operating condition controllers which may be used to change one or more of the operating conditions during exposure of the sensors to the eluent from the chromatography analyzer to form a response pattern which is then compared with a library of previously established patterns. Gas and liquid chromatographic embodiments are disclosed. In the gas embodiment, the operating condition controllers include heated filaments which may convert electrochemically inactive components to electrochemically active products. In the liquid chromatography embodiment, low-power, liquid-phase equivalents of heated filaments are used with appropriate sensors. The library response patterns may be divided into subsets and the formed pattern may be assigned for comparison only with the patterns of a particular subset.

  16. Mathematical method for the prediction of retention times of fatty acid methyl esters in temperature-programmed capillary gas chromatography.

    PubMed

    Torres, Alexandre G; Trugo, Nádia M F; Trugo, Luiz C

    2002-07-17

    An accurate method for identification of fatty acids in complex mixtures analyzed by temperature-programmed capillary gas chromatography is described. The method is based on a mathematical approach using regression curves obtained by plotting the relative retention times of fatty acid methyl esters (FAMEs) analyzed in isothermal and gradient temperature conditions. The method was applied to a complex biological sample (human milk), and it was possible to identify 64 fatty acids, including branched-chain and other fatty acids for which reference standards were not readily available. The identities of the majority of the peaks were confirmed by mass spectrometry. The relative residuals and the relative differences between estimated and measured relative retention times of individual FAMEs varied from 0.03 to 3.15% and from 0.0 to 2.9%, respectively. The method is useful for identification of fatty acids in routine analysis.

  17. /Chromatography+RECOVERY=superresolution chromatography

    NASA Astrophysics Data System (ADS)

    Kosarev, E. L.; Muranov, K. O.

    2003-04-01

    A method for improving the resolution of the chromatographic analysis based on deriving the point-spread function of a chromatographic column, i.e., a chromatogram of an individual compound, is described. The system of two data sets, namely, a chromatogram of a substance analyzed and a point-spread function of a chromatographic column in combination with the noise statistics, makes it possible to use the RECOVERY signal-reconstruction software package described in paper by Gelfgat et al. (Comp. Phys. Commun. 74 (1993) 335). The proposed method has been tested by chromatography of bovine serum albumin using gel filtration. The resultant resolution exceeds that reached using high-performance liquid chromatography (with the cost of the instruments being lower by a factor of 15-20).

  18. Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma, including metabolite concentrations at steady state.

    PubMed

    Vikingsson, Svante; Strömqvist, Malin; Svedberg, Anna; Hansson, Johan; Höiom, Veronica; Gréen, Henrik

    2016-08-01

    A novel, rapid and sensitive liquid chromatography tandem-mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi-quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C18 column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi-quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5-100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Fast, simple, and sensitive high-performance liquid chromatography method for measuring vitamins A and E in human blood plasma.

    PubMed

    Yuan, Chao; Burgyan, Maria; Bunch, Dustin R; Reineks, Edmunds; Jackson, Raymond; Steinle, Roxanne; Wang, Sihe

    2014-09-01

    Vitamins A and E are fat-soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high-performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α-tocopherol and γ-tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed-phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03-5.14, 0.32-36.02, and 0.10-9.99 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. Intr-aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Technology. Reference intervals were established using plasma samples collected from 51 healthy adult donors and were found to be 0.30-1.20, 6.0-23.0, and 0.3-3.2 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. In conclusion, we developed and validated a fast, simple, and sensitive high-performance liquid chromatography method for measuring the major forms of vitamins A and E in human plasma.

  20. A fast liquid chromatography-mass spectrometry (LC-MS) method for quantification of major polar metabolites in plants.

    PubMed

    Liu, Zhiqian; Rochfort, Simone

    2013-01-01

    Current liquid chromatography (LC) based methods for the analysis of polar plant metabolites require multiple runs using complex mobile phases and a combination of different columns. Here we describe a fast liquid chromatography-mass spectrometry (LC-MS) method for the determination of major polar metabolites in plants that requires only a single run using a single column. The method takes advantage of the ability to acquire both positive and negative data in an ion trap mass spectrometer (MS) and also the accurate mass capability of the orbitrap MS. The separation of polar compounds is achieved with a polar, reversed-phase column (Synergi Hydro-RP). A single analysis with a 25min runtime is able to reliably determine the level of nearly all essential amino acids, several major organic acids and several major sugars in plant materials, as exemplified by analysis of a perennial ryegrass extract. The level of detection on column was below 0.1ng (average 0.03ng) for most amino acids, below 5ng (average 2.3ng) for organics acids and below 1ng (average 0.64ng) for sugars. The levels of quantified metabolites in ryegrass varied from 22μg/g dry weight for histidine to 41mg/g dry weight for sucrose.

  1. A dielectric affinity microbiosensor

    NASA Astrophysics Data System (ADS)

    Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

    2010-01-01

    We present an affinity biosensing approach that exploits changes in dielectric properties of a polymer due to its specific, reversible binding with an analyte. The approach is demonstrated using a microsensor comprising a pair of thin-film capacitive electrodes sandwiching a solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid), a synthetic polymer with specific affinity to glucose. Binding with glucose induces changes in the permittivity of the polymer, which can be measured capacitively for specific glucose detection, as confirmed by experimental results at physiologically relevant concentrations. The dielectric affinity biosensing approach holds the potential for practical applications such as long-term continuous glucose monitoring.

  2. Dating of seals produced with stamp-pad ink using gas chromatography method.

    PubMed

    Li, Biao

    2014-09-01

    Seals are often placed on important documents, and determining the date when a seal was affixed can be important to assess the authenticity of a document. In this study, thirty-seven different brands of stamp-pad inks and three different types of paper were used to make diachronic samples of the seals. The volatile components of stamp-pad ink were determined, and the stamp-pad ink was classified by gas chromatography with a flame ionization detector. Calibration curves were created to show the relationship between the content of volatile stamp-pad ink components and the age of stamp-pad ink entries stored under natural aging, UV-induced aging, and heating aging conditions. The experimental results showed that GC was useful in the analysis of stamp-pad ink and applicable for determining the relative age of stamp-pad ink entries in some cases where the seal was placed on the document within 3 months.

  3. Novel materials and methods for solid-phase extraction and liquid chromatography

    SciTech Connect

    Ambrose, Diana

    1997-06-24

    This report contains a general introduction which discusses solid-phase extraction and solid-phase micro-extraction as sample preparation techniques for high-performance liquid chromatography, which is also evaluated in the study. This report also contains the Conclusions section. Four sections have been removed and processed separately: silicalite as a sorbent for solid-phase extraction; a new, high-capacity carboxylic acid functionalized resin for solid-phase extraction; semi-micro solid-phase extraction of organic compounds from aqueous and biological samples; and the high-performance liquid chromatographic determination of drugs and metabolites in human serum and urine using direct injection and a unique molecular sieve.

  4. Affine dynamics with torsion

    NASA Astrophysics Data System (ADS)

    Gültekin, Kemal

    2016-03-01

    In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schrödinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor.

  5. Liquid chromatography-tandem mass spectrometry screening method for the simultaneous detection of stimulants and diuretics in urine.

    PubMed

    Hsu, Ku Fu; Chien, Kuei-Yu; Chang-Chien, Guo Ping; Lin, Su Fan; Hsu, Pei Hsuan; Hsu, Mei-Chich

    2011-11-01

    This study established a simultaneous screening method based on solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the detection of 23 stimulants and 23 diuretics in human urine. An electrospray ionization source and multiple reaction monitoring were used for data acquisition. All stimulants and diuretics were separated in less than 12.52 min. The limits of detection were in the range of 25-500 ng/mL for stimulants and 25-125 ng/mL for diuretics. To evaluate the performance of this method, urine samples were collected from 1627 athletes in Taiwan, and 7 positive samples were found. This LC-MS-MS method not only meets the minimum required performance limits set by the World Anti-Doping Agency but also provides a fast way to analyze the authentic urine samples in doping control laboratories.

  6. Rapid and sensitive method for analysis of nitrate in meat samples using ultra performance liquid chromatography-mass spectrometry.

    PubMed

    Siddiqui, Masoom Raza; Wabaidur, Saikh Mohammad; ALOthman, Zeid A; Rafiquee, M Z A

    2015-12-05

    A sensitive and selective ultra performance liquid chromatography-mass spectrometric method has been developed for the quantitative analysis of nitrate in meat samples. Selected ion reaction (SIR) mode was adopted to identify and quantify the nitrate. Chromatographic analyses were performed on a BEH C-18 column with a mobile phase consisting of a surfactant (Cetylpyridinium chloride) and acetonitrile in equal ratio (50/50, v/v) at a flow rate of 0.4 mL min(-1). The limit of detection and limit of quantitation of the developed method was found to be 0.0599 and 0.1817 mg kg(-1), respectively. The linearity of the proposed method was checked in the concentration range of 0.5-10 mg kg(-1) with an excellent correlation coefficient (r) of 0.997. The recovery of the nitrate in the meat samples were in the range of 98.02-98.99%.

  7. A Supercritical Fluid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Quantification of Metformin and Gliclazide in Human Plasma

    PubMed Central

    Agrawal, Y. K.; Gogoi, P. J.; Manna, K.; Bhatt, H. G.; Jain, V. K.

    2010-01-01

    Present study reports the development and validation of a simultaneous estimation of metformin and gliclazide in human plasma using supercritical fluid chromatography followed by tandem mass spectrometry. Acetonitrile:water (80:20) mixture was used as a mobile phase along with liquid CO2 in supercritical fluid chromatography and phenformin as an internal standard. The modified plasma samples were analyzed by electro-spray ionization method in selective reaction monitoring mode in tandem mass spectrometry. Supercritical fluid chromatographic separation was performed using nucleosil C18 containing column as a stationary phase. The separated products were identified by characteristic peaks and specific fragments peaks in tandem mass spectrometry as m/z 130 to 86 for metformin, m/z 324 to 110 for gliclazide and m/z 206 to 105 for phenformin. The present method was found linear in the concentration ranges of 6.0-3550 ng/ml and 7.5-7500 ng/ml for metformin and gliclazide, respectively. Pharmacokinetic study was performed after an oral administration of dispersible tablets containing 500 mg of metformin and 80 mg of gliclazide using same techniques. PMID:20582190

  8. A convenient method for epichlorohydrin determination in water using headspace-solid-phase microextraction and gas chromatography.

    PubMed

    Lasa, M; Garcia, R; Millán, E

    2006-08-01

    A simple procedure for epychlorohydrin determination in water is presented. In order to optimize the epichlorohydrin extraction conditions in water using headspace (HS)-solid-phase microextraction (SPME), followed by gas chromatography, an experimental design in two steps is performed. Firstly, a 2(5-2) fractional factorial design for screening the significant variables is used. Secondly, a central composite design for optimizing them is carried out. The best experimental conditions are the followings: poly(dimethysiloxane)-divinylbenzene coating fiber; 20 min extraction time; 5 degrees C extraction temperature; 300 g/L sodium chloride; and 20 mL HS volume in a 40-mL vial. Using the previous extraction conditions with gas chromatography (GC)-flame ionization detection equipment, a limit of detection (LOD) of 1.8 microg/L and a relative standard deviation (RSD) of 3.8% (for 25 microg/L) are obtained. With a GC electron capture detection equipment the RSD is 6.6% (for 5 microg/L), and the LOD found is lower (0.08 microg/L). The method is applied to the analysis of water from four treatment plants at the entrance and effluent stream. The standard addition method is used to quantitate the epichlorohydrin that is found in the raw water of the three wastewater treatment plants.

  9. Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis.

    PubMed

    Amelina, Hanna; Apraiz, Itxaso; Sun, Wei; Cristobal, Susana

    2007-06-01

    Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS), and they are involved in beta-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.

  10. Retention mechanism assessment and method development for the analysis of iohexol and its related compounds in hydrophilic interaction liquid chromatography.

    PubMed

    Jovanović, Marko; Rakić, Tijana; Jančić-Stojanović, Biljana; Ivanović, Darko; Medenica, Mirjana

    2014-07-01

    Hydrophilic interaction liquid chromatography (HILIC) has emerged in recent years as a valuable alternative to reversed-phase liquid chromatography in the analysis of polar compounds. Research in HILIC is divided into two directions: the assessment of the retention mechanism and retention behavior, and the development of HILIC methods. In this work, four polar neutral analytes (iohexol and its related compounds A, B, and C) were analyzed on two silica and two diol columns in HILIC mode with the aim to investigate thoroughly the retention mechanisms and retention behavior of polar neutral compounds on these four columns. The adsorption and partition contribution to the overall HILIC retention mechanism was investigated by fitting the retention data to linear (adsorption and partition) and nonlinear (mixed-retention and quadratic) theoretical models. On the other hand, the establishment of empirical second-order polynomial retention models on the basis of D-optimal design made possible the estimation of the simultaneous influence of several mobile-phase-related factors. Furthermore, these models were also used as the basis for the application of indirect modeling of the selectivity factor and a grid point search approach in order to achieve the optimal separation of analytes. After the optimization goals had been set, the grids were searched and the optimal conditions were identified. Finally, the optimized method was subjected to validation.

  11. DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin.

    PubMed

    Werner, P A; Galbraith, R M; Arnaud, P

    1983-10-01

    Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

  12. A Method for the Quantitation of Trace Levels of Dimethyl Sulfoxide in Urine by High Performance Liquid Chromatography

    DTIC Science & Technology

    1989-05-01

    HIGH PERFORMANCE LIQUID CHROMATOGRAPHY by...for the sample cleanup and concentration, followed by separation by reversed phase high performance liquid chromatography . EXPERIMENTAL Materials...DIMETHYL SULFOXIDE IN URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 4. AUTHORS (Last name, first name, middle initial. If military, show rank, e.g.

  13. Biomonitoring method for bisphenol A in human urine by ultra-high-performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Anderson, David J; Brozek, Eric M; Cox, Kyley J; Porucznik, Christina A; Wilkins, Diana G

    2014-03-15

    An ultra-high-performance liquid chromatography-tandem mass spectrometry method for the measurement of total bisphenol A in human urine was developed and validated. The method utilized liquid/liquid extraction with 1-chlorobutane and a human urine aliquot size of 800μL. Chromatography was performed on an Acquity UPLC(®) system with a Kinetex(®) Phenyl-Hexyl column. Mass spectrometric analysis was with negative electrospray ionization on a Quattro Premier XE™. The surrogate matrix method was used for the preparation of calibration standards in synthetic urine due to the presence of BPA in control human urine. The validated calibration range was 0.75-20ng/mL with a limit of detection of 0.1ng/mL. The internal standard was d16-bisphenol A. Method validation utilized quality control samples at three concentrations in both synthetic urine and human urine. Bisphenol A mono-glucuronide was fortified in synthetic urine in each analytical run to monitor the enzymatic conversion of the glucuronide conjugate to BPA by β-glucuronidase. Validated method parameters included linearity, accuracy, precision, integrity of dilution, selectivity, re-injection reproducibility, recovery/matrix effect, solution stability, and matrix stability in human urine. Acceptance criteria for analytical standards and QCs were ±20% of nominal concentration. Matrix stability in human urine was validated after 24h at ambient temperature, after three freeze/thaw cycles, and after frozen storage at -20°C and -80°C for up to 218 days. The method has been applied to the analysis of over 1750 human urine samples from a biomonitoring study. The median and mean urine BPA concentrations were 2.71ng/mL and 4.75ng/mL, respectively.

  14. A fast, simple, and reliable hydrophilic interaction liquid chromatography method for the determination of ascorbic and isoascorbic acids.

    PubMed

    Barros, Ana I R N A; Silva, Ana P; Gonçalves, Berta; Nunes, Fernando M

    2010-03-01

    A reliable method for the determination of total vitamin C must be able to resolve ascorbic acid (AA) and the epimeric isoascorbic acid (IAA) and determine the sum of AA and its oxidized form dehydroascorbic acid. AA and IAA are polar molecules with a low retention time in conventional reversed phase systems, and hence of difficult resolution. Hydrophilic interaction chromatography using a TSKgel Amide-80 stationary phase with isocratic elution was successful in resolving the two epimers. The column was compatible with injections of high concentrations of metaphosphoric acid, tris(2-carboxyethyl)-phosphine, and EDTA without drift of baseline and retention time. Total AA and IAA were extracted, stabilized, and reduced in one step at 40 °C, using 5% m-phosphoric acid, 2 mM of EDTA, and 2 mM of tris(2-carboxyethyl)-phosphine as reducing agent. This simple, fast, and robust hydrophilic interaction chromatography-DAD method was applied for the analysis of food products namely fruit juices, chestnut, and ham and also in pharmaceutical and multivitamin tablets. Method validation was performed on the food products, including parameters of precision, accuracy, linearity, limit of detection, and quantification (LOQ). The absence of matrix interferences was assessed by the standard addition method and Youden calibration. The method was fast, accurate, and precise with a LOQ(AA) of 1.5 mg/L and LOQ(IAA) of 3.7 mg/L. The simple experimental procedure, completed in 1 h, the possibility of using IAA as an internal standard, and low probability of artifacts are the major advantages of the proposed method for the routine determination of these compounds in a large number of samples.

  15. Applying Chromatography.

    ERIC Educational Resources Information Center

    Klein, Jessie W.; Patev, Paul

    1998-01-01

    Presents three experiments to introduce students to different kinds of chromatography: (1) paper chromatography; (2) gel filtration chromatography; and (3) reverse-phase liquid chromatography. Written in the form of a laboratory manual, explanations of each of the techniques, materials needed, procedures, and a glossary are included. (PVD)

  16. A method to assess genomic DNA methylation using high-performance liquid chromatography/electrospray ionization mass spectrometry.

    PubMed

    Friso, Simonetta; Choi, Sang-Woon; Dolnikowski, Gregory G; Selhub, Jacob

    2002-09-01

    Eukaryotic DNA is methylated at some cytosine residues, and this epigenetic feature performs critical functions. We developed a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The DNA was enzymatically hydrolyzed by sequential digestion with three enzymes. DNA hydrolyzates were subsequently separated by reversed-phase high-perfor