Enrichment and Analysis of Non-enzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron Transfer Dissociation Mass Spectrometry

PubMed Central

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

2008-01-01

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. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus. PMID:17488106

Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

PubMed

Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Svensson, Susanne; Ohlson, Sten; Isaksson, Roland

2013-07-01

In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have a major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Purification of swine haptoglobin by affinity chromatography.

PubMed Central

Eurell, T E; Hall, W F; Bane, D P

1990-01-01

A globin-agarose affinity chromatography technique was used to purify swine haptoglobin. This technique provides a highly specific, single-step purification method without the contamination of extraneous serum proteins reported by previous studies. Complex formation between the haptoglobin isolate and swine hemoglobin confirmed that biological activity was maintained during the purification process. Immunoelectrophoretic and Ouchterlony immunodiffusion methods revealed that the swine haptoglobin isolate cross-reacted with polyvalent antisera against human haptoglobin. Images Fig. 2. Fig. 3. PMID:2123414

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

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

An affinity chromatography-gel filtration device for preparing thyroid microsomal antigen.

PubMed

Wang, L; Zheng, W F

1987-09-24

On the basis of conventional differential centrifugation for preparing crude thyroid microsomal antigen (TMAg), we have employed Sepharose 4B gel filtration and affinity chromatography separately to study the elution pattern in terms of absorbance and antigenic activity. The result indicates that thyroglobulin (TG) exists in two forms in crude TMAg, i.e., 'free TG' and 'membrane-bound TG'. TMAg is present in two forms in the eluate: (1) the TM fragment or TMAg polymer, which is produced at a higher rate and has greater antigenic activity, but which is less pure; (2) soluble TMAg, which is produced at a lower rate and has less antigenic activity, but which is more pure. We have developed an affinity chromatography-gel filtration (AC-GF) device which is a combination of affinity chromatography and a Sepharose 4B column. Sephadex G-50 is placed between the rubber stopper and Sepharose 4B in the GF column to ensure intactness of the entire system. With such a device, the AC removes the contaminated TG from TM homogenate, and allows the latter to pass directly from AC to GF for rechromatography. This device extracts the full advantages of both methods and each compensates for any deficiency of the other. Using this one-step procedure, one has the greatest chance of removing TG and obtaining TM fragments of TMAg polymers of higher antigenic activity, as well as separating small amounts of more purified soluble TMAg. Thus, the newly developed method meets the need of large quantities of TMAg for practical application, and at the same time the more purified preparations can be used for analytical purposes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.

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-cmore » 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.« less

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)

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

PubMed

Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

2017-06-23

The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

PubMed

Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

2012-11-01

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Fragment screening for drug leads by weak affinity chromatography (WAC-MS).

PubMed

Ohlson, Sten; Duong-Thi, Minh-Dao

2018-02-23

Fragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of K d and irrelevant non-specific interactions are abundant in this area of transient interactions. Fortunately, there are methods that can study weak interactions quite efficiently of which NMR, surface plasmon resonance (SPR) and X-ray crystallography are the most prominent. Now, a new technology based on zonal affinity chromatography, weak affinity chromatography (WAC), has been introduced which has remedied many of the problems with other technologies. By combining WAC with mass spectrometry (WAC-MS), it is a powerful tool to identify binders quantitatively in terms of affinity and kinetics either from fragment libraries or from complex mixtures of biological extracts. As WAC-MS can be multiplexed by analysing mixtures of fragments (20-100 fragments) in one sample, this approach yields high throughput, where a whole library of e.g. >2000 fragments can be analysed quantitatively within a day. WAC-MS is easy to perform, where the robustness and quality of HPLC is fully utilized. This review will highlight the rationale behind the application of WAC-MS for fragment screening in drug discovery. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

PubMed

Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

2016-04-05

Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

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.

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

PubMed Central

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

2013-01-01

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. PMID:24225840

Phospholipid bilayer affinities and solvation characteristics by electrokinetic chromatography with a nanodisc pseudostationary phase.

PubMed

Penny, William M; Steele, Harmen B; Ross, J B Alexander; Palmer, Christopher P

2017-03-01

Phospholipid bilayer nanodiscs composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and synthetic maleic acid-styrene copolymer belts have been introduced as a pseudostationary phase (PSP) in electrokinetic chromatography and demonstrated good performance. The nanodiscs provide a suitable migration range and high theoretical plate counts. Using this nanodisc pseudostationary phase, the affinity of the bilayer structure for probe solutes was determined and characterized. Good correlation is observed between retention factors and octanol water partition coefficients for particular categories of solutes, but the general correlation is weak primarily because the nanodiscs show stronger affinity than octanol for hydrogen bond donors. This suggests that a more appropriate application of this technology is to measure and characterize interactions between solutes and lipid bilayers directly. Linear solvation energy relationship analysis of the nanodisc-solute interactions in this study demonstrates that the nanodiscs provide a solvation environment with low cohesivity and weak hydrogen bond donating ability, and provide relatively strong hydrogen bond acceptor strength. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

PubMed Central

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

2011-01-01

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. PMID:21936497

Multiparameter cell affinity chromatography: separation and analysis in a single microfluidic channel.

PubMed

Li, Peng; Gao, Yan; Pappas, Dimitri

2012-10-02

The ability to sort and capture more than one cell type from a complex sample will enable a wide variety of studies of cell proliferation and death and the analysis of disease states. In this work, we integrated a pneumatic actuated control layer to an affinity separation layer to create different antibody-coating regions on the same fluidic channel. The comparison of different antibody capture capabilities to the same cell line was demonstrated by flowing Ramos cells through anti-CD19- and anti-CD71-coated regions in the same channel. It was determined that the cell capture density on the anti-CD19 region was 2.44 ± 0.13 times higher than that on the anti-CD71-coated region. This approach can be used to test different affinity molecules for selectivity and capture efficiency using a single cell line in one separation. Selective capture of Ramos and HuT 78 cells from a mixture was also demonstrated using two antibody regions in the same channel. Greater than 90% purity was obtained on both capture areas in both continuous flow and stop flow separation modes. A four-region antibody-coated device was then fabricated to study the simultaneous, serial capture of three different cell lines. In this case the device showed effective capture of cells in a single separation channel, opening up the possibility of multiple cell sorting. Multiparameter sequential blood sample analysis was also demonstrated with high capture specificity (>97% for both CD19+ and CD4+ leukocytes). The chip can also be used to selectively treat cells after affinity separation.

Adsorption of endotoxins on Ca2+ -iminodiacetic acid by metal ion affinity chromatography.

PubMed

Lopes, André Moreni; Romeu, Jorge Sánchez; Meireles, Rolando Páez; Perera, Gabriel Marquez; Morales, Rolando Perdomo; Pessoa, Adalberto; Cárdenas, Lourdes Zumalacárregui

2012-11-01

Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol/L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU/mL and 100 000 EU/mL. This new protocol represents a substantial advantage in time, effort, and production costs.

[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.

Chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozyme.

PubMed

Yamada, H; Fukumura, T; Ito, Y; Imoto, T

1985-04-01

Preparation of chitin-coated Celite as an affinity adsorbent for high-performance liquid chromatography of lysozymes and its application to separation of N-bromosuccinimide-oxidized lysozymes are described. By pH gradient elution, two diastereomers of oxindolealanine-62-lysozyme, delta 1-acetoxytryptophan-62-lysozyme (intermediate product in the reaction in acetate buffer), and native lysozyme were all separated within 40 min.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.

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 appliedmore » 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.« less

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.

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

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. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

PubMed

Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

2001-08-24

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

PubMed

de Aquino, Luciana Cristina Lins; de Sousa, Heloisa Ribeiro Tunes; Miranda, Everson Alves; Vilela, Luciano; Bueno, Sônia Maria Alves

2006-04-13

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

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.

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

PubMed

Serpa, Gisele; Augusto, Elisabeth Fátima Pires; Tamashiro, Wirla Maria Silva Cunha; Ribeiro, Mariana Borçoe; Miranda, Everson Alves; Bueno, Sônia Maria Alves

2005-02-25

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

PubMed

Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

2016-09-01

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

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

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

PubMed

Martinović, Tamara; Andjelković, Uroš; Klobučar, Marko; Černigoj, Urh; Vidič, Jana; Lučić, Marina; Pavelić, Krešimir; Josić, Djuro

2017-11-01

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selective affinity chromatography of DNA polymerases with associated 3' to 5' exonuclease activities.

PubMed

Lee, M Y; Whyte, W A

1984-05-01

The use of 5'-AMP as a ligand for the affinity chromatography of DNA polymerases with intrinsic 3' to 5' exonuclease activities was investigated. The basis for this is that 5'-AMP would be expected to act as a ligand for the associated 3' to 5' exonuclease. The requirements for binding of Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus DNA polymerase delta, all of which have associated 3' to 5' exonuclease activities, to several commercially available 5'-AMP supports with different linkages of 5'-AMP to either agarose or cellulose were examined. The DNA polymerases which possessed 3' to 5' exonuclease activities were bound to agarose types in which the 5'-phosphoryl group and the 3'-hydroxyl group of the AMP were unsubstituted. Bound enzyme could be eluted by either an increase in ionic strength or competitive binding of nucleoside 5'-monophosphates. Magnesium was found to reinforce the binding of the enzyme to these affinity supports. DNA polymerase alpha, which does not have an associated 3' to 5' exonuclease activity, did not bind to any of these columns. These differences can be used to advantage for the purification of DNA polymerases that have associated 3' to 5' exonuclease activities, as well as a means for establishing the association of 3' to 5' exonuclease activities with DNA polymerases.

ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

2010-01-01

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

PubMed Central

DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.

2016-01-01

A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

Method development of enantiomer separations by affinity capillary electrophoresis, cyclodextrin electrokinetic chromatography and capillary electrophoresis-mass spectrometry.

PubMed

Tanaka, Yoshihide

2002-07-01

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 mumol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

Temperature effect on affinity chromatography of two lectins from the seeds of Ricinus communis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, H.W.; Davis, D.S.; Wei, C.H.

1976-06-01

Specific adsorption capacity of Sepharose 4B in affinity chromatography for two purified galactose-binding lectins, designated as III/sub L/ and III/sub H/, from the seed of Ricinus communis (castor bean) was measured from 7 to 24/sup 0/C. The adsorption coefficients for these two protein fractions as a function of temperature were also obtained. It was found that there is a characteristic transition of adsorption coefficient at 18/sup 0/C for both lectins. Adsorption coefficients between Sepharose 4B and these two lectins were also expressed in terms of ..delta..G, ..delta..H, and ..delta..S. It is suggested that the difference in the temperature dependence ofmore » the binding energy of these two lectins may be used for their separation at selected temperature.« less

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.

PubMed

Zhou, Jian-Liang; An, Jing-Jing; Li, Ping; Li, Hui-Jun; Jiang, Yan; Cheng, Jie-Fei

2009-03-20

We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

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.

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

PubMed Central

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. PMID:26641240

A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments.

PubMed

Moulder, Robert; Filén, Jan-Jonas; Salmi, Jussi; Katajamaa, Mikko; Nevalainen, Olli S; Oresic, Matej; Aittokallio, Tero; Lahesmaa, Riitta; Nyman, Tuula A

2005-07-01

The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

PubMed

Salehi, Nasrin; Peng, Ching-An

2016-07-08

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

PubMed

Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

1998-07-03

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

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.

Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

PubMed Central

de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra

2016-01-01

Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

2D Affine and Projective Shape Analysis.

PubMed

Bryner, Darshan; Klassen, Eric; Huiling Le; Srivastava, Anuj

2014-05-01

Current techniques for shape analysis tend to seek invariance to similarity transformations (rotation, translation, and scale), but certain imaging situations require invariance to larger groups, such as affine or projective groups. Here we present a general Riemannian framework for shape analysis of planar objects where metrics and related quantities are invariant to affine and projective groups. Highlighting two possibilities for representing object boundaries-ordered points (or landmarks) and parameterized curves-we study different combinations of these representations (points and curves) and transformations (affine and projective). Specifically, we provide solutions to three out of four situations and develop algorithms for computing geodesics and intrinsic sample statistics, leading up to Gaussian-type statistical models, and classifying test shapes using such models learned from training data. In the case of parameterized curves, we also achieve the desired goal of invariance to re-parameterizations. The geodesics are constructed by particularizing the path-straightening algorithm to geometries of current manifolds and are used, in turn, to compute shape statistics and Gaussian-type shape models. We demonstrate these ideas using a number of examples from shape and activity recognition.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of Quantitative Performance of Sequential Immobilized Metal Affinity Chromatographic Enrichment for Phosphopeptides

PubMed Central

Sun, Zeyu; Hamilton, Karyn L.; Reardon, Kenneth F.

2014-01-01

We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium-dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. While our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semi-quantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. PMID:24096195

Monocarboxylate and alpha-ketoglutarate carriers from bovine heart mitochondria. Purification by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate.

PubMed

Bolli, R; Nałecz, K A; Azzi, A

1989-10-25

2-Cyano-4-hydroxycinnamate was covalently linked, through a diazo bond, to Sepharose 4B, which had been elongated with a hydrophobic spacer. A Triton X-100 extract from bovine heart mitochondria was pre-purified by hydroxylapatite chromatography and passed through the 2-cyano-4-hydroxycinnamate affinity resin in the presence of 0.7% deoxycholate. At pH 6 and in the presence of 0.2 M sodium chloride, a single polypeptide with an Mr of 34,000 was eluted. Subsequently, at pH 8 and in the presence of 2-cyano-4-hydroxycinnamate, another single protein with an Mr of 31,500 was released. Both proteins were reconstituted into phospholipid vesicles and their transport activities were measured. High, delta pH-dependent, 2-cyanocinnamate-sensitive pyruvate uptake was measured in vesicles containing only the 34-kDa protein. alpha-Ketobutyrate and other alpha-ketomonocarboxylic acids were competitive inhibitors of the pyruvate uptake, whereas di- and tricarboxylates had only small effects. alpha-Ketoglutarate-alpha-ketoglutarate exchange could only be measured in vesicles containing the 31.5-kDa protein. The molecular weight of this protein and its functional properties were similar to those of the alpha-ketoglutarate carrier isolated by a different method (Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369). 2-Cyano-4-hydroxycinnamate inhibited the alpha-ketoglutarate exchange in a noncompetitive manner with an apparent Ki of 0.7 mM. It is concluded that by the described affinity chromatography procedure, two mitochondrial carriers transporting alpha-ketoacids, i.e. the monocarboxylate and the alpha-ketoglutarate carrier, could be purified in a functionally active state.

Affinity approaches in RNAi-based therapeutics purification.

PubMed

Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

2016-05-15

The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

PubMed

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

2016-02-05

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 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL 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. Copyright © 2015 Elsevier B.V. All rights reserved.

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model.

PubMed

Boto, R E F; Anyanwu, U; Sousa, F; Almeida, P; Queiroz, J A

2009-09-01

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright (c) 2009 John Wiley & Sons, Ltd.

Purification of native M. vogae and H. contortus tubulin by TOG affinity chromatography.

PubMed

Munguía, Beatriz; Teixeira, Ramiro; Veroli, Victoria; Melian, Elisa; Saldaña, Jenny; Minteguiaga, Mahia; Señorale, Mario; Marín, Mónica; Domínguez, Laura

2017-11-01

Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 μg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites. Copyright © 2017 Elsevier Inc. All rights reserved.

Dye-ligand affinity systems.

PubMed

Denizli, A; Pişkin, E

2001-10-30

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

Lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (LIMACS): a novel method to analyze protein-lipid interaction.

PubMed

Bieberich, Erhard

2011-04-26

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

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.

Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

PubMed

Leedman, P J; Newman, J D; Harrison, L C

1989-07-01

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

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

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

PubMed

Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

2016-01-01

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

DEVELOPMENT OF SULFHYDRYL-REACTIVE SILICA FOR PROTEIN IMMOBILIZATION IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Wa, Chunling; Hage, David S.

2008-01-01

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77–81% of this protein to maleimide- or iodacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, non-specific binding, stability and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations. PMID:17297940

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. Copyright © 2013 Elsevier B.V. All rights reserved.

High-throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry.

PubMed

Razavi, Morteza; Frick, Lauren E; LaMarr, William A; Pope, Matthew E; Miller, Christine A; Anderson, N Leigh; Pearson, Terry W

2012-12-07

We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.

STUDIES OF VERAPAMIL BINDING TO HUMAN SERUM ALBUMIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

PubMed Central

Mallik, Rangan; Yoo, Michelle J.; Chen, Sike; Hage, David S.

2008-01-01

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body. PMID:18980867

Affinity purification and mass spectrometry: an attractive choice to investigate protein-protein interactions in plant immunity

USDA-ARS?s Scientific Manuscript database

Affinity purification of protein complexes from biological tissues, followed by liquid chromatography- tandem mass spectrometry (AP-MS/MS), has ballooned in recent years due to sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, improvements in affinity purifica...

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

PubMed

Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

2010-12-10

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.

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. Copyright © 2014 John Wiley & Sons, Ltd.

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

PubMed

Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

1993-12-01

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

PubMed Central

Kaur, Jasvir; Reinhardt, Dieter P.

2012-01-01

Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

Employing immuno-affinity for the analysis of various microbial metabolites of the mycotoxin deoxynivalenol.

PubMed

Zhu, Yan; Hassan, Yousef I; Shao, Suqin; Zhou, Ting

2018-06-29

Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly detected in grains infested with Fusarium species. The maximum tolerated levels of DON in the majority of world's countries are restricted to 0.75 mg kg -1 within the human food chain and to less than 1-5 mg kg -1 in animal feed depending on the feed material and/or animal species due to DON's short and long-term adverse effects on human health and animal productivity. The ability to accurately analyze DON and some of its fungal/bacterial metabolites is increasingly gaining a paramount importance in food/feed analysis and research. In this study, we used the immuno-affinity approach to enrich and detect DON and three of its bacterial metabolites, namely 3-epi-DON, 3-keto-DON, and deepoxy-DON (DOM-1). The optimized enrichment step coupled with high performance liquid chromatography can accurately and reproducibly quantify the aforementioned metabolites in feed matrixes (silage extract as an example in this case). It minimizes any background interface and provides a fast and easy-to-operate protocol for the analytical determination of such metabolites. More importantly, the presented data demonstrates the ability of the utilized monoclonal antibody, generated originally to capture DON in Enzyme-Linked Immunosorbent Assays (ELISA), to cross react with three less/non-toxic DON metabolites. This raises the concerns about the genuine need to account for such cross-reactivity when DON contamination is assessed through an immuno-affinity based analyses using the investigated antibody. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Analysis of Structural Features Contributing to Weak Affinities of Ubiquitin/Protein Interactions.

PubMed

Cohen, Ariel; Rosenthal, Eran; Shifman, Julia M

2017-11-10

Ubiquitin is a small protein that enables one of the most common post-translational modifications, where the whole ubiquitin molecule is attached to various target proteins, forming mono- or polyubiquitin conjugations. As a prototypical multispecific protein, ubiquitin interacts non-covalently with a variety of proteins in the cell, including ubiquitin-modifying enzymes and ubiquitin receptors that recognize signals from ubiquitin-conjugated substrates. To enable recognition of multiple targets and to support fast dissociation from the ubiquitin modifying enzymes, ubiquitin/protein interactions are characterized with low affinities, frequently in the higher μM and lower mM range. To determine how structure encodes low binding affinity of ubiquitin/protein complexes, we analyzed structures of more than a hundred such complexes compiled in the Ubiquitin Structural Relational Database. We calculated various structure-based features of ubiquitin/protein binding interfaces and compared them to the same features of general protein-protein interactions (PPIs) with various functions and generally higher affinities. Our analysis shows that ubiquitin/protein binding interfaces on average do not differ in size and shape complementarity from interfaces of higher-affinity PPIs. However, they contain fewer favorable hydrogen bonds and more unfavorable hydrophobic/charge interactions. We further analyzed how binding interfaces change upon affinity maturation of ubiquitin toward its target proteins. We demonstrate that while different features are improved in different experiments, the majority of the evolved complexes exhibit better shape complementarity and hydrogen bond pattern compared to wild-type complexes. Our analysis helps to understand how low-affinity PPIs have evolved and how they could be converted into high-affinity PPIs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recent applications of hydrophilic interaction liquid chromatography in pharmaceutical analysis.

PubMed

Zhang, Qian; Yang, Feng-Qing; Ge, Liya; Hu, Yuan-Jia; Xia, Zhi-Ning

2017-01-01

Hydrophilic interaction liquid chromatography, an alternative liquid chromatography mode, is of particular interest in separating hydrophilic and polar ionic compounds. Compared with traditional liquid chromatography techniques, hydrophilic interaction liquid chromatography offers specific advantages mainly including: (1) relatively green and water-soluble mobile phase composition, which enhances the solubility of hydrophilic and polar ionic compounds; (2) no need for ion-pairing reagents and high content of organic solvent, which benefits mass spectrometry detection; (3) high orthogonality to reverse-phase liquid chromatography, well adapted to two-dimensional liquid chromatography for complicated samples. Therefore, hydrophilic interaction liquid chromatography has been rapidly developed in many areas over the past decades. This review summarizes the recent progress (from 2012 to July 2016) of hydrophilic interaction liquid chromatography in pharmaceutical analysis, with the focus on detecting chemical drugs in various matrices, charactering active compounds of natural products and assessing biotherapeutics through typical structure unit. Moreover, the retention mechanism and behavior of analytes in hydrophilic interaction liquid chromatography as well as some novel hydrophilic interaction liquid chromatography columns used for pharmaceutical analysis are also described. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Supercritical fluid chromatography for lipid analysis in foodstuffs.

PubMed

Donato, Paola; Inferrera, Veronica; Sciarrone, Danilo; Mondello, Luigi

2017-01-01

The task of lipid analysis has always challenged separation scientists, and new techniques in chromatography were often developed for the separation of lipids; however, no single technique or methodology is yet capable of affording a comprehensive screening of all lipid species and classes. This review acquaints the role of supercritical fluid chromatography within the field of lipid analysis, from the early developed capillary separations based on pure CO 2 , to the most recent techniques employing packed columns under subcritical conditions, including the niche multidimensional techniques using supercritical fluids in at least one of the separation dimensions. A short history of supercritical fluid chromatography will be introduced first, from its early popularity in the late 1980s, to the sudden fall and oblivion until the last decade, experiencing a regain of interest within the chromatographic community. Afterwards, the subject of lipid nomenclature and classification will be briefly dealt with, before discussing the main applications of supercritical fluid chromatography for food analysis, according to the specific class of lipids. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biomonitoring of selenoprotein P in human serum by fast affinity chromatography coupled to ICP-MS.

PubMed

Heitland, Peter; Köster, Helmut D

2018-04-01

Most of the Se in human serum is bound to selenoprotein P (SEPP1) in which Se is present in form of selenocysteine. The SEPP1 is a new possible biomarker for the Se status and for this reason we developed a fast, simple and reliable method for the quantitative determination of SEPP1 in serum by affinity chromatography coupled to ICP-MS. It is possible to separate SEPP1 from other selenoproteins in serum in only 5 min, which allows high sample throughput in clinical laboratories. Measured and certified concentrations of total Se and Se(SEPP1) are in good agreement for the reference material SRM 1950. The SEPP1 concentration was stable in serum samples of 3 persons for a minimum of 2 weeks. Further results of method validation were described including internal and external quality assurance. The analytical method was applied for a biomonitoring study of the SEPP1 and total Se concentration in human serum of 50 occupationally non-exposed persons living in northern Germany. Concentration ranges and mean concentrations for Se(SEPP1) are 31.1-59.7 and 46.2 μg/L, respectively. The corresponding values for total Se are 62-120 and 83.5 μg/L. The mean percentage of total Se in serum present as SEPP1 is 58%. Copyright © 2018 Elsevier GmbH. All rights reserved.

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

PubMed Central

Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

2013-01-01

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

Simultaneous analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography.

PubMed

Taguchi, Kaori; Fukusaki, Eiichiro; Bamba, Takeshi

2014-10-03

Chromatography techniques usually use a single state in the mobile phase, such as liquid, gas, or supercritical fluid. Chromatographers manage one of these techniques for their purpose but are sometimes required to use multiple methods, or even worse, multiple techniques when the target compounds have a wide range of chemical properties. To overcome this challenge, we developed a single method covering a diverse compound range by means of a "unified" chromatography which completely bridges supercritical fluid chromatography and liquid chromatography. In our method, the phase state was continuously changed in the following order; supercritical, subcritical and liquid. Moreover, the gradient of the mobile phase starting at almost 100% CO2 was replaced with 100% methanol at the end completely. As a result, this approach achieved further extension of the polarity range of the mobile phase in a single run, and successfully enabled the simultaneous analysis of fat- and water-soluble vitamins with a wide logP range of -2.11 to 10.12. Furthermore, the 17 vitamins were exceptionally separated in 4min. Our results indicated that the use of dense CO2 and the replacement of CO2 by methanol are practical approaches in unified chromatography covering diverse compounds. Additionally, this is a first report to apply the novel approach to unified chromatography, and can open another door for diverse compound analysis in a single chromatographic technique with single injection, single column and single system. Copyright © 2014. Published by Elsevier B.V.

Analysis of Explosives in Soil Using Solid Phase Microextraction and Gas Chromatography: Environmental Analysis

DTIC Science & Technology

2006-01-01

ENVIRONMENTAL ANALYSIS Analysis of Explosives in Soil Using Solid Phase Microextraction and Gas Chromatography Howard T. Mayfield Air Force Research...Abstract: Current methods for the analysis of explosives in soils utilize time consuming sample preparation workups and extractions. The method detection...chromatography/mass spectrometry to provide a con- venient and sensitive analysis method for explosives in soil. Keywords: Explosives, TNT, solid phase

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).

Binding of TEM-1 beta-lactamase to beta-lactam antibiotics by frontal affinity chromatography.

PubMed

Chen, Xiu; Li, Yuhua; Zhang, Yan; Yang, Jianting; Bian, Liujiao

2017-04-15

TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718×10 3 , 6.624×10 3 and 2.244×10 3 (mol/L), respectively at 288K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

PubMed

Forier, Cynthia; Boschetti, Egisto; Ouhammouch, Mohamed; Cibiel, Agnès; Ducongé, Frédéric; Nogré, Michel; Tellier, Michel; Bataille, Damien; Bihoreau, Nicolas; Santambien, Patrick; Chtourou, Sami; Perret, Gérald

2017-03-17

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Enrichment of high affinity subclasses and glycoforms from serum-derived IgG using FcγRs as affinity ligands.

PubMed

Boesch, Austin W; Kappel, James H; Mahan, Alison E; Chu, Thach H; Crowley, Andrew R; Osei-Owusu, Nana Y; Alter, Galit; Ackerman, Margaret E

2018-05-01

As antibodies continue to gain predominance in drug discovery and development pipelines, efforts to control and optimize their activity in vivo have matured to incorporate sophisticated abilities to manipulate engagement of specific Fc binding partners. Such efforts to promote diverse functional outcomes include modulating IgG-Fc affinity for FcγRs to alternatively potentiate or reduce effector functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. While a number of natural and engineered Fc features capable of eliciting variable effector functions have been demonstrated in vitro and in vivo, elucidation of these important functional relationships has taken significant effort through use of diverse genetic, cellular and enzymatic techniques. As an orthogonal approach, we demonstrate use of FcγR as chromatographic affinity ligands to enrich and therefore simultaneously identify favored binding species from a complex mixture of serum-derived pooled polycloncal human IgG, a load material that contains the natural repertoire of Fc variants and post-translational modifications. The FcγR-enriched IgG was characterized for subclass and glycoform composition and the impact of this bioseparation step on antibody activity was measured in cell-based effector function assays including Natural Killer cell activation and monocyte phagocytosis. This work demonstrates a tractable means to rapidly distinguish complex functional relationships between two or more interacting biological agents by leveraging affinity chromatography followed by secondary analysis with high-resolution biophysical and functional assays and emphasizes a platform capable of surveying diverse natural post-translational modifications that may not be easily produced with high purity or easily accessible with recombinant expression techniques. © 2018 Wiley Periodicals, Inc.

A highly selective dispersive liquid-liquid microextraction approach based on the unique fluorous affinity for the extraction and detection of per- and polyfluoroalkyl substances coupled with high performance liquid chromatography tandem-mass spectrometry.

PubMed

Wang, Juan; Shi, Yali; Cai, Yaqi

2018-04-06

In the present study, a highly selective fluorous affinity-based dispersive liquid-liquid microextraction (DLLME) technique was developed for the extraction and analysis of per- and polyfluoroalkyl substances (PFASs) followed by high performance liquid chromatography tandem-mass spectrometry. Perfluoro-tert-butanol with multiple C-F bonds was chosen as the extraction solvent, which was injected into the aqueous samples with a dispersive solvent (acetonitrile) in a 120:800 (μL, v/v) mixture for PFASs enrichment. The fluorous affinity-based extraction mechanism was confirmed by the significantly higher extraction recoveries for PFASs containing multiple fluorine atoms than those for compounds with fewer or no fluorine atoms. The extraction recoveries of medium and long-chain PFASs (CF 2  > 5) exceeded 70%, except perfluoroheptanoic acid, while those of short-chain PFASs were lower than 50%, implying that the proposed DLLME may not be suitable for their extraction due to weak fluorous affinity. This highly fluoroselective DLLME technique can greatly decrease the matrix effect that occurs in mass spectrometry detection when applied to the analysis of urine samples. Under the optimum conditions, the relative recoveries of PFASs with CF 2  > 5 ranged from 80.6-121.4% for tap water, river water and urine samples spiked with concentrations of 10, 50 and 100 ng/L. The method limits of quantification for PFASs in water and urine samples were in the range of 0.6-8.7 ng/L. Furthermore, comparable concentrations of PFASs were obtained via DLLME and solid-phase extraction, confirming that the developed DLLME technique is a promising method for the extraction of PFASs in real samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Using Affinity Chromatography to Investigate Novel Protein–Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course

PubMed Central

2009-01-01

Inquiry-driven lab exercises require students to think carefully about a question, carry out an investigation of that question, and critically analyze the results of their investigation. Here, we describe the implementation and assessment of an inquiry-based laboratory exercise in which students obtain and analyze novel data that contribute to our understanding of macromolecular trafficking between the nucleus and cytoplasm in eukaryotic cells. Although many of the proteins involved in nucleocytoplasmic transport are known, the physical interactions between some of these polypeptides remain uncharacterized. In this cell and molecular biology lab exercise, students investigate novel protein–protein interactions between factors involved in nuclear RNA export. Using recombinant protein expression, protein extraction, affinity chromatography, SDS-polyacrylamide gel electrophoresis, and Western blotting, undergraduates in a sophomore-level lab course identified a previously unreported association between the soluble mRNA transport factor Mex67 and the C-terminal region of the yeast nuclear pore complex protein Nup1. This exercise immersed students in the process of investigative science, from proposing and performing experiments through analyzing data and reporting outcomes. On completion of this investigative lab sequence, students reported enhanced understanding of the scientific process, increased proficiency with cellular and molecular methods and content, greater understanding of data analysis and the importance of appropriate controls, an enhanced ability to communicate science effectively, and an increased enthusiasm for scientific research and for the lab component of the course. The modular nature of this exercise and its focus on asking novel questions about protein–protein interactions make it easily transferable to undergraduate lab courses performed in a wide variety of contexts. PMID:19723816

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.

Isolation and purification of wheat germ agglutinin and analysis of its properties

NASA Astrophysics Data System (ADS)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

A Space Affine Matching Approach to fMRI Time Series Analysis.

PubMed

Chen, Liang; Zhang, Weishi; Liu, Hongbo; Feng, Shigang; Chen, C L Philip; Wang, Huili

2016-07-01

For fMRI time series analysis, an important challenge is to overcome the potential delay between hemodynamic response signal and cognitive stimuli signal, namely the same frequency but different phase (SFDP) problem. In this paper, a novel space affine matching feature is presented by introducing the time domain and frequency domain features. The time domain feature is used to discern different stimuli, while the frequency domain feature to eliminate the delay. And then we propose a space affine matching (SAM) algorithm to match fMRI time series by our affine feature, in which a normal vector is estimated using gradient descent to explore the time series matching optimally. The experimental results illustrate that the SAM algorithm is insensitive to the delay between the hemodynamic response signal and the cognitive stimuli signal. Our approach significantly outperforms GLM method while there exists the delay. The approach can help us solve the SFDP problem in fMRI time series matching and thus of great promise to reveal brain dynamics.

Using cell membrane chromatography and HPLC-TOF/MS method for in vivo study of active components from roots of Aconitum carmichaeli

PubMed Central

Cao, Yan; Chen, Xiao-Fei; Lü, Di-Ya; Dong, Xin; Zhang, Guo-Qing; Chai, Yi-Feng

2012-01-01

An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high Performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis ofthe analysis of the first dimension, retention components of the urine sample were collected into 30 fractions (one fraction per minute). Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine (Songorine, 14-benzoylneoline, Deoxyaconitine, etc.) exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the System suggest that the CMC can be applied to in vivo study. PMID:29403691

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

PubMed

Szamel, M; Goppelt, M; Resch, K

1985-12-19

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Coupled liquid chromatography-gas chromatography for the rapid analysis of gamma-oryzanol in rice lipids.

PubMed

Miller, Andreas; Frenzel, Thomas; Schmarr, Hans-Georg; Engel, Karl-Heinz

2003-01-24

An approach based on on-line coupled liquid chromatography-gas chromatography (LC-GC) was developed for the rapid analysis of gamma-oryzanol in rice. Total lipids were extracted from rice and subjected to LC-GC without any prior purification. gamma-Oryzanol was pre-separated by HPLC from rice lipids and transferred on-line to GC analysis in order to separate its major constituents. 24-methylenecycloartanyl ferulate, cycloartenyl ferulate, campesteryl ferulate, beta-sitosteryl ferulate and campestanyl ferulate. The identities of the compounds were confirmed by off-line GC-MS analysis. Total gamma-oryzanol content could be quantified by HPLC-UV detection and the distribution of gamma-oryzanol constituents could be determined by on-line coupled GC analysis. The proposed methodology paves the way for high-throughput investigations providing information on natural variations in gamma-oryzanol content and its composition in different rice varieties.

Selection of a high-affinity and in vivo bioactive ssDNA aptamer against angiotensin II peptide.

PubMed

Heiat, Mohammad; Ranjbar, Reza; Latifi, Ali Mohammad; Rasaee, Mohammad Javad

2016-08-01

Unique features of aptamers have attracted interests for a broad range of applications. Aptamers are able to specifically bind to targets and inhibit their functions. This study, aimed to isolate the high affinity ssDNA aptamers against bio-regulator peptide angiotensin II (Ang II) and investigate their bioactivity in cellular and animal models. To isolate ssDNA aptamers, 12 rounds of affinity chromatography SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure were carried out. The SPR (surface plasmon resonance) and ELONA (enzyme linked oligonucleotide assay) analysis were used to determine the affinity and specificity of aptamers. The ability of selected aptamers to inhibit the proliferative effect of Ang II on human aortic vascular smooth muscle cells (HA-VSMCs) and their performance on Wistar rat urinary system and serum electrolyte levels were investigated. Two full-length aptamers (FLC112 and FLC125) with high affinity of respectively 7.52±2.44E-10 and 5.87±1.3E-9M were isolated against Ang II. The core regions of these aptamers (CRC112 and CRC125) also showed affinity of 5.33±1.15E-9 and 4.11±1.09E-9M. In vitro analysis revealed that FLC112 and FLC125 can inhibit the proliferative effect of Ang II on HA-VSMCs (P<0.05). They also significantly reduced the serum sodium level and increased the urine volume (P<0.05). The core regions of aptamers did not show high inhibitory potential against Ang II. It can be a spotlight that ssDNA aptamers have high potential for blocking Ang II. In conclusion, it appears that the researches focusing on high affinity and bioactive aptamers may lead to excellent results in blocking Ang II activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

PubMed

Horejsí, V; Tichá, M; Kocourek, J

1977-09-29

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

An inorganic boronate affinity in-needle monolithic device for specific capture of cis-diol containing compounds.

PubMed

Jin, Shanxia; Zhang, Wei; Yang, Qin; Dai, Lili; Zhou, Ping

2018-02-01

In this work, inorganic boronate affinity monolith was prepared by in situ synthesis in 0.33mm i.d. stainless steel needle through sol-gel process using tetraethoxysilane and tetrabutyl orthotitanate as the co-precursors. The morphology, structure and composition of the monolith were characterized. In contrast to conventional boronate affinity materials, inorganic boric acid was used as affinity ligand. Different compounds were used for the evaluation of the boronate affinity of this inorganic monolithic material. The monolith exhibited good selectivity towards cis-diol containing compounds. Recovery of greater than 90% was achieved for in-needle extraction of catechol under neutral conditions. Owing to the hydrophilic property of the monolith, the procedure of affinity chromatography could be performed in aqueous solution. This monolithic in-needle device will be useful for boronate affinity extraction of small-volume samples. Copyright © 2017 Elsevier B.V. All rights reserved.

What reaches the antenna? How to calibrate odor flux and ligand-receptor affinities.

PubMed

Andersson, Martin N; Schlyter, Fredrik; Hill, Sharon Rose; Dekker, Teun

2012-06-01

Physiological studies on olfaction frequently ignore the airborne quantities of stimuli reaching the sensory organ. We used a gas chromatography-calibrated photoionization detector to estimate quantities released from standard Pasteur pipette stimulus cartridges during repeated puffing of 27 compounds and verified how lack of quantification could obscure olfactory sensory neuron (OSN) affinities. Chemical structure of the stimulus, solvent, dose, storage condition, puff interval, and puff number all influenced airborne quantities. A model including boiling point and lipophilicity, but excluding vapor pressure, predicted airborne quantities from stimuli in paraffin oil on filter paper. We recorded OSN responses of Drosophila melanogaster, Ips typographus, and Culex quinquefasciatus, to known quantities of airborne stimuli. These demonstrate that inferred OSN tuning width, ligand affinity, and classification can be confounded and require stimulus quantification. Additionally, proper dose-response analysis shows that Drosophila AB3A OSNs are not promiscuous, but highly specific for ethyl hexanoate, with other earlier proposed ligands 10- to 10 000-fold less potent. Finally, we reanalyzed published Drosophila OSN data (DoOR) and demonstrate substantial shifts in affinities after compensation for quantity and puff number. We conclude that consistent experimental protocols are necessary for correct OSN classification and present some simple rules that make calibration, even retroactively, readily possible.

[Immuno-affinity chromatographic purification: the study of methods to test citrinin in monascus products by high performance liquid chromatography].

PubMed

Qiu, Wen-qian; Liu, Xiao-xia; Zheng, Kui-cheng; Fu, Wu-sheng

2012-08-01

To establish a method to test citrinin (CIT) in monascus products by immuno-affinity chromatography (IAC)-high performance liquid chromatography (HPLC), and to detect the content of CIT in monascus products in Fujian province. IAC-HPLC was applied to detect the CIT content in monascus products. The conditions to use HPLC were as follows: C(18) reversed-phase chromatographic column, 150.0 mm×4.6mm×3 µm; mobile phase: the volume ratio of acetonitrile and 0.1% phosphoric acid solution at 65:35; isocratic elution; column temperature: 28°C; flow velocity: 0.8 ml/min; fluorescence detector, excitation wavelength (λ(ex)) was 331 nm and emission wavelength (λ(em)) was 500 nm. The standard curved was established by the linear regression of peak area (Y) to CIT content (X, ng/ml). The accuracy and precision of the method would then be verified. And 32 kinds of monascus products were determined and their color values were compared by this method. The standard curve established in this study was Y = 4634.8X-136.42, r = 1.000; whose limits of detection was 20 µg/kg and the limits of qualification was 64 µg/kg. In the range between 200 and 800 µg/kg, the standard recovery rate was 98.9% - 110.0% (n = 3), and the relative standard deviation (RSD) was 0.51% - 1.76%. Out of the 32 samples, CIT was detected from 11 samples of monascus rice, 9 samples of monascus powder and 5 samples of monascus pigments, the content was around 0.212 - 14.500 mg/kg. 4 out of 7 functional monascus samples were detected out CIT, whose content at 0.142 - 0.275 mg/kg. The method to detect CIT in monascus products by IAC-HPLC has been established.

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

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.

Optimization of Immobilized Gallium (III) Ion Affinity Chromatography for Selective Binding and Recovery of Phosphopeptides from Protein Digests

PubMed Central

Aryal, Uma K.; Olson, Douglas J.H.; Ross, Andrew R.S.

2008-01-01

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides. PMID:19183793

Prostate cell membrane chromatography-liquid chromatography-mass spectrometry for screening of active constituents from Uncaria rhynchophylla.

PubMed

He, Jianyu; Han, Shengli; Yang, Fangfang; Zhou, Nan; Wang, Sicen

2013-01-01

Uncaria rhynchophylla is a traditional Chinese medicinal herb used to treat hypertension and convulsive disorders such as epilepsy. Rat prostate cell membrane chromatography combined with liquid chromatography-mass spectrometry (LC-MS) was used to identify active constituents from U. rhynchophylla extracts. Four compounds (corynoxeine, isorhynchophylline, isocorynoxeine and rhynchophylline) were discovered. Competitive binding assay results indicated that the four compounds were in direct competition at a single common binding site and interacted with α1A adrenergic receptors (α1A-AR) in a manner similar to tamsulosin. Affinity constant values of the four compounds binding with α1A-AR were also measured using rat prostate cell membrane chromatography (CMC). Finally, their pharmacodynamic effects were tested on rat caudal arteries. This CMC combined LC-MS system offers a means of drug discovery by screening natural medicinal herbs for new pharmacologically active molecules targeting specific receptors.

Impact of comprehensive two-dimensional gas chromatography with mass spectrometry on food analysis.

PubMed

Tranchida, Peter Q; Purcaro, Giorgia; Maimone, Mariarosa; Mondello, Luigi

2016-01-01

Comprehensive two-dimensional gas chromatography with mass spectrometry has been on the separation-science scene for about 15 years. This three-dimensional method has made a great positive impact on various fields of research, and among these that related to food analysis is certainly at the forefront. The present critical review is based on the use of comprehensive two-dimensional gas chromatography with mass spectrometry in the untargeted (general qualitative profiling and fingerprinting) and targeted analysis of food volatiles; attention is focused not only on its potential in such applications, but also on how recent advances in comprehensive two-dimensional gas chromatography with mass spectrometry will potentially be important for food analysis. Additionally, emphasis is devoted to the many instances in which straightforward gas chromatography with mass spectrometry is a sufficiently-powerful analytical tool. Finally, possible future scenarios in the comprehensive two-dimensional gas chromatography with mass spectrometry food analysis field are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Approaches to High-Performance Preparative Chromatography of Proteins

NASA Astrophysics Data System (ADS)

Sun, Yan; Liu, Fu-Feng; Shi, Qing-Hong

Preparative liquid chromatography is widely used for the purification of chemical and biological substances. Different from high-performance liquid chromatography for the analysis of many different components at minimized sample loading, high-performance preparative chromatography is of much larger scale and should be of high resolution and high capacity at high operation speed and low to moderate pressure drop. There are various approaches to this end. For biochemical engineers, the traditional way is to model and optimize a purification process to make it exert its maximum capability. For high-performance separations, however, we need to improve chromatographic technology itself. We herein discuss four approaches in this review, mainly based on the recent studies in our group. The first is the development of high-performance matrices, because packing material is the central component of chromatography. Progress in the fabrication of superporous materials in both beaded and monolithic forms are reviewed. The second topic is the discovery and design of affinity ligands for proteins. In most chromatographic methods, proteins are separated based on their interactions with the ligands attached to the surface of porous media. A target-specific ligand can offer selective purification of desired proteins. Third, electrochromatography is discussed. An electric field applied to a chromatographic column can induce additional separation mechanisms besides chromatography, and result in electrokinetic transport of protein molecules and/or the fluid inside pores, thus leading to high-performance separations. Finally, expanded-bed adsorption is described for process integration to reduce separation steps and process time.

Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

PubMed

Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet

2017-12-01

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

PubMed

Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

2006-04-01

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

Lipidomic analysis of biological samples: Comparison of liquid chromatography, supercritical fluid chromatography and direct infusion mass spectrometry methods.

PubMed

Lísa, Miroslav; Cífková, Eva; Khalikova, Maria; Ovčačíková, Magdaléna; Holčapek, Michal

2017-11-24

Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography - mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements

Supercritical Fluid Chromatography in Natural Product Analysis - An Update.

PubMed

Gibitz Eisath, Nora; Sturm, Sonja; Stuppner, Hermann

2018-04-01

The wide chemical diversity of natural products has challenged analysts all over the world and has been a driving force for the development of innovative technologies since decades. In the last years, supercritical fluid chromatography (SFC) has finally emerged from the shadow of liquid chromatography (LC) and gas chromatography (GC) and has become a powerful tool in modern natural product analysis. Whereas in the past the technique had mainly been restricted to a small group of nonpolar compounds, it has largely expanded its suitability in the last years and has demonstrated possibilities without boundaries. This mini-review, focused on the latest applications, provides a brief update on the current status of SFC in natural product analysis with the aim to demonstrate its applicability for both polar and nonpolar plant constituents. The approaches cover the whole range of polarity, including carotenoids, flavonoids, water-unstable ginkgolides, and even highly polar triterpene saponins with several sugar residues. Georg Thieme Verlag KG Stuttgart · New York.

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

PubMed

Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro

2011-01-01

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification.

PubMed

Shin, Min Jae; Tan, Lihan; Jeong, Min Ho; Kim, Ji-Heung; Choe, Woo-Seok

2011-08-05

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification. Copyright © 2011 Elsevier B.V. All rights reserved.

Affinity Purification of Proteins in Tag-Free Form: Split Intein-Mediated Ultrarapid Purification (SIRP).

PubMed

Guan, Dongli; Chen, Zhilei

2017-01-01

Proteins purified using affinity-based chromatography often exploit a recombinant affinity tag. Existing methods for the removal of the extraneous tag, needed for many applications, suffer from poor efficiency and/or high cost. Here we describe a simple, efficient, and potentially low-cost approach-split intein-mediated ultrarapid purification (SIRP)-for both the purification of the desired tagged protein from Escherichia coli lysate and removal of the tag in less than 1 h. The N- and C-fragment of a self-cleaving variant of a naturally split DnaE intein from Nostoc punctiforme are genetically fused to the N-terminus of an affinity tag and a protein of interest (POI), respectively. The N-intein/affinity tag is used to functionalize an affinity resin. The high affinity between the N- and C-fragment of DnaE intein enables the POI to be purified from the lysate via affinity to the resin, and the intein-mediated C-terminal cleavage reaction causes tagless POI to be released into the flow-through. The intein cleavage reaction is strongly inhibited by divalent ions (e.g., Zn 2+ ) under non-reducing conditions and is significantly enhanced by reducing conditions. The POI is cleaved efficiently regardless of the identity of the N-terminal amino acid except in the cases of threonine and proline, and the N-intein-functionalized affinity resin can be regenerated for multiple cycles of use.

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

Preliminary Crystallographic Study of Hemoglobin from Buffalo (Bubalus bubalis): A Low Oxygen Affinity Species.

PubMed

Balasubramanian, Moovarkumudalvan; Moorthy, Ponnuraj Sathya; Neelagandan, Kamariah; Ponnuswamy, Mondikalipudur Nanjappa Gounder

2009-01-01

Hemoglobin is a tetrameric, iron-containing metalloprotein, which plays a vital role in the transportation of oxygen from lungs to tissues and carbon dioxide back to lungs. Though good amount of work has already been done on hemoglobins, the scarcity of data on three dimensional structures pertaining to low oxygen affinity hemoglobins from mammalian species, motivated our group to work on this problem specifically. Herein, we report the preliminary crystallographic analysis of buffalo hemoglobin, which belongs to low oxygen affinity species. The buffalo blood was collected, purified by anion exchange chromatography and crystallized with PEG 3350 using 50mM phosphate buffer at pH 6.7 as a precipitant by hanging drop vapor diffusion method. Data collection was carried out using mar345dtb image plate detector system. Buffalo hemoglobin crystallizes in orthorhombic space group P2(1)2(1)2(1) with one whole biological molecule (alpha2beta2) in the asymmetric unit with cell dimensions a=63.064A, b=74.677A, c=110.224A.

LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

PubMed Central

2015-01-01

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. PMID:25134008

Two-Dimensional Liquid Chromatography Analysis of Polystyrene/Polybutadiene Block Copolymers.

PubMed

Lee, Sanghoon; Choi, Heejae; Chang, Taihyun; Staal, Bastiaan

2018-05-15

A detailed characterization of a commercial polystyrene/polybutadiene block copolymer material (Styrolux) was carried out using two-dimensional liquid chromatography (2D-LC). The Styrolux is prepared by statistical linking reaction of two different polystyrene- block-polybutadienyl anion precursors with a multivalent linking agent. Therefore, it is a mixture of a number of branched block copolymers different in molecular weight, composition, and chain architecture. While individual LC analysis, including size exclusion chromatography, interaction chromatography, or liquid chromatography at critical condition, is not good enough to resolve all the polymer species, 2D-LC separations coupling two chromatography methods were able to resolve all polymer species present in the sample; at least 13 block copolymer species and a homopolystyrene blended. Four different 2D-LC analyses combining a different pair of two LC methods provide their characteristic separation results. The separation characteristics of the 2D-LC separations are compared to elucidate the elution characteristics of the block copolymer species.

Comprehensive two-dimensional liquid chromatography for polyphenol analysis in foodstuffs.

PubMed

Cacciola, Francesco; Farnetti, Sara; Dugo, Paola; Marriott, Philip John; Mondello, Luigi

2017-01-01

Polyphenols are a class of plant secondary metabolites that are recently drawing a special interest because of their broad spectrum of pharmacological effects. As they are characterized by an enormous structural variability, the identification of these molecules in food samples is a difficult task, and sometimes having only a limited number of commercially available reference materials is not of great help. One-dimensional liquid chromatography is the most widely applied analytical approach for their analysis. In particular, the hyphenation of liquid chromatography to mass spectrometry has come to play an influential role by allowing relatively fast tentative identification and accurate quantification of polyphenolic compounds at trace levels in vegetable media. However, when dealing with very complex real-world food samples, a single separation system often does not provide sufficient resolving power for attaining rewarding results. Comprehensive two-dimensional liquid chromatography is a technique of great analytical impact, since it offers much higher peak capacities than separations in a single dimension. In the present review, we describe applications in the field of comprehensive two-dimensional liquid chromatography for polyphenol analysis in real-world food samples. Comprehensive two-dimensional liquid chromatography applications to nonfood matrices fall outside the scope of the current report and will not be discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of salts on protein-surface interactions: applications for column chromatography.

PubMed

Tsumoto, Kouhei; Ejima, Daisuke; Senczuk, Anna M; Kita, Yoshiko; Arakawa, Tsutomu

2007-07-01

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. Copyright 2007 Wiley-Liss, Inc.

Improved Thermal Modulator for Gas Chromatography

NASA Technical Reports Server (NTRS)

Hasselbrink, Ernest Frederick, Jr.; Hunt, Patrick J.; Sacks, Richard D.

2008-01-01

An improved thermal modulator has been invented for use in a variant of gas chromatography (GC). The variant in question denoted as two-dimensional gas chromatography (2DGC) or GC-GC involves the use of three series-connected chromatographic columns, in the form of capillary tubes coated interiorly with suitable stationary phases (compounds for which different analytes exhibit different degrees of affinity). The two end columns are relatively long and are used as standard GC columns. The thermal modulator includes the middle column, which is relatively short and is not used as a standard GC column: instead, its temperature is modulated to affect timed adsorption and desorption of analyte gases between the two end columns in accordance with a 2DGC protocol.

A rapid solution-based method for determining the affinity of heroin hapten-induced antibodies to heroin, its metabolites, and other opioids.

PubMed

Torres, Oscar B; Duval, Alexander J; Sulima, Agnieszka; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

2018-06-01

We describe for the first time a method that utilizes microscale thermophoresis (MST) technology to determine polyclonal antibody affinities to small molecules. Using a novel type of heterologous MST, we have accurately measured a solution-based binding affinity of serum antibodies to heroin which was previously impossible with other currently available methods. Moreover, this mismatch approach (i.e., using a cross-reactive hapten tracer) has never been reported in the literature. When compared with equilibrium dialysis combined with ultra-performance liquid chromatography/tandem mass spectrometry (ED-UPLC/MS/MS), this novel MST method yields similar binding affinity values for polyclonal antibodies to the major heroin metabolites 6-AM and morphine. Additionally, we herein report the method of synthesis of this novel cross-reactive hapten, MorHap-acetamide-a useful analog for the study of heroin hapten-antibody interactions. Using heterologous MST, we were able to determine the affinities, down to nanomolar accuracies, of polyclonal antibodies to various abused opioids. While optimizing this method, we further discovered that heroin is protected from serum esterase degradation by the presence of these antibodies in a concentration-dependent manner. Lastly, using affinity data for a number of structurally different opioids, we were able to dissect the moieties that are crucial to antibody binding. The novel MST method that is presented herein can be extended to the analysis of any ligand that is prone to degradation and can be applied not only to the development of vaccines to substances of abuse but also to the analysis of small molecule/protein interactions in the presence of serum. Graphical abstract Strategy for the determination of hapten-induced antibody affinities using Microscale thermophoresis.

Application of gas chromatography to analysis of spirit-based alcoholic beverages.

PubMed

Wiśniewska, Paulina; Śliwińska, Magdalena; Dymerski, Tomasz; Wardencki, Waldemar; Namieśnik, Jacek

2015-01-01

Spirit-based beverages are alcoholic drinks; their production processes are dependent on the type and origin of raw materials. The composition of this complex matrix is difficult to analyze, and scientists commonly choose gas chromatography techniques for this reason. With a wide selection of extraction methods and detectors it is possible to provide qualitative and quantitative analysis for many chemical compounds with various functional groups. This article describes different types of gas chromatography techniques and their most commonly used associated extraction techniques (e.g., LLE, SPME, SPE, SFE, and SBME) and detectors (MS, TOFMS, FID, ECD, NPD, AED, O or EPD). Additionally, brief characteristics of internationally popular spirit-based beverages and application of gas chromatography to the analysis of selected alcoholic drinks are presented.

Enrichment of Glycoproteins using Nano-scale Chelating Con A Monolithic Capillary Chromatography

PubMed Central

Feng, Shun; Yang, Na; Pennathur, Subramaniam; Goodison, Steve; Lubman, David M.

2009-01-01

Immobilized lectin chromatography can be employed for glycoprotein enrichment, but commonly used columns have limitations of yield and resolution. In order to improve efficiency and to make the technique applicable to minimal sample material, we have developed a nano-scale chelating Concanavalin A (Con A) monolithic capillary prepared using GMA-EDMA (glycidyl methacrylate–co-ethylene dimethacrylate) as polymeric support. Con A was immobilized on Cu(II)-charged iminodiacetic acid (IDA) regenerable sorbents by forming a IDA:Cu(II):Con A sandwich affinity structure that has high column capacity as well as stability. When compared with conventional Con A lectin chromatography, the monolithic capillary enabled the better reproducible detection of over double the number of unique N-glycoproteins in human urine samples. Utility for analysis of minimal biological samples was confirmed by the successful elucidation of glycoprotein profiles in mouse urine samples at the microliter scale. The improved efficiency of the nano-scale monolithic capillary will impact the analysis of glycoproteins in complex biological samples, especially where only limited material may be available. PMID:19366252

Group type analysis of asphalt by column liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, C.; Yang, J.; Xue, Y.

2008-07-01

An improved analysis method for characterization of asphalt was established. The method is based on column chromatography technique. The asphalts were separated into four groups: saturates, aromatics, resins, and asphaltenes, quantitatively. About 0.1 g of sample was required in each analysis. About 20 mL of n-heptanes was used to separate out saturates first. Then about 35 mL of n-heptanes/dichloromethane (.5, v/v) mixture was used to separate out aromatics. About 30 mL of dichloromethane/tetrahydrofuran (1/3, v/v) mixture was used to separate out resin. The quality of the separation was confirmed by infrared spectra (IR) and {sup 1}H NMR analysis. The modelmore » compounds, tetracosan for saturates, dibenz(o)anthracen for aromatics, and acetanilide for resins were used for verification. The IR and {sup 1}H NMR analysis of the prepared fractions from the column liquid chromatography were in good agreement that of pure reagents.« less

A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model

USDA-ARS?s Scientific Manuscript database

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with differ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

PubMed Central

Murphy, Patrick J. M.; Stone, Orrin J.; Anderson, Michelle E.

2011-01-01

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media

Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

PubMed

Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

2018-04-25

Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

CHEMICAL ANALYSIS OF WET SCRUBBERS UTILIZING ION CHROMATOGRAPHY

EPA Science Inventory

The report describes the key elements required to develop a sampling and analysis program for a wet scrubber using ion chromatography as the main analytical technique. The first part of the report describes a sampling program for two different types of wet scrubbers: the venturi/...

Comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for analysis of toad skin.

PubMed

Li, Jia-Fu; Yan, Xia; Wu, Yun-Long; Fang, Mei-Juan; Wu, Zhen; Qiu, Ying-Kun

2017-04-15

An analytical two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D NPLC × RPLC) system was constructed with a newly developed thermal evaporation assisted adsorption (TEAA) interface. This novel TEAA interface with heating temperature above solvent boiling point allowed fast removal of organic NPLC solvent and successfully solved the solvent incompatibility problem between NPLC and RPLC. The system achieved rapid on-line solvent exchange between the two dimensions within a short modulation time of 190 s and was applied in the analysis of an extract from the skin of Bufo bufo gargarizans. This is the first time to realize the on-line comprehensive analysis of a moderate polar natural product by coupling NPLC with reversed phase ultra-high performance liquid chromatography (UHPLC). To be highlighted, with the TEAA interface, the 2D NPLC × RPLC system provided excellent resolution and orthogonality (75.2%), when compared with that of 2D RPLC × RPLC. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

PubMed Central

Chung, Wai Keen; Freed, Alexander S.; Holstein, Melissa A.; McCallum, Scott A.; Cramer, Steven M.

2010-01-01

NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems. PMID:20837551

Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis.

PubMed

King, Cory; Patel, Rekha; Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Gu, Zhenyu; Liu, Hongcheng

2018-05-15

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen. Copyright © 2018 Elsevier B.V. All rights reserved.

High oxygen affinity hemoglobins.

PubMed

Mangin, O

2017-02-01

High oxygen affinity hemoglobins are responsible for rare and heterogeneous autosomic dominant genetic diseases. They cause pure erythrocytosis, sometimes accountable for hyperviscosity and thrombosis, or hemolysis. Differential diagnoses must be first ruled out. The diagnosis is based on the identification of a decreased P50, and their possible characterization by cation exchange-high performance liquid chromatography and capillary electrophoresis. Finally, genetic studies of the responsible globin chain gene will confirm the mutation. The prognosis mainly relies on the P50 decrease rate and on the hemoglobin cooperativity impairment. Disease management should be personalized, and it should primarily depend on smoking cessation and physical activity. Phlebotomy and platelet aggregation inhibitors' prescriptions can be discussed. There is no contraindication to flights, high-altitude conditions, or pregnancy. Nevertheless, blood donation must be prohibited. Copyright © 2016 Société Nationale Française de Médecine Interne (SNFMI). Published by Elsevier SAS. All rights reserved.

Principles of Micellar Electrokinetic Capillary Chromatography Applied in Pharmaceutical Analysis

PubMed Central

Hancu, Gabriel; Simon, Brigitta; Rusu, Aura; Mircia, Eleonora; Gyéresi, Árpád

2013-01-01

Since its introduction capillary electrophoresis has shown great potential in areas where electrophoretic techniques have rarely been used before, including here the analysis of pharmaceutical substances. The large majority of pharmaceutical substances are neutral from electrophoretic point of view, consequently separations by the classic capillary zone electrophoresis; where separation is based on the differences between the own electrophoretic mobilities of the analytes; are hard to achieve. Micellar electrokinetic capillary chromatography, a hybrid method that combines chromatographic and electrophoretic separation principles, extends the applicability of capillary electrophoretic methods to neutral analytes. In micellar electrokinetic capillary chromatography, surfactants are added to the buffer solution in concentration above their critical micellar concentrations, consequently micelles are formed; micelles that undergo electrophoretic migration like any other charged particle. The separation is based on the differential partitioning of an analyte between the two-phase system: the mobile aqueous phase and micellar pseudostationary phase. The present paper aims to summarize the basic aspects regarding separation principles and practical applications of micellar electrokinetic capillary chromatography, with particular attention to those relevant in pharmaceutical analysis. PMID:24312804

Affinity Proteomics in the mountains: Alpbach 2015.

PubMed

Taussig, Michael J

2016-09-25

The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. Copyright © 2016 Elsevier B.V. All rights reserved.

Repertoire Analysis of Antibody CDR-H3 Loops Suggests Affinity Maturation Does Not Typically Result in Rigidification

PubMed Central

Jeliazkov, Jeliazko R.; Sljoka, Adnan; Kuroda, Daisuke; Tsuchimura, Nobuyuki; Katoh, Naoki; Tsumoto, Kouhei; Gray, Jeffrey J.

2018-01-01

Antibodies can rapidly evolve in specific response to antigens. Affinity maturation drives this evolution through cycles of mutation and selection leading to enhanced antibody specificity and affinity. Elucidating the biophysical mechanisms that underlie affinity maturation is fundamental to understanding B-cell immunity. An emergent hypothesis is that affinity maturation reduces the conformational flexibility of the antibody’s antigen-binding paratope to minimize entropic losses incurred upon binding. In recent years, computational and experimental approaches have tested this hypothesis on a small number of antibodies, often observing a decrease in the flexibility of the complementarity determining region (CDR) loops that typically comprise the paratope and in particular the CDR-H3 loop, which contributes a plurality of antigen contacts. However, there were a few exceptions and previous studies were limited to a small handful of cases. Here, we determined the structural flexibility of the CDR-H3 loop for thousands of recent homology models of the human peripheral blood cell antibody repertoire using rigidity theory. We found no clear delineation in the flexibility of naïve and antigen-experienced antibodies. To account for possible sources of error, we additionally analyzed hundreds of human and mouse antibodies in the Protein Data Bank through both rigidity theory and B-factor analysis. By both metrics, we observed only a slight decrease in the CDR-H3 loop flexibility when comparing affinity matured antibodies to naïve antibodies, and the decrease was not as drastic as previously reported. Further analysis, incorporating molecular dynamics simulations, revealed a spectrum of changes in flexibility. Our results suggest that rigidification may be just one of many biophysical mechanisms for increasing affinity. PMID:29545810

Recent Advance in Liquid Chromatography/Mass Spectrometry Techniques for Environmental Analysis in Japan

PubMed Central

Suzuki, Shigeru

2014-01-01

The techniques and measurement methods developed in the Environmental Survey and Monitoring of Chemicals by Japan’s Ministry of the Environment, as well as a large amount of knowledge archived in the survey, have led to the advancement of environmental analysis. Recently, technologies such as non-target liquid chromatography/high resolution mass spectrometry and liquid chromatography with micro bore column have further developed the field. Here, the general strategy of a method developed for the liquid chromatography/mass spectrometry (LC/MS) analysis of environmental chemicals with a brief description is presented. Also, a non-target analysis for the identification of environmental pollutants using a provisional fragment database and “MsMsFilter,” an elemental composition elucidation tool, is presented. This analytical method is shown to be highly effective in the identification of a model chemical, the pesticide Bendiocarb. Our improved micro-liquid chromatography injection system showed substantially enhanced sensitivity to perfluoroalkyl substances, with peak areas 32–71 times larger than those observed in conventional LC/MS. PMID:26819891

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

PubMed Central

Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G.

2017-01-01

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays. PMID:28607052

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.

Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.

PubMed

Konziase, Benetode

2015-08-01

We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

PubMed

Fonseca, L P; Cabral, J M

1996-01-01

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices.

PubMed

Jannot, Guillaume; Vasquez-Rifo, Alejandro; Simard, Martin J

2011-01-01

During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2'-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.

Size-exclusion chromatography system for macromolecular interaction analysis

DOEpatents

Stevens, Fred J.

1988-01-01

A low pressure, microcomputer controlled system employing high performance liquid chromatography (HPLC) allows for precise analysis of the interaction of two reversibly associating macromolecules such as proteins. Since a macromolecular complex migrates faster than its components during size-exclusion chromatography, the difference between the elution profile of a mixture of two macromolecules and the summation of the elution profiles of the two components provides a quantifiable indication of the degree of molecular interaction. This delta profile is used to qualitatively reveal the presence or absence of significant interaction or to rank the relative degree of interaction in comparing samples and, in combination with a computer simulation, is further used to quantify the magnitude of the interaction in an arrangement wherein a microcomputer is coupled to analytical instrumentation in a novel manner.

Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography.

PubMed

Venkatramani, C J; Al-Sayah, Mohammad; Li, Guannan; Goel, Meenakshi; Girotti, James; Zang, Lisa; Wigman, Larry; Yehl, Peter; Chetwyn, Nik

2016-02-01

A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC-SFC in pharmaceutical analysis is presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

PubMed

Sandra, Koen; Steenbeke, Mieke; Vandenheede, Isabel; Vanhoenacker, Gerd; Sandra, Pat

2017-11-10

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development. Copyright © 2017 Elsevier B.V. All rights reserved.

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

Host-Guest Complexes with Protein-Ligand-Like Affinities: Computational Analysis and Design

PubMed Central

Moghaddam, Sarvin; Inoue, Yoshihisa

2009-01-01

It has recently been discovered that guests combining a nonpolar core with cationic substituents bind cucurbit[7]uril (CB[7]) in water with ultra-high affinities. The present study uses the Mining Minima algorithm to study the physics of these extraordinary associations and to computationally test a new series of CB[7] ligands designed to bind with similarly high affinity. The calculations reproduce key experimental observations regarding the affinities of ferrocene-based guests with CB[7] and β-cyclodextrin and provide a coherent view of the roles of electrostatics and configurational entropy as determinants of affinity in these systems. The newly designed series of compounds is based on a bicyclo[2.2.2]octane core, which is similar in size and polarity to the ferrocene core of the existing series. Mining Minima predicts that these new compounds will, like the ferrocenes, bind CB[7] with extremely high affinities. PMID:19133781

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins.

PubMed

Dong, Dexian; Gui, Yanli; Chen, Dezhao; Li, Rongxiu

2008-01-01

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. 2008 John Wiley & Sons, Ltd

Polymer Analysis by Liquid Chromatography/Electrospray Ionization Time-of-Flight Mass Spectrometry.

PubMed

Nielen, M W; Buijtenhuijs, F A

1999-05-01

Hyphenation of liquid chromatography (LC) techniques with electrospray ionization (ESI) orthogonal acceleration time-of-flight (oa-TOF) mass spectrometry (MS) provides both MS-based structural information and LC-based quantitative data in polymer analysis. In one experimental setup, three different LC modes are interfaced with MS:  size-exclusion chromatography (SEC/MS), gradient polymer elution chromatography (GPEC/MS), and liquid chromatography at the critical point of adsorption (LCCC/MS). In SEC/MS, both absolute mass calibration of the SEC column based on the polymer itself and determination of monomers and end groups from the mass spectra are achieved. GPEC/MS shows detailed chemical heterogeneity of the polymer and the chemical composition distribution within oligomer groups. In LCCC/MS, the retention behavior is primarily governed by chemical heterogeneities, such as different end group functionalities, and quantitative end group calculations can be easily made. The potential of these methods and the benefit of time-of-flight analyzers in polymer analysis are discussed using SEC/MS of a polydisperse poly(methyl methacrylate) sample, GPEC/MS of dipropoxylated bisphenol A/adipic acid polyester resin, LCCC/MS of alkylated poly(ethylene glycol), and LCCC/MS of terephthalic acid/neopentyl glycol polyester resin.

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

PubMed

Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

2016-08-26

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Hb Potomac (101 Glu replaced by Asp): speculations on placental oxygen transport in carriers of high-affinity hemoglobins.

PubMed

Charache, S; Jacobson, R; Brimhall, B; Murphy, E A; Hathaway, P; Winslow, R; Jones, R; Rath, C; Simkovich, J

1978-02-01

Blood from a woman with unexplained erythrocytosis had increased oxygen affinity, but no abnormality could be detected by electrophoresis or chromatography of her hemolysate. Separation of the tryptic peptides of her beta chains disclosed two half-sized peaks in the regions of beta T-11. The faster of these was abnormal, with the structure beta 101 Glu replaced by Asp. The new hemoglobin was called "Potomac." Three of the proband's four surviving siblings and both of her children were carriers. Differences in the ratio of carrier: normal children born to male of female carriers of 23 other high-affinity hemoglobins were not significant. The high proportion of carriers in this kindred was probably due to chance alone, and not because high maternal oxygen affinity interfered with oxygen transport to fetuses with normal hemoglobin.

Affinity Interaction between Hexamer Peptide Ligand HWRGWV and Immunoglobulin G Studied by Quartz Crystal Microbalance and Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Shen, Fei

Immunoglobulins (Ig), also referred to as antibodies, act as protective agents against pathogens trying to invade an organism. Human immunoglobulin G (hIgG), as the most prominent immunoglobulin presented in serum and other human fluids, has broad applications in fields like immunotherapy and clinical diagnostics. Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG purifaction and detection. However, drawbacks associated with these two protein ligands have motivated searches for alternative affinity ligands. The hexamer peptide ligand HWRGWV identified from a one-bead-one-peptide combinatorial library synthesized on chromatography resins has demonstrated high affinity and specificity to the Fc fragment of hIgG. A chromatography resin with HWRGWV can purify human IgG (hIgG) from complete minimum essential medium (cMEM) with purities and yields as high as 95%, which are comparable to using Protein A as affinity ligand (4). As a short peptide ligand, HWRGWV can be produced at relatively low costs under good manufacturing practices (GMP) conditions, it is highly robust, less immunogenic and allows for milder elution conditions for the bound antibody (3, 5). Although this short peptide ligand has exhibited promising properties for IgG capture and purification, limited information is available on the intrinsic mechanisms of affinity interaction between the peptide ligand and target protein. In this study, the affinity interaction between hIgG and peptide ligand immobilized on solid surfaces was studied by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). Compared with previous methods employed for the peptide characterization, QCM and SPR can provide direct measurements of equilibrium adsorption isotherms and rates of adsorption, allowing a complete kinetic and thermodynamics analyses of the ligand-target interactions. New methods were developed to modify gold and silica surfaces of QCM and SPR

Chemical and technical challenges in the analysis of central carbon metabolites by liquid-chromatography mass spectrometry.

PubMed

Siegel, David; Permentier, Hjalmar; Reijngoud, Dirk-Jan; Bischoff, Rainer

2014-09-01

This review deals with chemical and technical challenges in the analysis of small-molecule metabolites involved in central carbon and energy metabolism via liquid-chromatography mass-spectrometry (LC-MS). The covered analytes belong to the prominent pathways in biochemical carbon oxidation such as glycolysis or the tricarboxylic acid cycle and, for the most part, share unfavorable properties such as a high polarity, chemical instability or metal-affinity. The topic is introduced by selected examples on successful applications of metabolomics in the clinic. In the core part of the paper, the structural features of important analyte classes such as nucleotides, coenzyme A thioesters or carboxylic acids are linked to "problematic hotspots" along the analytical chain (sample preparation and-storage, separation and detection). We discuss these hotspots from a chemical point of view, covering issues such as analyte degradation or interactions with metals and other matrix components. Based on this understanding we propose solutions wherever available. A major notion derived from these considerations is that comprehensive carbon metabolomics inevitably requires multiple, complementary analytical approaches covering different chemical classes of metabolites. Copyright © 2013 Elsevier B.V. All rights reserved.

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

PubMed

White, Jim F; Grisshammer, Reinhard

2010-09-07

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives

PubMed Central

Edelmann, Mariola J.

2011-01-01

Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide. PMID:22174558

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

PubMed Central

O'Herrin, Sean M.; Lebowitz, Michael S.; Bieler, Joan G.; al-Ramadi, Basel K.; Utz, Ursula; Bothwell, Alfred L.M.; Schneck, Jonathan P.

1997-01-01

Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis

Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.

PubMed

Bresolin, I T L; Borsoi-Ribeiro, M; Tamashiro, W M S C; Augusto, E F P; Vijayalakshmi, M A; Bueno, S M A

2010-04-01

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics.

PubMed

Kahsay, Getu; Song, Huiying; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin

2014-01-01

This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

PubMed

Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

2017-06-23

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

Advances in silver ion chromatography for the analysis of fatty acids and triacylglycerols-2001 to 2011.

PubMed

Momchilova, Svetlana M; Nikolova-Damyanova, Boryana M

2012-01-01

An effort is made to critically present the achievements in silver ion chromatography during the last decade. Novelties in columns, mobile-phase compositions and detectors are described. Recent applications of silver ion chromatography in the analysis of fatty acids and triacylglycerols are presented while stressing novel analytical strategies or new objects. The tendencies in the application of the method in complementary ways with reversed-phase chromatography, chiral chromatography and, especially, mass detection are outlined.

Preparation of high affinity antibody for ribavirin with new haptens and residue analysis in chicken muscle, eggs and duck muscle.

PubMed

Wang, Zhaopeng; Yu, Xuezhi; Ma, Licai; Liu, Hebing; Ding, Shuangyang; Wang, Zhanhui; Zhang, Xiya; Shen, Jianzhong; Wen, Kai

2018-05-23

In this work, high affinity polyclonal antibodies for ribavirin (RBV) from new haptens were prepared and were used to analyse RBV residues in chicken muscle, eggs and duck muscle. The new haptens were synthesised with different spacers, and the best antibody was obtained with an IC 50 value as low as 0.61 ng/mL in indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivities with another five antiviral drugs including amantadine, rimantadine, moroxydine, zanamivir and oseltamivir were less than 0.1%, which indicated the good specificity of the antibody. An ELISA was developed based on the antibody and applied to detect RBV in multi-food matrices. The sample preparation prior to detection only needed simple dilution after trichloroacetic acid extraction. The limits of detection were 1.07, 1.18 and 1.03 μg/kg in chicken muscle, eggs and duck muscle, respectively. Recoveries ranged from 89.0% to 112.7% with coefficients of variation below 13.0%. Ten blind samples of chicken muscle were analysed simultaneously by ELISA and liquid chromatography-tandem mass spectrometry, and a good correlation between the methods was observed. The results indicated that the high affinity antibody could be applied for the simple and fast detection of RBV in multi-food matrices.

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.

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

PubMed

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

2000-10-01

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.

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.

Improved full analytical polygon-based method using Fourier analysis of the three-dimensional affine transformation.

PubMed

Pan, Yijie; Wang, Yongtian; Liu, Juan; Li, Xin; Jia, Jia

2014-03-01

Previous research [Appl. Opt.52, A290 (2013)] has revealed that Fourier analysis of three-dimensional affine transformation theory can be used to improve the computation speed of the traditional polygon-based method. In this paper, we continue our research and propose an improved full analytical polygon-based method developed upon this theory. Vertex vectors of primitive and arbitrary triangles and the pseudo-inverse matrix were used to obtain an affine transformation matrix representing the spatial relationship between the two triangles. With this relationship and the primitive spectrum, we analytically obtained the spectrum of the arbitrary triangle. This algorithm discards low-level angular dependent computations. In order to add diffusive reflection to each arbitrary surface, we also propose a whole matrix computation approach that takes advantage of the affine transformation matrix and uses matrix multiplication to calculate shifting parameters of similar sub-polygons. The proposed method improves hologram computation speed for the conventional full analytical approach. Optical experimental results are demonstrated which prove that the proposed method can effectively reconstruct three-dimensional scenes.

Liquid chromatography/tandem mass spectrometry with fluorous derivatization method for selective analysis of sialyl oligosaccharides.

PubMed

Sakaguchi, Yohei; Hayama, Tadashi; Yoshida, Hideyuki; Itoyama, Miki; Todoroki, Kenichiro; Yamaguchi, Masatoshi; Nohta, Hitoshi

2014-12-15

A separation-oriented derivatization method using a specific fluorous affinity between perfluoroalkyl-containing compounds was applied to selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis of sialyl oligosaccharides. The perfluoroalkyl-labeled sialyl oligosaccharides could be selectively retained on an LC column with the perfluoroalkyl-modified stationary phase and effectively distinguished from non-derivatized species. Sialyl oligosaccharides (3'-sialyllactose, 6'-sialyllactose, sialyllacto-N-tetraose a, sialyllacto-N-tetraose b, sialyllacto-N-tetraose c, and disialyllacto-N-tetraose) were derivatized with 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecylamine via amidation in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (condensation reagent). The obtained derivatives were directly injected onto the fluorous LC column without any pretreatments and then detected by positive electrospray ionization MS/MS. The method enabled accurate determination of the sialyl oligosaccharides in biological samples such as human urine and human milk, because there was no interference with matrix-induced effects during LC/MS/MS analysis. The limits of detection of the examined sialyl oligosaccharides, defined as signal-to-noise (S/N) = 3, were in the range 0.033-0.13 nM. Accuracy in the range 95.6-108% was achieved, and the precision (relative standard deviation) was within 9.4%. This method enabled highly selective and sensitive analysis of sialyl oligosaccharides, enabling accurate measurement of even their trace amounts in biological matrices. The proposed method may prove to be a powerful tool for the analysis of various sialyl oligosaccharides. Copyright © 2014 John Wiley & Sons, Ltd.

Evidence of land plant affinity for the Devonian fossil Protosalvinia (Foerstia)

USGS Publications Warehouse

Romankiw, L.A.; Hatcher, P.G.; Roen, J.B.

1988-01-01

The Devonian plant fossil Protosalvinia (Foerstia) has been examined by solid-state 13C nuclear magnetic resonance spectroscopy (NMR) and pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). Results of these studies reveal that the chemical structure of Protosalvinia is remarkably similar to that of coalified wood. A well-defined phenolic carbon peak in the NMR spectra and the appearance of phenol and alkylated phenols in pyrolysis products are clearly indicative of lignin-like compounds. These data represent significant new information on the chemical nature of Protosalvinia and provide the first substantial organic geochemical evidence for land plant affinity. -Authors

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

Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

PubMed Central

Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

2012-01-01

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

Use and practice of achiral and chiral supercritical fluid chromatography in pharmaceutical analysis and purification.

PubMed

Lemasson, Elise; Bertin, Sophie; West, Caroline

2016-01-01

The interest of pharmaceutical companies for complementary high-performance chromatographic tools to assess a product's purity or enhance this purity is on the rise. The high-throughput capability and economic benefits of supercritical fluid chromatography, but also the "green" aspect of CO2 as the principal solvent, render supercritical fluid chromatography very attractive for a wide range of pharmaceutical applications. The recent reintroduction of new robust instruments dedicated to supercritical fluid chromatography and the progress in stationary phase technology have also greatly benefited supercritical fluid chromatography. Additionally, it was shown several times that supercritical fluid chromatography could be orthogonal to reversed-phase high-performance liquid chromatography and could efficiently compete with it. Supercritical fluid chromatography is an adequate tool for small molecules of pharmaceutical interest: synthetic intermediates, active pharmaceutical ingredients, impurities, or degradation products. In this review, we first discuss about general chromatographic conditions for supercritical fluid chromatography analysis to better suit compounds of pharmaceutical interest. We also discuss about the use of achiral and chiral supercritical fluid chromatography for analytical purposes and the recent applications in these areas. The use of preparative supercritical fluid chromatography by pharmaceutical companies is also covered. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A dual protease approach for expression and affinity purification of recombinant proteins.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

A Dual Protease Approach for Expression and Affinity Purification of Recombinant Proteins

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2016-01-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification. PMID:27105777

Determination of lipid bilayer affinities and solvation characteristics by electrokinetic chromatography using polymer-bound lipid bilayer nanodiscs.

PubMed

Penny, William M; Palmer, Christopher P

2018-03-01

Styrene-maleic acid polymer-bound lipid bilayer nanodiscs have been investigated and characterized by electrokinetic chromatography. Linear solvation energy relationship analysis was employed to characterize the changes in solvation environment of nanodiscs of varied belt to lipid ratio, belt polymer chemistry and molecular weight, and lipid composition. Increases in the lipid to belt polymer ratio resulted in smaller, more cohesive nanodiscs with greater electrophoretic mobility. Nanodisc structures with belt polymers of different chemistry and molecular weight were compared and showed only minor changes in solvent characteristics and selectivity consistent with changes in structure of the lipid bilayer. Seven phospholipid and sphingomyelin nanodiscs of different lipid composition were characterized. Changes in lipid head group structure had a significant effect on bilayer-solute interactions. In most cases, changes in alkyl tail structure had no discernible effect on solvation environment aside from those explained by changes in the gel-liquid transition temperature. Comparison to vesicles of similar lipid composition show only minor differences in solvation environment, likely due to differences in lipid composition and bilayer curvature. Together these results provide evidence that the dominant solute-nanodisc interactions are with the lipid bilayer and that head group chemistry has a greater impact on bilayer-solute interactions than alkyl tail or belt polymer structure. Nanodisc electrokinetic chromatography is demonstrated to allow characterization of solute interactions with lipid bilayers of varied composition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

PubMed

Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

2015-07-01

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

Separation techniques: Chromatography

PubMed Central

Coskun, Ozlem

2016-01-01

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

Industrial application of green chromatography - II. Separation and analysis of preservatives in skincare products using subcritical water chromatography.

PubMed

Yang, Y; Kapalavavi, B; Gujjar, L; Hadrous, S; Marple, R; Gamsky, C

2012-10-01

Several high-temperature liquid chromatography (HTLC) and subcritical water chromatography (SBWC) methods have been successfully developed in this study for separation and analysis of preservatives contained in Olay skincare creams. Efficient separation and quantitative analysis of preservatives have been achieved on four commercially available ZirChrom and Waters XBridge columns at temperatures ranging from 100 to 200°C. The quantification results obtained by both HTLC and SBWC methods developed for preservatives analysis are accurate and reproducible. A large number of replicate HTLC and SBWC runs also indicate no significant system building-up or interference for skincare cream analysis. Compared with traditional HPLC separation carried out at ambient temperature, the HTLC methods can save up to 90% methanol required in the HPLC mobile phase. However, the SBWC methods developed in this project completely eliminated the use of toxic organic solvents required in the HPLC mobile phase, thus saving a significant amount of money and making the environment greener. Although both homemade and commercial systems can accomplish SBWC separations, the SBWC methods using the commercial system for preservative analysis are recommended for industrial applications because they can be directly applied in industrial plant settings. © 2012 The Authors ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

High-pressure liquid chromatography analysis of antibiotic susceptibility disks.

PubMed Central

Hagel, R B; Waysek, E H; Cort, W M

1979-01-01

The analysis of antibiotic susceptibility disks by high-pressure liquid chromatography (HPLC) was investigated. Methods are presented for the potency determination of mecillinam, ampicillin, carbenicillin, and cephalothin alone and in various combinations. Good agreement between HPLC and microbiological data is observed for potency determinations with recoveries of greater than 95%. Relative standard deviations of lower than 2% are recorded for each HPLC method. HPLC methods offer improved accuracy and greater precision when compared to the standard microbiological methods of analysis for susceptibility disks. PMID:507793

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

PubMed

Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

2014-01-01

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers.

Application in pesticide analysis: Liquid chromatography - A review of the state of science for biomarker discovery and identification

EPA Science Inventory

Book Chapter 18, titled Application in pesticide analysis: Liquid chromatography - A review of the state of science for biomarker discovery and identification, will be published in the book titled High Performance Liquid Chromatography in Pesticide Residue Analysis (Part of the C...

Identification and Quantification of Pesticides in Environmental Waters With Solid Phase Microextraction and Analysis Using Field-Portable Gas Chromatography-Mass Spectrometry

DTIC Science & Technology

2004-06-10

Microextraction and Analysis using Field-Portable Gas Chromatography-Mass Spectrometry Name of Candidate: CPT Michael J. Nack...and Analysis using Field-Portable Gas Chromatography-Mass Spectrometry Beyond brief excerpts is with the permission of the copyright owner, and...Pesticides in Environmental Waters with Solid Phase Microextraction and Analysis using Field-Portable Gas Chromatography-Mass Spectrometry

Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications.

PubMed

Shetty, Dinesh; Khedkar, Jayshree K; Park, Kyeng Min; Kim, Kimoon

2015-12-07

The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.

Lp-dual affine surface area

NASA Astrophysics Data System (ADS)

Wei, Wang; Binwu, He

2008-12-01

According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

Qualitative Analysis of Analgesic Tablets: An Experiment Employing High Pressure Liquid Chromatography.

ERIC Educational Resources Information Center

Beaver, Rodney W.; And Others

1983-01-01

Describes an experiment on the qualitative analysis of several over-the-counter analgesic tablets. Background information, procedures used (including high pressure liquid chromatography), and typical student results are included. (JN)

Analysis of lignans in Magnoliae Flos by turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry.

PubMed

Zhou, Xuan; Chen, Cen; Ye, Xiaolan; Song, Fenyun; Fan, Guorong; Wu, Fuhai

2016-04-01

In this study, a method coupling turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry was developed for analyzing the lignans in Magnoliae Flos. By the online pretreatment of turbulent flow chromatography solid-phase extraction, the impurities removal and analytes concentration were automatically processed, and the lignans were separated rapidly and well. Seven lignans of Magnoliae Flos including epieudesmin, magnolin, 1-irioresinol-B-dimethyl ether, epi-magnolin, fargesin aschantin, and demethoxyaschantin were identified by comparing their retention behavior, UV spectra, and mass spectra with those of reference substances or literature data. The developed method was validated, and the good results showed that the method was not only automatic and rapid, but also accurate and reliable. The turbulent flow chromatography with online solid-phase extraction and high-performance liquid chromatography with tandem mass spectrometry method holds a high potential to become an effective method for the quality control of lignans in Magnoliae Flos and a useful tool for the analysis of other complex mixtures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Introduction of structural affinity handles as a tool in selective nucleic acid separations

NASA Technical Reports Server (NTRS)

Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

2011-01-01

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

NASA Technical Reports Server (NTRS)

Knisley, Keith A.; Rodkey, L. Scott

1986-01-01

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study

PubMed Central

Jamalapuram, Seshulatha; Vuppala, Pradeep K.; Mesangeau, Christophe; McCurdy, Christopher R.; Avery, Bonnie A.

2014-01-01

A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLCTM BEH HILIC column (1.7 µm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats. PMID:22406103

Use of T-2 toxin-immobilized amine-activated beads as an efficient affinity purification matrix for the isolation of specific IgY.

PubMed

Edupuganti, Soujanya Ratna; Edupuganti, Om Prakash; O'Kennedy, Richard; Defrancq, Eric; Boullanger, Stéphanie

2013-04-01

An affinity purification method that isolates T-2 toxin-specific IgY utilizing a T-2-toxin-immobilized column was developed. The T-2 toxin was covalently coupled via a carbonyldiimidazole-activated hydroxyl functional group to amine-activated sepharose beads. The affinity-purified IgY was characterized by gel electrophoresis, fast protein liquid chromatography, enzyme-linked immunosorbant assay, surface plasmon resonance and mass spectrometry. A competitive inhibition ELISA (CI-ELISA) was performed using affinity-purified IgY with a T-2 toxin detection sensitivity of 30 ng/mL, which falls within the maximum permissible limit of 100 ng/mL. The cross reactivity of IgY towards deoxynivalenol, zearalenone, fumonisin B1 and HT-2 was significantly reduced after affinity purification. A surface plasmon resonance (SPR)-based inhibition assay was also applied for quantitative determination of T-2 toxin in spiked wheat samples. The results obtained indicate the feasibility of utilizing this IgY-based assay for the detection of T-2 toxin in food samples.

Classification of neocortical interneurons using affinity propagation.

PubMed

Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

2013-01-01

In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

Using Single Drop Microextraction for Headspace Analysis with Gas Chromatography

ERIC Educational Resources Information Center

Riccio, Daniel; Wood, Derrick C.; Miller, James M.

2008-01-01

Headspace (HS) gas chromatography (GC) is commonly used to analyze samples that contain non-volatiles. In 1996, a new sampling technique called single drop microextraction, SDME, was introduced, and in 2001 it was applied to HS analysis. It is a simple technique that uses equipment normally found in the undergraduate laboratory, making it ideal…

[High-performance liquid-liquid chromatography in beverage analysis].

PubMed

Bricout, J; Koziet, Y; de Carpentrie, B

1978-01-01

Liquid liquid chromatography was performed with columns packed with stationary phases chemically bonded to silica microparticules. These columns show a high efficiency and are used very easily. Flavouring compounds like aromatic aldehydes which have a low volatility were analyzed in brandy using a polar phase alkylnitrile. Sapid substances like amarogentin in Gentiana lutea or glyryrrhizin in Glycyrrhiza glabra were determined by reversed phase chromatography. Finally ionizable substances like synthetic dyes can be analyzed by paired ion chromatography witha non polar stationary phase.

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

Structural analysis of commercial ceramides by gas chromatography-mass spectrometry.

PubMed

Bleton, J; Gaudin, K; Chaminade, P; Goursaud, S; Baillet, A; Tchapla, A

2001-05-11

A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.

Comparative Normal/Failing Rat Myocardium Cell Membrane Chromatographic Analysis System for Screening Specific Components That Counteract Doxorubicin-Induced Heart Failure from Acontium carmichaeli

PubMed Central

2015-01-01

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K+ channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models. PMID:24731167

Measurements of the talus in the assessment of population affinity.

PubMed

Bidmos, Mubarak A; Dayal, Manisha R; Adegboye, Oyelola A

2018-06-01

As part of their routine work, forensic anthropologists are expected to report population affinity as part of the biological profile of an individual. The skull is the most widely used bone for the estimation of population affinity but it is not always present in a forensic case. Thus, other bones that preserve well have been shown to give a good indication of either the sex or population affinity of an individual. In this study, the potential of measurements of the talus was investigated for the purpose of estimating population affinity in South Africans. Nine measurements from two hundred and twenty tali of South African Africans (SAA) and South African Whites (SAW) from the Raymond A. Dart Collection of Human Skeletons were used. Direct and step-wise discriminant function and logistic regression analyses were carried out using SPSS and SAS. Talar length was the best single variable for discriminating between these two groups for males while in females the head height was the best single predictor. Average accuracies for correct population affinity classification using logistic regression analysis were higher than those obtained from discriminant function analysis. This study was the first of its type to employ discriminant function analyses and logistic regression analyses to estimate the population affinity of an individual from the talus. Thus these equations can now be used by South African anthropologists when estimating the population affinity of dismembered or damaged or incomplete skeletal remains of SAA and SAW. Copyright © 2018 Elsevier B.V. All rights reserved.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterisation of odorants in roasted stem tea using gas chromatography-mass spectrometry and gas chromatography-olfactometry analysis.

PubMed

Sasaki, Tetsuya; Koshi, Erina; Take, Harumi; Michihata, Toshihide; Maruya, Masachika; Enomoto, Toshiki

2017-04-01

Roasted stem tea has a characteristic flavour, which is obtained by roasting tea stems, by-product of green tea production. This research aims to understand the characteristic odorants in roasted stem tea by comparing it to roasted leaf tea. We revealed potent odorants in commercial roasted stem tea using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry with aroma extract dilution analysis (AEDA). The difference between roasted stem and leaf tea derived from the same tea plants were investigated using GC-MS. Pyrazine compounds exhibited a roasted odour and high flavour dilution (FD) factors, as determined via AEDA. Roasted stem tea was richer in these pyrazines than roasted leaf tea. Geraniol and linalool exhibited high FD factors and a floral odour, and roasted stem tea was richer in these compounds than roasted leaf tea. These results may have a positive impact on the development of tea products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gas-liquid chromatography in lunar organic analysis.

NASA Technical Reports Server (NTRS)

Gehrke, C. W.

1972-01-01

Gas-liquid chromatography (GLC) is a powerful and sensitive method for the separation and detection of organic compounds at nanogram levels. The primary requirement for successful analyses is that the compounds of interest must be volatile under the chromatographic conditions employed. Nonvolatile organic compounds must be converted to volatile derivatives prior to analysis. The derivatives of choice must be both amenable to chromatographic separation and be relatively stable. The condition of volatility necessitates the development of efficient derivatization reactions for important groups of compounds as amino acids, carbohydrates, nucleosides, etc. Trimethylsilylation and trifluoroacetylation represent specific areas of recent prominence. Some relevant practical aspects of GLC are discussed.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

PubMed Central

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

Diagnosis of gastroenterological diseases by metabolome analysis using gas chromatography-mass spectrometry.

PubMed

Yoshida, Masaru; Hatano, Naoya; Nishiumi, Shin; Irino, Yasuhiro; Izumi, Yoshihiro; Takenawa, Tadaomi; Azuma, Takeshi

2012-01-01

Recently, metabolome analysis has been increasingly applied to biomarker detection and disease diagnosis in medical studies. Metabolome analysis is a strategy for studying the characteristics and interactions of low molecular weight metabolites under a specific set of conditions and is performed using mass spectrometry and nuclear magnetic resonance spectroscopy. There is a strong possibility that changes in metabolite levels reflect the functional status of a cell because alterations in their levels occur downstream of DNA, RNA, and protein. Therefore, the metabolite profile of a cell is more likely to represent the current status of a cell than DNA, RNA, or protein. Thus, owing to the rapid development of mass spectrometry analytical techniques metabolome analysis is becoming an important experimental method in life sciences including the medical field. Here, we describe metabolome analysis using liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis-mass spectrometry, and matrix assisted laser desorption ionization-mass spectrometry. Then, the findings of studies about GC-MS-based metabolome analysis of gastroenterological diseases are summarized, and our research results are also introduced. Finally, we discuss the realization of disease diagnosis by metabolome analysis. The development of metabolome analysis using mass spectrometry will aid the discovery of novel biomarkers, hopefully leading to the early detection of various diseases.

Affinity and Efficacy Studies of Tetrahydrocannabinolic Acid A at Cannabinoid Receptor Types One and Two.

PubMed

McPartland, John M; MacDonald, Christa; Young, Michelle; Grant, Phillip S; Furkert, Daniel P; Glass, Michelle

2017-01-01

Introduction: Cannabis biosynthesizes Δ 9 -tetrahydrocannabinolic acid (THCA-A), which decarboxylates into Δ 9 -tetrahydrocannabinol (THC). There is growing interest in the therapeutic use of THCA-A, but its clinical application may be hampered by instability. THCA-A lacks cannabimimetic effects; we hypothesize that it has little binding affinity at cannabinoid receptor 1 (CB 1 ). Materials and Methods: Purity of certified reference standards were tested with high performance liquid chromatography (HPLC). Binding affinity of THCA-A and THC at human (h) CB 1 and hCB 2 was measured in competition binding assays, using transfected HEK cells and [ 3 H]CP55,940. Efficacy at hCB 1 and hCB 2 was measured in a cyclic adenosine monophosphase (cAMP) assay, using a Bioluminescence Resonance Energy Transfer (BRET) biosensor. Results: The THCA-A reagent contained 2% THC. THCA-A displayed small but measurable binding at both hCB 1 and hCB 2 , equating to approximate K i values of 3.1μM and 12.5μM, respectively. THC showed 62-fold greater affinity at hCB 1 and 125-fold greater affinity at hCB 2 . In efficacy tests, THCA-A (10μM) slightly inhibited forskolin-stimulated cAMP at hCB 1 , suggestive of weak agonist activity, and no measurable efficacy at hCB 2 . Discussion: The presence of THC in our THCA-A certified standard agrees with decarboxylation kinetics (literature reviewed herein), which indicate contamination with THC is nearly unavoidable. THCA-A binding at 10μM approximated THC binding at 200nM. We therefore suspect some of our THCA-A binding curve was artifact-from its inevitable decarboxylation into THC-and the binding affinity of THCA-A is even weaker than our estimated values. We conclude that THCA-A has little affinity or efficacy at CB 1 or CB 2 .

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. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry.

PubMed

Liu, Ning; Lu, Xin; Yang, YuHan; Yao, Chen Xi; Ning, BaoMing; He, Dacheng; He, Lan; Ouyang, Jin

2015-10-01

A new approach for monitoring the binding affinity between drugs and alpha 1-acid glycoprotein in real time was developed based on a combination of drug-protein reaction followed by Venturi easy ambient sonic-spray ionization mass spectrometry determination of the free drug concentrations. A known basic drug, propranolol was used to validate the new built method. Binding constant values calculated by venturi easy ambient sonic-spray ionization mass spectrometry was in good accordance with a traditional ultrafiltration combined with high performance liquid chromatography method. Then six types of basic drugs were used as the samples to conduct the real time analysis. Upon injection of alpha 1-acid glycoprotein to the drug mixture, the ion chromatograms were extracted to show the changes in the free drug concentrations in real time. By observing the drop-out of six types of drugs during the whole binding reaction, the binding affinities of different drugs were distinguished. A volume shift validating experiment and an injection delay correcting experiment were also performed to eliminate extraneous factors and verify the reliability of our experiment. Therefore, the features of Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS) and the experimental results indicate that our technique is likely to become a powerful tool for monitoring drug-AGP binding affinity in real time. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid quantitative analysis of 8-iso-prostaglandin-F(2alpha) using liquid chromatography-tandem mass spectrometry and comparison with an enzyme immunoassay method.

PubMed

Dahl, Jeffrey H; van Breemen, Richard B

2010-09-15

A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the measurement of urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)), a biomarker of lipid peroxidation. Because urine contains numerous F(2) prostaglandin isomers, each with identical mass and similar mass spectrometric fragmentation patterns, chromatographic separation of 8-iso-PGF(2alpha) from its isomers is necessary for its quantitative analysis using MS/MS. We were able to achieve this separation using an isocratic LC method with a run time of less than 9min, which is at least threefold faster than previous methods, while maintaining sensitivity, accuracy, precision, and reliability. The limits of detection and quantitation were 53 and 178pg/ml urine, respectively. We compared our method with a commercially available affinity purification and enzyme immunoassay kit and found both assays to be in agreement. Despite the high sensitivity of the enzyme immunoassay method, it is more expensive and has a narrower dynamic range than LC-MS/MS. Our method was optimized for rapid measurement of 8-iso-PGF(2alpha) in urine, and it is ideally suited for clinical sample analysis. 2010 Elsevier Inc. All rights reserved.

Analysis of new psychoactive substances in human urine by ultra-high performance supercritical fluid and liquid chromatography: Validation and comparison.

PubMed

Borovcová, Lucie; Pauk, Volodymyr; Lemr, Karel

2018-05-01

New psychoactive substances represent serious social and health problem as tens of new compounds are detected in Europe annually. They often show structural proximity or even isomerism, which complicates their analysis. Two methods based on ultra high performance supercritical fluid chromatography and ultra high performance liquid chromatography with mass spectrometric detection were validated and compared. A simple dilute-filter-and-shoot protocol utilizing propan-2-ol or methanol for supercritical fluid or liquid chromatography, respectively, was proposed to detect and quantify 15 cathinones and phenethylamines in human urine. Both methods offered fast separation (<3 min) and short total analysis time. Precision was well <15% with a few exceptions in liquid chromatography. Limits of detection in urine ranged from 0.01 to 2.3 ng/mL, except for cathinone (5 ng/mL) in supercritical fluid chromatography. Nevertheless, this technique distinguished all analytes including four pairs of isomers, while liquid chromatography was unable to resolve fluoromethcathinone regioisomers. Concerning matrix effects and recoveries, supercritical fluid chromatography produced more uniform results for different compounds and at different concentration levels. This work demonstrates the performance and reliability of supercritical fluid chromatography and corroborates its applicability as an alternative tool for analysis of new psychoactive substances in biological matrixes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

COMPARATIVE EVALUATION OF GC/MS (GAS CHROMATOGRAPHY/MASS SPECTROMETRY) DATA ANALYSIS PROCESSING

EPA Science Inventory

Mass spectra obtained by fused silica capillary gas chromatography/mass spectrometry/data system (GC/MS/DS) analysis of mixtures of organic chemicals adsorbed on Tenax GC cartridges was subjected to manual and automated interpretative techniques. Synthetic mixtures (85 chemicals ...

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.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Comprehensive two-dimensional gas chromatography applied to illicit drug analysis.

PubMed

Mitrevski, Blagoj; Wynne, Paul; Marriott, Philip J

2011-11-01

Multidimensional gas chromatography (MDGC), and especially its latest incarnation--comprehensive two-dimensional gas chromatography (GC × GC)--have proved advantageous over and above classic one-dimensional gas chromatography (1D GC) in many areas of analysis by offering improved peak capacity, often enhanced sensitivity and, especially in the case of GC × GC, the unique feature of 'structured' chromatograms. This article reviews recent advances in MDGC and GC × GC in drug analysis with special focus on ecstasy, heroin and cocaine profiling. Although 1D GC is still the method of choice for drug profiling in most laboratories because of its simplicity and instrument availability, GC × GC is a tempting proposition for this purpose because of its ability to generate a higher net information content. Effluent refocusing due to the modulation (compression) process, combined with the separation on two 'orthogonal' columns, results in more components being well resolved and therefore being analytically and statistically useful to the profile. The spread of the components in the two-dimensional plots is strongly dependent on the extent of retention 'orthogonality' (i.e. the extent to which the two phases possess different or independent retention mechanisms towards sample constituents) between the two columns. The benefits of 'information-driven' drug profiling, where more points of reference are usually required for sample differentiation, are discussed. In addition, several limitations in application of MDGC in drug profiling, including data acquisition rate, column temperature limit, column phase orthogonality and chiral separation, are considered and discussed. Although the review focuses on the articles published in the last decade, a brief chronological preview of the profiling methods used throughout the last three decades is given.

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Identification and measurement of chlorinated organic pesticides in water by electron-capture gas chromatography

USGS Publications Warehouse

Lamar, William L.; Goerlitz, Donald F.; Law, LeRoy M.

1965-01-01

Pesticides, in minute quantities, may affect the regimen of streams, and because they may concentrate in sediments, aquatic organisms, and edible aquatic foods, their detection and their measurement in the parts-per-trillion range are considered essential. In 1964 the U.S. Geological Survey at Menlo Park, Calif., began research on methods for monitoring pesticides in water. Two systems were selected--electron-capture gas chromatography and microcoulometric-titration gas chromatography. Studies on these systems are now in progress. This report provides current information on the development and application of an electron-capture gas chromatographic procedure. This method is a convenient and extremely sensitive procedure for the detection and measurement of organic pesticides having high electron affinities, notably the chlorinated organic pesticides. The electron-affinity detector is extremely sensitive to these substances but it is not as sensitive to many other compounds. By this method, the chlorinated organic pesticide may be determined on a sample of convenient size in concentrations as low as the parts-per-trillion range. To insure greater accuracy in the identifications, the pesticides reported were separated and identified by their retention times on two different types of gas chromatographic columns.

Direct optimization, affine gap costs, and node stability.

PubMed

Aagesen, Lone

2005-09-01

The outcome of a phylogenetic analysis based on DNA sequence data is highly dependent on the homology-assignment step and may vary with alignment parameter costs. Robustness to changes in parameter costs is therefore a desired quality of a data set because the final conclusions will be less dependent on selecting a precise optimal cost set. Here, node stability is explored in relationship to separate versus combined analysis in three different data sets, all including several data partitions. Robustness to changes in cost sets is measured as number of successive changes that can be made in a given cost set before a specific clade is lost. The changes are in all cases base change cost, gap penalties, and adding/removing/changing affine gap costs. When combining data partitions, the number of clades that appear in the entire parameter space is not remarkably increased, in some cases this number even decreased. However, when combining data partitions the trees from cost sets including affine gap costs were always more similar than the trees were from cost sets without affine gap costs. This was not the case when the data partitions were analyzed independently. When data sets were combined approximately 80% of the clades found under cost sets including affine gap costs resisted at least one change to the cost set.

[Column chromatography purification and analysis of biodiesel by transesterification].

PubMed

Liu, Yang; Yi, Huai-feng; Chen, Yu; Wu, Yu-long; Yang, Ming-de; Chen, Zeng; Tong, Jun-mao

2012-02-01

In the present paper, crude biodiesel prepared with sorbifolia oil as raw material by transesterification was purified by column chromatography, then the composition of biodiesel was analyzed by gas chromatography, FTIR, GC-MS and 1H NMR. Column chromatography can separate the crude biodiesel into two fractions: petroleum ether eluted fraction (A1) and methanol eluted fraction (A2). Petroleum ether eluted fraction was mainly biodiesel fraction, which was produced from sorbifolia oil by transesterification, including methyl linoleate, methyl cis-9-octadecenoate and so on; methanol eluted fraction was mainly glycerol fraction, which came from the side reaction of transesterification. The results show that the purity of refined biodiesel increased from 77.51% to 93.872, and the product recovery rate reached up to 91.04% after the purification by column chromatography. The results obtained by FTIR and 1H NMR further showed that the column chromatography can effectively improve the purity of biodiesel. This paper provides a basis for industrialization of purification of biodiesel.

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PubMed Central

Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.

PubMed

Park, Seong-Jun; Ahn, Hee-Sung; Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

Adjoint affine fusion and tadpoles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urichuk, Andrew, E-mail: andrew.urichuk@uleth.ca; Walton, Mark A., E-mail: walton@uleth.ca; International School for Advanced Studies

2016-06-15

We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are writtenmore » for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.« less

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization

Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry.

PubMed

Chen, Guilin; Huang, Bill X; Guo, Mingquan

2018-05-21

Medicinal plants have played an important role in maintaining human health for thousands of years. However, the interactions between the active components in medicinal plants and some certain biological targets during a disease are still unclear in most cases. To conduct the high-throughput screening for small active molecules that can interact with biological targets, which is of great theoretical significance and practical value. The ultrafiltration mass spectrometry (UF-LC/MS) is a powerful bio-analytical method by combining affinity ultrafiltration and liquid chromatography-mass spectrometry (LC/MS), which could rapidly screen and identify small active molecules that bind to biological targets of interest at the same time. Compared with other analytical methods, affinity UF-LC/MS has the characteristics of fast, sensitive and high throughput, and is especially suitable for the complicated extracts of medicinal plants. In this review, the basic principle, characteristics and some most recent challenges in UF-LC/MS have been demonstrated. Meanwhile, the progress and applications of affinity UF-LC/MS in the discovery of the active components from natural medicinal plants and the interactions between small molecules and biological target proteins are also briefly summarised. In addition, the future directions for UF-LC/MS are also prospected. Affinity UF-LC/MS is a powerful tool in studies on the interactions between small active molecules and biological protein targets, especially in the high-throughput screening of active components from the natural medicinal plants. Copyright © 2018 John Wiley & Sons, Ltd.

Gas Chromatography

NASA Astrophysics Data System (ADS)

Qian, Michael C.

Gas chromatography (GC) has many applications in the analysis of food products. GC has been used for the determination of fatty acids, triglycerides, cholesterol, gases, water, alcohols, pesticides, flavor compounds, and many more. While GC has been used for other food components such as sugars, oligosaccharides, amino acids, peptides, and vitamins, these substances are more suited to analysis by high performance liquid chromatography. GC is ideally suited to the analysis of volatile substances that are thermally stable. Substances such as pesticides and flavor compounds that meet these criteria can be isolated from a food and directly injected into the GC. For compounds that are thermally unstable, too low in volatility, or yield poor chromatographic separation due to polarity, a derivatization step must be done before GC analysis. The two parts of the experiment described here include the analysis of alcohols that requires no derivatization step, and the analysis of fatty acids which requires derivatization. The experiments specify the use of capillary columns, but the first experiment includes conditions for a packed column.

Comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones.

PubMed

Carnes, Stephanie; O'Brien, Stacey; Szewczak, Angelica; Tremeau-Cayel, Lauriane; Rowe, Walter F; McCord, Bruce; Lurie, Ira S

2017-09-01

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite-5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

PubMed

Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

2017-01-01

In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

Exploring Girls' Science Affinities Through an Informal Science Education Program

NASA Astrophysics Data System (ADS)

Todd, Brandy; Zvoch, Keith

2017-10-01

This study examines science interests, efficacy, attitudes, and identity—referred to as affinities, in the context of an informal science outreach program for girls. A mixed methods design was used to explore girls' science affinities before, during, and after participation in a cohort-based summer science camp. Multivariate analysis of survey data revealed that girls' science affinities varied as a function of the joint relationship between family background and number of years in the program, with girls from more affluent families predicted to increase affinities over time and girls from lower income families to experience initial gains in affinities that diminish over time. Qualitative examination of girls' perspectives on gender and science efficacy, attitudes toward science, and elements of science identities revealed a complex interplay of gendered stereotypes of science and girls' personal desires to prove themselves knowledgeable and competent scientists. Implications for the best practice in fostering science engagement and identities in middle school-aged girls are discussed.

Quantifying protein microstructure and electrostatic effects on the change in Gibbs free energy of binding in immobilized metal affinity chromatography.

PubMed

Pathange, Lakshmi P; Bevan, David R; Zhang, Chenming

2008-03-01

Electrostatic forces play a major role in maintaining both structural and functional properties of proteins. A major component of protein electrostatics is the interactions between the charged or titratable amino acid residues (e.g., Glu, Lys, and His), whose pK(a) (or the change of the pK(a)) value could be used to study protein electrostatics. Here, we report the study of electrostatic forces through experiments using a well-controlled model protein (T4 lysozyme) and its variants. We generated 10 T4 lysozyme variants, in which the electrostatic environment of the histidine residue was perturbed by altering charged and neutral amino acid residues at various distances from the histidine (probe) residue. The electrostatic perturbations were theoretically quantified by calculating the change in free energy (DeltaDeltaG(E)) using Coulomb's law. On the other hand, immobilized metal affinity chromatography (IMAC) was used to quantify these perturbations in terms of protein binding strength or change in free energy of binding (DeltaDeltaG(B)), which varies from -0.53 to 0.99 kcal/mol. For most of the variants, there is a good correlation (R(2) = 0.97) between the theoretical DeltaDeltaG(E) and experimental DeltaDeltaG(B) values. However, there are three deviant variants, whose histidine residue was found to be involved in site-specific interactions (e.g., ion pair and steric hindrance), which were further investigated by molecular dynamics simulation. This report demonstrates that the electrostatic (DeltaDeltaG(Elec)) and microstructural effects (DeltaDeltaG(Micro)) in a protein can be quantified by IMAC through surface histidine mediated protein-metal ion interaction and that the unique microstructure around a histidine residue can be identified by identifying the abnormal binding behaviors during IMAC.

Non-affine deformations in polymer hydrogels

PubMed Central

Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

2012-01-01

Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

Analysis and Identification of Acid-Base Indicator Dyes by Thin-Layer Chromatography

ERIC Educational Resources Information Center

Clark, Daniel D.

2007-01-01

Thin-layer chromatography (TLC) is a very simple and effective technique that is used by chemists by different purposes, including the monitoring of the progress of a reaction. TLC can also be easily used for the analysis and identification of various acid-base indicator dyes.

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

PubMed Central

Field, Christopher R.; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C.; Rose-Pehrsson, Susan L.

2014-01-01

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples. PMID:25145416

Quantitative detection of trace explosive vapors by programmed temperature desorption gas chromatography-electron capture detector.

PubMed

Field, Christopher R; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C; Rose-Pehrsson, Susan L

2014-07-25

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

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)

Lp-mixed affine surface area

NASA Astrophysics Data System (ADS)

Wang, Weidong; Leng, Gangsong

2007-11-01

According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Meta-analysis of cannabinoid ligand binding affinity and receptor distribution: interspecies differences

PubMed Central

McPartland, J M; Glass, M; Pertwee, R G

2007-01-01

A meta-analysis, unlike a literature review, synthesizes previous studies into new results. Pooled data from 211 studies measured ligand binding affinities at human (Hs) or rat (Rn) cannabinoid receptors CB1 and CB2. Cochrane methods were modified for this non-clinical analysis. Meta-regression detected data heterogeneity arising from methodological factors: use of sectioned tissues, lack of PMSF and choice of radioligand. Native brain tissues exhibited greater affinity (lower nM) than transfected cells, but the trend fell short of significance, as did the trend between centrifugation and filtration methods. Correcting for heterogeneity, mean Ki values for Δ9-tetrahydrocannabinol differed significantly between HsCB1 and RnCB1 (25.1 and 42.6 nM, respectively) but not between HsCB1 and HsCB2 (25.1 and 35.2). Mean Kd values for HsCB1, RnCB1 and HsCB2 of CP55,940 (2.5, 0.98, 0.92) and WIN55,212-2 (16.7, 2.4, 3.7) differed between HsCB1 and RnCB1 and between HsCB1 and HsCB2. SR141716A differed between HsCB1 and RnCB1 (2.9 and 1.0 nM). Anandamide at HsCB1, RnCB1 and HsCB2 (239.2, 87.7, 439.5) fell short of statistical differences due to heterogeneity. We consider these Kd and Ki values to be the most valid estimates in the literature. Sensitivity analyses did not support the numerical validity of cannabidiol, cannabinol, 2-arachidonoyl glycerol and all ligands at RnCB2. Aggregate rank order analysis of CB1 distribution in the brain (pooled from 119 autoradiographic, immunohistochemical and in situ hybridization studies) showed denser HsCB1 expression in cognitive regions (cerebral cortex) compared to RnCB1, which was relatively richer in movement-associated areas (cerebellum, caudate-putamen). Implications of interspecies differences are discussed. PMID:17641667

Cytotoxicity of seven bisphenol analogues compared to bisphenol A and relationships with membrane affinity data.

PubMed

Russo, Giacomo; Capuozzo, Antonella; Barbato, Francesco; Irace, Carlo; Santamaria, Rita; Grumetto, Lucia

2018-06-01

Bisphenol A (BPA) is a chemical used in numerous industrial applications. Due to its well ascertained toxicity as endocrine disruptor, industries have started to replace it with other bisphenols whose alleged greater safety is scarcely supported by literature studies. In this study, the toxicity of seven BPA analogues was evaluated using both in silico and in vitro techniques, as compared to BPA toxicity. Furthermore, their affinity indexes for phospholipids (i.e. phospholipophilicity) were determined by immobilized artificial membrane liquid chromatography (IAM-LC) and possible relationships with in vitro toxic activity were also investigated. The results on four different cell cultures yielded similar ranking of toxicity for the bisphenols considered, with IC 50 values confirming their poor acute toxicity. As compared to BPA, bisphenol AF, bisphenol B, bisphenol M, and bisphenol A diglycidyl ether resulted more toxic, while bisphenol S, bisphenol F and bisphenol E were found as the less toxic congeners. These results are partly consistent with the scale of phospholipid affinity showing that toxicity increases at increasing membrane affinity. Therefore, phospholipophilicity determination can be assumed as a useful preliminary tool to select less toxic congeners to surrogate BPA in industrial applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

High Performance Liquid Chromatography of Some Analgesic Compounds: An Instrumental Analysis Experiment.

ERIC Educational Resources Information Center

Haddad, Paul; And Others

1983-01-01

Background information, procedures, and results are provided for an experiment demonstrating techniques of solvent selection, gradient elution, pH control, and ion-pairing in the analysis of an analgesic mixture using reversed-phase liquid chromatography on an octadecylsilane column. Although developed using sophisticated/expensive equipment, less…

ANALYSIS OF FERRIC AND FERROUS IONS IN SOIL EXTRACTS BY ION CHROMATOGRAPHY

EPA Science Inventory

A method using ion chromatography (IC) for the analysis of ferrous (Fe 2+) and ferric (Fe 3+) ions in soil extracts has been developed. This method uses an ion exchange column with detection at 520 nm after post-column derivatization. Selectivity is achieved by using an anionic...

Two-column sequential injection chromatography--new approach for fast and effective analysis and its comparison with gradient elution chromatography.

PubMed

Chocholous, Petr; Satínský, Dalibor; Sklenárová, Hana; Solich, Petr

2010-05-23

This work presents novel approach in low-pressure chromatography flow systems--two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab, USA) with two commercially available monolithic columns the "first column" Chromolith Flash RP-18e (25 mm x 4.6 mm i.d. with guard column 5 mm x 4.6 mm i.d.) and the "second column" Chromolith RP-18e (10 mm x 4.6 mm i.d.) and CCD UV-vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min(-1) (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min(-1) (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 microL of filtered supernatant was injected on each column (2 x 10 microL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 microg m

Fluorogen-Activating-Proteins as Universal Affinity Biosensors for Immunodetection

PubMed Central

Gallo, Eugenio; Vasilev, Kalin V.; Jarvik, Jonathan

2014-01-01

Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. PMID:24122476

[Analysis of microalbuminuria with immunonephelometry and high performance liquid chromatography. Evaluation of new criteria].

PubMed

Markó, Lajos; Molnár, Gergo Attila; Wagner, Zoltán; Koszegi, Tamás; Matus, Zoltán; Mohás, Márton; Kuzma, Mónika; Szijártó, István András; Wittmann, István

2008-01-13

Evaluation study, the rate of microalbuminuria positivity among the immunonephelometrically negative patients decreased to 14.5% by high performance liquid chromatography and the decrease in the number of microalbuminuria positive cases by high performance liquid chromatography could be observed mainly in the diabetic and hypertensive group (49% vs. 7.5%), while slighter decrease could be observed in the non-diabetic hypertensive group (37% vs. 26.5%). Applying the traditional criteria, the strongest predictor was the male gender by the logistic regression analysis. In 28% of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established using high performance liquid chromatography. Almost in one-third of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established by high performance liquid chromatography for which diagnosis three constitutive urine examinations are still needed. New criteria determined by the Heart Outcomes Prevention Evaluation study can be used neither in case of diabetic and hypertensive patients, nor in the case of non-diabetic hypertensive patients. The gender as the most important predictor of microalbuminuria cannot be ignored.

Establishment of analysis method for methane detection by gas chromatography

NASA Astrophysics Data System (ADS)

Liu, Xinyuan; Yang, Jie; Ye, Tianyi; Han, Zeyu

2018-02-01

The study focused on the establishment of analysis method for methane determination by gas chromatography. Methane was detected by hydrogen flame ionization detector, and the quantitative relationship was determined by working curve of y=2041.2x+2187 with correlation coefficient of 0.9979. The relative standard deviation of 2.60-6.33% and the recovery rate of 96.36%∼105.89% were obtained during the parallel determination of standard gas. This method was not quite suitable for biogas content analysis because methane content in biogas would be over the measurement range in this method.

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

Gel compression considerations for chromatography scale-up for protein C purification.

PubMed

He, W; Bruley, D F; Drohan, W N

1998-01-01

This work is to establish theoretical and experimental relationships for the scale-up of Immobilized Metal Affinity Chromatography (IMAC) and Immuno Affinity Chromatography for the low cost production of large quantities of Protein C. The external customer requirements for this project have been established for Protein C deficient people with the goal of providing prophylactic patient treatment. Deep vein thrombosis is the major symptom for protein C deficiency creating the potential problem of embolism transport to important organs, such as, lung and brain. Gel matrices for protein C separation are being analyzed to determine the relationship between the material properties of the gel and the column collapse characteristics. The fluid flow rate and pressure drop is being examined to see how they influence column stability. Gel packing analysis includes two considerations; one is bulk compression due to flow rate, and the second is gel particle deformation due to fluid flow and pressure drop. Based on the assumption of creeping flow, Darcy's law is being applied to characterize the flow through the gel particles. Biot's mathematical description of three-dimensional consolidation in porous media is being used to develop a set of system equations. Finite difference methods are being utilized to obtain the equation solutions. In addition, special programs such as finite element approaches, ABAQUS, will be studied to determine their application to this particular problem. Experimental studies are being performed to determine flow rate and pressure drop correlation for the chromatographic columns with appropriate gels. Void fraction is being measured using pulse testing to allow Reynolds number calculations. Experimental yield stress is being measured to compare with the theoretical calculations. Total Quality Management (TQM) tools have been utilized to optimize this work. For instance, the "Scatter Diagram" has been used to evaluate and select the appropriate gels and

Detailed analysis of petroleum hydrocarbon attenuation in biopiles by high-performance liquid chromatography followed by comprehensive two-dimensional gas chromatography.

PubMed

Mao, Debin; Lookman, Richard; Van De Weghe, Hendrik; Van Look, Dirk; Vanermen, Guido; De Brucker, Nicole; Diels, Ludo

2009-02-27

Enhanced bioremediation of petroleum hydrocarbons in two biopiles was quantified by high-performance liquid chromatography (HPLC) followed by comprehensive two-dimensional gas chromatography (GCXGC). The attenuation of 34 defined hydrocarbon classes was calculated by HPLC-GCXGC analysis of representative biopile samples at start-up and after 18 weeks of biopile operation. In general, a-cyclic alkanes were most efficiently removed from the biopiles, followed by monoaromatic hydrocarbons. Cycloalkanes and polycyclic aromatic hydrocarbons (PAHs) were more resistant to degradation. A-cyclic biomarkers farnesane, trimethyl-C13, norpristane, pristane and phytane dropped to only about 10% of their initial concentrations. On the other hand, C29-C31 hopane concentrations remained almost unaltered after 18 weeks of biopile operation, confirming their resistance to biodegradation. They are thus reliable indicators to estimate attenuation potential of petroleum hydrocarbons in biopile processed soils.

Fast liquid chromatography combined with mass spectrometry for the analysis of metabolites and proteins in human body fluids.

PubMed

Kortz, Linda; Helmschrodt, Christin; Ceglarek, Uta

2011-03-01

In the last decade various analytical strategies have been established to enhance separation speed and efficiency in high performance liquid chromatography applications. Chromatographic supports based on monolithic material, small porous particles, and porous layer beads have been developed and commercialized to improve throughput and separation efficiency. This paper provides an overview of current developments in fast chromatography combined with mass spectrometry for the analysis of metabolites and proteins in clinical applications. Advances and limitations of fast chromatography for the combination with mass spectrometry are discussed. Practical aspects of, recent developments in, and the present status of high-throughput analysis of human body fluids for therapeutic drug monitoring, toxicology, clinical metabolomics, and proteomics are presented.

Characterization of self-affinity in the global regime

NASA Astrophysics Data System (ADS)

Neimark, Alexander V.

1994-11-01

Methods for characterization of self-affine surfaces and measurements of their roughness exponents H are developed. It is shown that for smoothed surfaces, which underwent particular coarse graining or averaging of the small-scale fluctuations, the excess surface area Sex and the mean square root radius of curvature ac are related by two distinct asymptotic power laws if ac is well below or well above a certain crossover scale acr. In the local regime of self-affinity, when ac<affinity, when ac>>acr, Sex~(ac/acr)-2(1-H)/(2-H). The former scaling relationship is consistent with the well known definition of local fractal dimensions dloc=dtop+1-H. The latter scaling relationship offers alternatives for characterization of self-affinity over large scales by means of excess dimensions defined as dex=dtop+2(1-H)/(2-H) and can be used for determination of roughness exponents from the measurements provided in the global regime. The thermodynamic method of fractal analysis, proposed earlier for self-similar surfaces (A.V. Neimark, Pis'ma Zh. Eksp. Teor. Fiz. 51, 535 (1990) [JETP Lett. 51, 607 (1990)]; Physica A 191, 258 (1992)), is extended for self-affine surfaces for determination of fractal dimensions and roughness exponents from adsorption and capillary experimental data.

Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing.

PubMed

Heider, Susanne; Muzard, Julien; Zaruba, Marianne; Metzner, Christoph

2017-07-01

Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein

Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution

PubMed Central

Minakuchi, Kazunobu; Murata, Dai; Okubo, Yuji; Nakano, Yoshiyuki; Yoshida, Shinichi

2013-01-01

Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin. PMID:23868198

Identification of atmospheric organic sources using the carbon hollow tube-gas chromatography method and factor analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cobb, G.P.; Braman, R.S.; Gilbert, R.A.

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 qualitymore » control method using the hollow tube-gas chromatography analysis is discussed.« less

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

PubMed Central

Gargano, Andrea F.G.; Schoenmakers, Peter J.

2017-01-01

Abstract Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations. PMID:29027363

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Fundamentals of affinity cell separations.

PubMed

Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

2018-03-01

Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

PubMed

Goubran-Botros, H; Nanak, E; Abdul Nour, J; Birkenmeir, G; Vijayalakshmi, M A

1992-04-24

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

PubMed

Barnidge, David R; Jelinek, Diane F; Muddiman, David C; Kay, Neil E

2005-01-01

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

Advanced proteomic liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Fang; Smith, Richard D.; Shen, Yufeng

2012-10-26

Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.

Affinity purification–mass spectrometry and network analysis to understand protein-protein interactions

PubMed Central

Morris, John H; Knudsen, Giselle M; Verschueren, Erik; Johnson, Jeffrey R; Cimermancic, Peter; Greninger, Alexander L; Pico, Alexander R

2015-01-01

By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification–mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography–MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one’s familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2–4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one’s data and find the right questions. PMID:25275790

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)

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies.

PubMed

Moureaux, T; Leydecker, M T; Meyer, C

1989-02-15

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).

High-Throughput Analysis of Sucrose Fatty Acid Esters by Supercritical Fluid Chromatography/Tandem Mass Spectrometry

PubMed Central

Hori, Katsuhito; Tsumura, Kazunobu; Fukusaki, Eiichiro; Bamba, Takeshi

2014-01-01

Supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry was applied to the profiling of sucrose fatty acid esters (SEs). The SFC conditions (column and modifier gradient) were optimized for the effective separation of SEs. In the column test, a silica gel reversed-phase column was selected. Then, the method was used for the detailed characterization of commercial SEs and the successful analysis of SEs containing different fatty acids. The present method allowed for fast and high-resolution separation of monoesters to tetra-esters within a shorter time (15 min) as compared to the conventional high-performance liquid chromatography. The applicability of our method for the analysis of SEs was thus demonstrated. PMID:26819875

Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

ERIC Educational Resources Information Center

Parsons, Julie; Ridley, Kimberly

2012-01-01

Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

Using Single Drop Microextraction for Headspace Analysis with Gas Chromatography

NASA Astrophysics Data System (ADS)

Riccio, Daniel; Wood, Derrick C.; Miller, James M.

2008-07-01

Headspace (HS) gas chromatography (GC) is commonly used to analyze samples that contain non-volatiles. In 1996, a new sampling technique called single drop microextraction, SDME, was introduced, and in 2001 it was applied to HS analysis. It is a simple technique that uses equipment normally found in the undergraduate laboratory, making it ideal for instructional use, especially to illustrate HS analysis or as an alternative to solid-phase microextraction (SPME) to which it is very similar. The basic principles and practice of HS-GC using SDME are described, including a complete review of the literature. Some possible experiments are suggested using water and N -methylpyrrolidone (NMP) as solvents.

Supercritical fluid chromatography

NASA Astrophysics Data System (ADS)

Vigdergauz, M. S.; Lobachev, A. L.; Lobacheva, I. V.; Platonov, I. A.

1992-03-01

The characteristic features of supercritical fluid chromatography (SCFC) are examined and there is a brief historical note concerning the development of the method. Information concerning the use of supercritical fluid chromatography in the analysis of objects of different nature is presented in the form of a table. The roles of the mobile and stationary phases in the separation process and the characteristic features of the apparatus and of the use of the method in physicochemical research are discussed. The bibliography includes 364 references.

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.

Acylhydrazone bond dynamic covalent polymer gel monolithic column online coupling to high-performance liquid chromatography for analysis of sulfonamides and fluorescent whitening agents in food.

PubMed

Zhang, Chengjiang; Luo, Xialin; Wei, Tianfu; Hu, Yufei; Li, Gongke; Zhang, Zhuomin

2017-10-13

A new dynamic covalent polymer (DCP) gel was well designed and constructed based on imine chemistry. Polycondensation of 4,4'-biphenyldicarboxaldehyde and 1,3,5-benzenetricarbohydrazide via Schiff-base reaction resulted in an acylhydrazone bond gel (AB-gel) DCP. AB-gel DCP had three-dimensional network of interconnected nanoparticles with hierarchically porous structure. AB-gel DCP was successfully fabricated as a monolithic column by an in-situ chemical bonding method for online enrichment and separation purpose with excellent permeability. AB-gel DCP based monolithic column showed remarkable adsorption affinity towards target analytes including sulfonamides (SAs) and fluorescent whitening agents (FWAs) due to its strong π-π affinity, hydrophobic effect and hydrogen bonding interaction. Then, AB-gel DCP based monolithic column was applied for online separation and analysis of trace SAs and FWAs in food samples coupled with high-performance liquid chromatography (HPLC). Sulfathiazole (ST) and sulfadimidine (SM2) in one positive weever sample were actually found and determined with concentrations of 273.8 and 286.3μg/kg, respectively. 2,5-Bis(5-tert-butyl-2-benzoxazolyl) thiophene (FWA184) was actually quantified in one tea infusion sample with the concentration of 268.5ng/L. The spiked experiments suggested the good recoveries in range of 74.5-110% for SAs in weever and shrimp samples with relative standard deviations (RSDs) less than 9.7% and in range of 74.0-113% for FWAs in milk and tea infusion samples with RSDs less than 9.0%. AB-gel DCP monolithic column was proved to be a promising sample preparation medium for online separation and analysis of trace analytes in food samples with complex matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimizing separations in online comprehensive two-dimensional liquid chromatography.

PubMed

Pirok, Bob W J; Gargano, Andrea F G; Schoenmakers, Peter J

2018-01-01

Online comprehensive two-dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two-dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two-dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high-molecular-weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one-dimensional liquid chromatography, two-dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two-dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two-dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two-dimensional liquid chromatography separations. © 2017 The Authors. Journal of Separation Science published by WILEY-VCH Verlag GmbH & Co. KGaA.

Advanced proteomic liquid chromatography

PubMed Central

Xie, Fang; Smith, Richard D.; Shen, Yufeng

2012-01-01

Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput. PMID:22840822

Gas chromatography-vacuum ultraviolet spectroscopy for analysis of fatty acid methyl esters.

PubMed

Fan, Hui; Smuts, Jonathan; Bai, Ling; Walsh, Phillip; Armstrong, Daniel W; Schug, Kevin A

2016-03-01

A new vacuum ultraviolet (VUV) detector for gas chromatography was recently developed and applied to fatty acid methyl ester (FAME) analysis. VUV detection features full spectral acquisition in a wavelength range of 115-240nm, where virtually all chemical species absorb. VUV absorption spectra of 37 FAMEs, including saturated, monounsaturated, and polyunsaturated types were recorded. Unsaturated FAMEs show significantly different gas phase absorption profiles than saturated ones, and these classes can be easily distinguished with the VUV detector. Another advantage includes differentiating cis/trans-isomeric FAMEs (e.g. oleic acid methyl ester and linoleic acid methyl ester isomers) and the ability to use VUV data analysis software for deconvolution of co-eluting signals. As a universal detector, VUV also provides high specificity, sensitivity, and a fast data acquisition rate, making it a powerful tool for fatty acid screening when combined with gas chromatography. The fatty acid profile of several food oil samples (olive, canola, vegetable, corn, sunflower and peanut oils) were analyzed in this study to demonstrate applicability to real world samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nanomaterials as stationary phases and supports in liquid chromatography.

PubMed

Beeram, Sandya R; Rodriguez, Elliott; Doddavenkatanna, Suresh; Li, Zhao; Pekarek, Allegra; Peev, Darin; Goerl, Kathryn; Trovato, Gianfranco; Hofmann, Tino; Hage, David S

2017-10-01

The development of various nanomaterials over the last few decades has led to many applications for these materials in liquid chromatography (LC). This review will look at the types of nanomaterials that have been incorporated into LC systems and the applications that have been explored for such systems. A number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in LC, ranging from carbon nanotubes, fullerenes and nanodiamonds to metal nanoparticles and nanostructures based on silica, alumina, zirconia and titanium dioxide. Many ways have been described for incorporating these nanomaterials into LC systems. These methods have included covalent immobilization, adsorption, entrapment, and the synthesis or direct development of nanomaterials as part of a chromatographic support. Nanomaterials have been used in many types of LC. These applications have included the reversed-phase, normal-phase, ion-exchange, and affinity modes of LC, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. Both small and large analytes (e.g., dyes, drugs, amino acids, peptides and proteins) have been used to evaluate possible applications for these nanomaterial-based methods. The use of nanomaterials in columns, capillaries and planar chromatography has been considered as part of these efforts. Potential advantages of nanomaterials in these applications have included their good chemical and physical stabilities, the variety of interactions many nanomaterials can have with analytes, and their unique retention properties in some separation formats. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Near-affine-invariant texture learning for lung tissue analysis using isotropic wavelet frames.

PubMed

Depeursinge, Adrien; Van de Ville, Dimitri; Platon, Alexandra; Geissbuhler, Antoine; Poletti, Pierre-Alexandre; Müller, Henning

2012-07-01

We propose near-affine-invariant texture descriptors derived from isotropic wavelet frames for the characterization of lung tissue patterns in high-resolution computed tomography (HRCT) imaging. Affine invariance is desirable to enable learning of nondeterministic textures without a priori localizations, orientations, or sizes. When combined with complementary gray-level histograms, the proposed method allows a global classification accuracy of 76.9% with balanced precision among five classes of lung tissue using a leave-one-patient-out cross validation, in accordance with clinical practice.

VACUUM DISTILLATION COUPLED WITH GAS CHROMATOGRAPHY/MASS SPECTROMETRY FOR THE ANALYSIS OF ENVIRONMENTAL SAMPLES

EPA Science Inventory

A procedure is presented that uses a vacuum distillation/gas chromatography/mass spectrometry system for analysis of problematic matrices of volatile organic compounds. The procedure compensates for matrix effects and provides both analytical results and confidence intervals from...

APPLICATION OF CAPILLARY SUPERCRITICAL FLUID CHROMATOGRAPHY TO THE ANALYSIS OF A MIDDLE DISTILLATE FUEL

EPA Science Inventory

The paper describes the application of capillary supercritical fluid chromatography (SFC) to the analysis of a middle distillate fuel. Small diameter (50 micrometer i.d.) fused silica capillary columns coated with crosslinked 50% phenyl polymethylsiloxane provided high separation...

Quantitative Analysis of Tetramethylenedisulfotetramine ("Tetramine") Spiked into Beverages by Liquid Chromatography Tandem Mass Spectrometry with Validation by Gas Chromatography Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Owens, J; Hok, S; Alcaraz, A

Tetramethylenedisulfotetramine, commonly known as tetramine, is a highly neurotoxic rodenticide (human oral LD{sub 50} = 0.1 mg/kg) used in hundreds of deliberate food poisoning events in China. Here we describe a method for quantitation of tetramine spiked into beverages, including milk, juice, tea, cola, and water and cleaned up by C8 solid phase extraction and liquid-liquid extraction. Quantitation by high performance liquid chromatography tandem mass spectrometry (LC/MS/MS) was based upon fragmentation of m/z 347 to m/z 268. The method was validated by gas chromatography mass spectrometry (GC/MS) operated in SIM mode for ions m/z 212, 240, and 360. The limitmore » of quantitation was 0.10 {micro}g/mL by LC/MS/MS versus 0.15 {micro}g/mL for GC/MS. Fortifications of the beverages at 2.5 {micro}g/mL and 0.25 {micro}g/mL were recovered ranging from 73-128% by liquid-liquid extraction for GC/MS analysis, 13-96% by SPE and 10-101% by liquid-liquid extraction for LC/MS/MS analysis.« less

[Recent advances in analysis of petroleum geological samples by comprehensive two-dimensional gas chromatography].

PubMed

Gao, Xuanbo; Chang, Zhenyang; Dai, Wei; Tong, Ting; Zhang, Wanfeng; He, Sheng; Zhu, Shukui

2014-10-01

Abundant geochemical information can be acquired by analyzing the chemical compositions of petroleum geological samples. The information obtained from the analysis provides scientifical evidences for petroleum exploration. However, these samples are complicated and can be easily influenced by physical (e. g. evaporation, emulsification, natural dispersion, dissolution and sorption), chemical (photodegradation) and biological (mainly microbial degradation) weathering processes. Therefore, it is very difficult to analyze the petroleum geological samples and they cannot be effectively separated by traditional gas chromatography/mass spectrometry. A newly developed separation technique, comprehensive two-dimensional gas chromatography (GC x GC), has unique advantages in complex sample analysis, and recently it has been applied to petroleum geological samples. This article mainly reviews the research progres- ses in the last five years, the main problems and the future research about GC x GC applied in the area of petroleum geology.

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.

Multi-exemplar affinity propagation.

PubMed

Wang, Chang-Dong; Lai, Jian-Huang; Suen, Ching Y; Zhu, Jun-Yong

2013-09-01

The affinity propagation (AP) clustering algorithm has received much attention in the past few years. AP is appealing because it is efficient, insensitive to initialization, and it produces clusters at a lower error rate than other exemplar-based methods. However, its single-exemplar model becomes inadequate when applied to model multisubclasses in some situations such as scene analysis and character recognition. To remedy this deficiency, we have extended the single-exemplar model to a multi-exemplar one to create a new multi-exemplar affinity propagation (MEAP) algorithm. This new model automatically determines the number of exemplars in each cluster associated with a super exemplar to approximate the subclasses in the category. Solving the model is NP-hard and we tackle it with the max-sum belief propagation to produce neighborhood maximum clusters, with no need to specify beforehand the number of clusters, multi-exemplars, and superexemplars. Also, utilizing the sparsity in the data, we are able to reduce substantially the computational time and storage. Experimental studies have shown MEAP's significant improvements over other algorithms on unsupervised image categorization and the clustering of handwritten digits.

Analysis of particulate polycyclic aromatic hydrocarbons by on-line coupled supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry

NASA Astrophysics Data System (ADS)

Shimmo, Masahiko; Adler, Heidi; Hyötyläinen, Tuulia; Hartonen, Kari; Kulmala, Markku; Riekkola, Marja-Liisa

An on-line supercritical fluid extraction-liquid chromatography-gas chromatography-mass spectrometry (SFE-LC-GC-MS) method was developed for the analysis of the particulate polycyclic aromatic hydrocarbons (PAHs). The limits of detection of the system for the quantification standards were in the range of 0.25-0.57 ng, while the limits of determinations for filter samples varied from 0.02 to 0.04 ng m -3 (24 h sampling). The linearity was excellent from 5 to 300 ng ( R2>0.967). The analysis could be carried out in a closed system without tedious manual sample pretreatment and with no risk of errors by contamination or loss of the analytes. The results of the SFE-LC-GC-MS method were comparable with those for Soxhlet and shake-flask extractions with GC-MS. The new method was applied to the analysis of PAHs collected by high-volume filter in the Helsinki area to study the seasonal trend of the concentrations. The individual PAH concentrations varied from 0.015 to more than 1 ng m -3, while total PAH concentrations varied from 0.81 to 5.68 ng m -3. The concentrations were generally higher in winter than in summer. The mass percentage of the total PAHs in total suspended particulates ranged from 2.85×10 -3% in July to 15.0×10 -3% in December. Increased emissions in winter, meteorological conditions, and more serious artefacts during the sampling in summer season may explain the concentration profiles.

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[An examination of the determinants of social withdrawal and affinity for social withdrawal].

PubMed

Watanabe, Asami; Matsui, Yutaka; Takatsuka, Yusuke

2010-12-01

This study examined the determinants of social withdrawal using data from a survey by the Tokyo Metropolitan Government Office for Youth Affairs and Public Safety (2008). In addition, this study identified young people who showed an affinity for social withdrawal although they were not in a state of withdrawal, and examined the determinants of an affinity for social withdrawal. The results of stepwise discriminant analysis showed that factors such as social phobia, depression, violence, and emotional bonds with family differentiated between the general youth group and the social withdrawal group and the "affinity group". Social phobia, violence, and refusal to be interfered in self-decision making differentiated between the social withdrawal group and the "affinity group". This study shows that an "affinity group" should be cared as well as an actual withdrawal group.

Food and value motivation: Linking consumer affinities to different types of food products.

PubMed

de Boer, Joop; Schösler, Hanna

2016-08-01

This study uses the consumer affinity concept to examine the multiple motives that may shape consumers' relationships with food. The concept was applied in a study on four broad product types in the Netherlands, which cover a wide range of the market and may each appeal to consumers with different affinities towards foods. These product types may be denoted as 'conventional', 'efficient', 'gourmet' and 'pure'. A comparative analysis, based on Higgins' Regulatory Focus Theory, was performed to examine whether food-related value motivations could explain different consumer affinities for these product types. The affinities of consumers were measured by means of a non-verbal, visual presentation of four samples of food products in a nationwide survey (n = 742) among consumers who were all involved in food purchasing and/or cooking. The affinities found could be predicted fairly well from a number of self-descriptions relating to food and eating, which expressed different combinations of type of value motivation and involvement with food. The analysis demonstrated the contrasting role of high and low involvement as well as the potential complementarity of promotion- and prevention-focused value motivation. It is suggested that knowledge of the relationships between product types, consumer affinities and value motivation can help improve the effectiveness of interventions that seek to promote healthy and sustainable diets in developed countries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of small carbohydrates in several bioactive botanicals by gas chromatography with mass spectrometry and liquid chromatography with tandem mass spectrometry.

PubMed

Moldoveanu, Serban; Scott, Wayne; Zhu, Jeff

2015-11-01

Bioactive botanicals contain natural compounds with specific biological activity, such as antibacterial, antioxidant, immune stimulating, and taste improving. A full characterization of the chemical composition of these botanicals is frequently necessary. A study of small carbohydrates from the plant materials of 18 bioactive botanicals is further described. The study presents the identification of the carbohydrate using a gas chromatographic-mass spectrometric analysis that allows detection of molecules as large as maltotetraose, after changing them into trimethylsilyl derivatives. A number of carbohydrates in the plant (fructose, glucose, mannose, sucrose, maltose, xylose, sorbitol, and myo-, chiro-, and scyllo-inositols) were quantitated using a novel liquid chromatography with tandem mass spectrometric technique. Both techniques involved new method developments. The gas chromatography with mass spectrometric analysis involved derivatization and separation on a Rxi(®)-5Sil MS column with H2 as a carrier gas. The liquid chromatographic separation was obtained using a hydrophilic interaction type column, YMC-PAC Polyamine II. The tandem mass spectrometer used an electrospray ionization source in multiple reaction monitoring positive ion mode with the detection of the adducts of the carbohydrates with Cs(+) ions. The validated quantitative procedure showed excellent precision and accuracy allowing the analysis in a wide range of concentrations of the analytes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Affinity-aware checkpoint restart

DOE PAGES

Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

2014-12-08

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Affinity-aware checkpoint restart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saini, Ajay; Rezaei, Arash; Mueller, Frank

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by medium-pressure liquid chromatography combined with macroporous resin chromatography.

PubMed

Wang, Yun; Liu, Hui; Shen, Lifeng; Yao, Lan; Ma, Yinlian; Yu, Dingrong; Chen, Jianhong; Li, Puling; Chen, Ying; Zhang, Cun

2015-12-01

Gardeniae fructus is one of the most frequently used herbs in traditional Chinese medicine. In the present study, a process for the enrichment of six iridoid glycosides from Gardeniae fructus was developed using medium-pressure liquid chromatography combined with macroporous resin and reversed-phase chromatography. The purities of different fractions from Gardeniae fructus were assessed using quantitative high-performance liquid chromatography. After fractionation using HPD-100 column chromatography, a 30% ethanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis to separate and purify. Based on the orientation analysis results, six compounds-deacetyl asperulosidic acid methyl ester, gardenoside, ixoroside, scandoside methyl ester, genipin-1-O-β-d-gentiobioside, and geniposide-were successfully isolated and purified in three to four combined steps from Gardeniae fructus. The purities of these compounds were found by high-performance liquid chromatography analysis to be 97.9, 98.1, 95.5, 96.3, 97.1, and 98.7%, respectively. Moreover, their structures were elucidated by NMR spectroscopy and liquid chromatography with tandem mass spectrometry. The separation process was highly efficient, rapid, and accurate, making it a potential approach for the large-scale production of iridoids in the laboratory and providing several marker compounds for quality control. This procedure may be meaningful for the purification of other natural products used in traditional Chinese medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2017 Guralp Affinity Digitizer Evaluation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merchant, Bion J.

Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

Effect-directed analysis via hyphenated high-performance thin-layer chromatography for bioanalytical profiling of sunflower leaves.

PubMed

Móricz, Ágnes M; Ott, Péter G; Yüce, Imanuel; Darcsi, András; Béni, Szabolcs; Morlock, Gertrud E

2018-01-19

High-performance thin-layer chromatography (HPTLC) coupled with effect-directed analysis was used for non-targeted screening of sunflower leaf extract for components exhibiting antioxidant, antibacterial and/or cholinesterase enzyme inhibitory effects. The active compounds were characterized by HPTLC-electrospray ionization-high resolution mass spectrometry (ESI-HRMS) and HPTLC-Direct Analysis in Real Time (DART)-MS/MS. The latter ambient ionization technique (less soft than ESI) resulted in oxidation and fragmentation products and characteristic fragment ions. NMR spectroscopy after targeted isolation via preparative normal phase flash chromatography and semi-preparative reversed phase high-performance liquid chromatography supported the identification of two diterpenes to be (-)-kaur-16-en-19-oic acid and 15-α-angeloyloxy-ent-kaur-16-en-19-oic acid. Both compounds found to be multi-potent as they inhibited acetylcholinesterase and butyrylcholinesterase and showed antibacterial effects against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri bacteria. Kaurenoic acid was also active against the Gram-negative pepper pathogenic Xanthomonas euvesicatoria bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis.

PubMed

Hetzel, Terence; Loeker, Denise; Teutenberg, Thorsten; Schmidt, Torsten C

2016-10-01

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2 μm fully porous as well as the 2.7 μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2 μm fully porous particle packed column for ultra-fast liquid chromatography. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In situ search for organics by gas chromatography analysis: new derivatization / thermochemolysis approach

NASA Astrophysics Data System (ADS)

Geffroy, Claude; Buch, Arnaud; David, Marc; Aissat, Lyes; El Mufleh, Amel; Papot, S.; Sternberg, Robert

Many organic molecules are present in interstellar clouds and might be carried to the early Earth by comets and meteorites during the heavy bombardment phase in the first few hundred million years of the solar system. It has been suggested that extraterrestrial organic material may well represent an important part of the organic material available for the origin of life. Until samples, brought by future space missions, are available on Earth, in situ measurements are one of the way to get unaltered and non-contaminated samples for analysis. The analytical technique has to be robust, sensitive and non-specific due to the large scope of targets molecules. The only currently flight qualified technique of analysis of organic molecules in space is gas chromatography (Viking, Cassini-Huygens, SAM-MSL, COSAC-Rosetta). The main objective of this work is to present a new approach with multi step analysis using derivatisation and thermochemolysis reagents for a one pot in situ analysis of volatile and refractory organics in surface or sub-surface samples (Mars, comets).Indeed, no single technology enables to identify all organic compounds because naturally occurring molecules have different polarities, molecular weights, being extractible or recalcitrant, bonded trapped or adsorbed on minerals. Thus, we propose to wider the scope of chemical reagent already validated for in situ wet chemistry such as MTBSTFA (Rodier et al. 2001, 2002), DMF-DMA (Rodier et al. 2002), or TMAH (Rodier et al, 2005, Geffroy-Rodier et al; 2009) to analyze enantiomers of amino acids, carbohydrates and lipids in a one pot several steps sub system using a multi reagent and multi step approach. Thus using a new derivatizing agent, we successfully identified twenty one amino acids including twelve of the twenty proteinic amino acids without inhibiting following multi step thermochemolysis. *Geffroy-Rodier C, Grasset L, Sternberg R. Buch A. Amblès A. (2009) Thermochemolysis in search for organics in

Detection of martian amino acids by chemical derivatization coupled to gas chromatography: in situ and laboratory analysis.

PubMed

Rodier, C; Vandenabeele-Trambouze, O; Sternberg, R; Coscia, D; Coll, P; Szopa, C; Raulin, F; Vidal-Madjar, C; Cabane, M; Israel, G; Grenier-Loustalot, M F; Dobrijevic, M; Despois, D

2001-01-01

If there is, or ever was, life in our solar system beyond the Earth, Mars is the most likely place to search for. Future space missions will have then to take into account the detection of prebiotic molecules or molecules of biological significance such as amino acids. Techniques of analysis used for returned samples have to be very sensitive and avoid any chemical or biological contamination whereas in situ techniques have to be automated, fast and low energy consuming. Several possible methods could be used for in situ amino acid analyses on Mars, but gas chromatography would likely be the most suitable. Returned samples could be analyzed by any method in routine laboratory use such as gas chromatography, already successfully performed for analyses of organic matter including amino acids from martian meteorites. The derivatization step, which volatilizes amino acids to perform both in situ and laboratory analysis by gas chromatography, is discussed here. c2001 COSPAR. Published by Elsevier Science Ltd. All rights reserved.

Analysis of antimycin A by reversed-phase liquid chromatography/nuclear magnetic-resonance spectrometry

USGS Publications Warehouse

Ha, Steven T.K.; Wilkins, Charles L.; Abidi, Sharon L.

1989-01-01

A mixture of closely related streptomyces fermentation products, antimycin A, Is separated, and the components are identified by using reversed-phase high-performance liquid chromatography with directly linked 400-MHz proton nuclear magnetic resonance detection. Analyses of mixtures of three amino acids, alanine, glycine, and valine, are used to determine optimal measurement conditions. Sensitivity increases of as much as a factor of 3 are achieved, at the expense of some loss in chromatographic resolution, by use of an 80-μL NMR cell, Instead of a smaller 14-μL cell. Analysis of the antimycin A mixture, using the optimal analytical high performance liquid chromatography/nuclear magnetic resonance conditions, reveals it to consist of at least 10 closely related components.

Purification of human erythropoietin by affinity chromatography using cyclic peptide ligands.

PubMed

Kish, William S; Roach, Matthew K; Sachi, Hiroyuki; Naik, Amith D; Menegatti, Stefano; Carbonell, Ruben G

2018-05-15

Prior work described the identification and characterization of erythropoietin-binding cyclic peptides SLFFLH, VVFFVH, FSLLHH and FSLLSH (all of the form cyclo[(N α -Ac)Dap(A)-X 1 -X 6 -AE], wherein X 1 -X 6 is the listed sequences). In this work, the peptide ligands were synthesized on Toyopearl chromatographic resins and utilized for purifying recombinant human erythropoietin (rHuEPO) from complex sources. Elution buffer pH and composition were optimized to maximize the recovery of standard rHuEPO from the peptide resins. The peptide-based adsorbents were employed for separating rHuEPO from a mixture of albumin, myoglobin, and IgG to examine their selectivity. When using FSLLHH, the inclusion of low amounts of surfactants in the wash and elution buffers facilitated the recovery of rHuEPO with high yield and purity. Specifically, FSLLSH and VVFFVH afforded the most efficient separation of rHuEPO, with yield and purity of 85% and 95-97%, respectively. The affinity resins were also utilized to purify rHuEPO from spiked CHO cell culture fluid. In particular, FSLLSH provided the most successful separation from CHO, with yield and purity above 90%, and 1.0 log 10 reduction of host cell proteins. The influence of conductivity and pH in the CHO-rHuEPO load was investigated. Finally, FSLLSH-based resins were used to purify rHuEPO spiked into a Pichia pastoris cell culture fluid, resulting in product yield and purity of 96% and 84%, respectively, and 1.3 log 10 reduction of host DNA. These results compare well with values obtained using wheat germ agglutinin agarose and clearly indicate the potential of the cyclic peptide resins as a viable tool for rHuEPO purification. Copyright © 2018 Elsevier B.V. All rights reserved.

High-performance liquid chromatography coupled with tandem mass spectrometry technology in the analysis of Chinese Medicine Formulas: A bibliometric analysis (1997-2015).

PubMed

He, Xi-Ran; Li, Chun-Guang; Zhu, Xiao-Shu; Li, Yuan-Qing; Jarouche, Mariam; Bensoussan, Alan; Li, Ping-Ping

2017-01-01

There is a recognized challenge in analyzing traditional Chinese medicine formulas because of their complex chemical compositions. The application of modern analytical techniques such as high-performance liquid chromatography coupled with a tandem mass spectrometry has improved the characterization of various compounds from traditional Chinese medicine formulas significantly. This study aims to conduct a bibliometric analysis to recognize the overall trend of high-performance liquid chromatography coupled with tandem mass spectrometry approaches in the analysis of traditional Chinese medicine formulas, its significance and possible underlying interactions between individual herbs in these formulas. Electronic databases were searched systematically, and the identified studies were collected and analyzed using Microsoft Access 2010, Graph Pad 5.0 software and Ucinet software package. 338 publications between 1997 and 2015 were identified, and analyzed in terms of annual growth and accumulated publications, top journals, forms of traditional Chinese medicine preparations and highly studied formulas and single herbs, as well as social network analysis of single herbs. There is a significant increase trend in using high-performance liquid chromatography coupled with tandem mass spectrometry related techniques in analysis of commonly used forms of traditional Chinese medicine formulas in the last 3 years. Stringent quality control is of great significance for the modernization and globalization of traditional Chinese medicine, and this bibliometric analysis provided the first and comprehensive summary within this field. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

PubMed Central

Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

2013-01-01

Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

Gas chromatography in space

NASA Technical Reports Server (NTRS)

Akapo, S. O.; Dimandja, J. M.; Kojiro, D. R.; Valentin, J. R.; Carle, G. C.

1999-01-01

Gas chromatography has proven to be a very useful analytical technique for in situ analysis of extraterrestrial environments as demonstrated by its successful operation on spacecraft missions to Mars and Venus. The technique is also one of the six scientific instruments aboard the Huygens probe to explore Titan's atmosphere and surface. A review of gas chromatography in previous space missions and some recent developments in the current environment of fiscal constraints and payload size limitations are presented.

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: A COMPARISON OF THREE MODELS

EPA Science Inventory

A comparative analysis of how three COREPA models for ER binding affinity performed when used to predict potential estrogen receptor (ER) ligands is presented. Models I and II were developed based on training sets of 232 and 279 rat ER binding affinity measurements, respectively....

TRACE ANALYSIS OF FLUORESCEIN-DERIVATIZED PHENOXY ACID HERBICIDES BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY WITH LASER-INDUCTED FLUORESCENCE DETECTION

EPA Science Inventory

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection was used for the trace analysis of phenoxy acid herbicides. Capillary electrophoresis (CE) with LIF detection, which has not previously been used for pesticide analysis, overcomes the po...

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection †

PubMed Central

Karr, Dale B.; Waters, James K.; Emerich, David W.

1983-01-01

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis. PMID:16346443

Antisymmetric tensor generalizations of affine vector fields.

PubMed

Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

2016-02-01

Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation

PubMed Central

2017-01-01

Polymeric nanoparticles have become indispensable in modern society with a wide array of applications ranging from waterborne coatings to drug-carrier-delivery systems. While a large range of techniques exist to determine a multitude of properties of these particles, relating physicochemical properties of the particle to the chemical structure of the intrinsic polymers is still challenging. A novel, highly orthogonal separation system based on comprehensive two-dimensional liquid chromatography (LC × LC) has been developed. The system combines hydrodynamic chromatography (HDC) in the first-dimension to separate the particles based on their size, with ultrahigh-performance size-exclusion chromatography (SEC) in the second dimension to separate the constituting polymer molecules according to their hydrodynamic radius for each of 80 to 100 separated fractions. A chip-based mixer is incorporated to transform the sample by dissolving the separated nanoparticles from the first-dimension online in tetrahydrofuran. The polymer bands are then focused using stationary-phase-assisted modulation to enhance sensitivity, and the water from the first-dimension eluent is largely eliminated to allow interaction-free SEC. Using the developed system, the combined two-dimensional distribution of the particle-size and the molecular-size of a mixture of various polystyrene (PS) and polyacrylate (PACR) nanoparticles has been obtained within 60 min. PMID:28745485

Einstein’s gravity from a polynomial affine model

NASA Astrophysics Data System (ADS)

Castillo-Felisola, Oscar; Skirzewski, Aureliano

2018-03-01

We show that the effective field equations for a recently formulated polynomial affine model of gravity, in the sector of a torsion-free connection, accept general Einstein manifolds—with or without cosmological constant—as solutions. Moreover, the effective field equations are partially those obtained from a gravitational Yang–Mills theory known as Stephenson–Kilmister–Yang theory. Additionally, we find a generalization of a minimally coupled massless scalar field in General Relativity within a ‘minimally’ coupled scalar field in this affine model. Finally, we present a brief (perturbative) analysis of the propagators of the gravitational theory, and count the degrees of freedom. For completeness, we prove that a Birkhoff-like theorem is valid for the analyzed sector.

Membrane protein separation and analysis by supercritical fluid chromatography-mass spectrometry.

PubMed

Zhang, Xu; Scalf, Mark; Westphall, Michael S; Smith, Lloyd M

2008-04-01

Membrane proteins comprise 25-30% of the human genome and play critical roles in a wide variety of important biological processes. However, their hydrophobic nature has compromised efforts at structural characterization by both X-ray crystallography and mass spectrometry. The detergents that are generally used to solubilize membrane proteins interfere with the crystallization process essential to X-ray studies and cause severe ion suppression effects that hinder mass spectrometric analysis. In this report, the use of supercritical fluid chromatography-mass spectrometry for the separation and analysis of integral membrane proteins and hydrophobic peptides is investigated. It is shown that detergents are rapidly and effectively separated from the proteins and peptides, yielding them in a state suitable for direct mass spectrometric analysis.

[Serological affinity of some species of nonpathogenic corynebacteria].

PubMed

Furtat, I M; Nohina, T M; Mikhal's'kyĭ, L O; Vedenieieva, O A

2002-01-01

Serological peculiarities of the species strains Corynebacterium glutamicum, C. ammoniagenes, C. vitaeruminis, C. variabilis and strain of Corynebacterium sp. (Brevibacterium stationis) UCM Ac-719 have been investigated with the help of immunoenzyme analysis ELISA with the use of mice immune serum, specific to C. ammoniagenes UCM Ac-732T, C. vitaeruminis UCM Ac-718T, C. variabilis UCM Ac-717T, C. glutamicum UCM Ac-733 and Corynebacterium sp. UCM Ac-719. It has been established that the species of nonpathogenic corynebacteria differ between themselves as to the degree of serological affinity. C. variabilis, C. ammoniagenes and C. glutamicum are the least similar as to this indication. Weak antigenic relations have been revealed in C. vitaeruminis and C. ammoniagenes. The latter displayed the higher, as compared with other strains, affinity for Corynebacterium sp. UCM Ac-719. The highest degree of serological affinity within the species was registered in strains C. glutamicum and C. variabilis. Data obtained evidence that the ELISA method permits conducting the high-reliability species diagnosis of nonpathogenic corynebacteria on the basis of their antigenic characteristics.

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

PubMed

Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

2014-02-01

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Personality Types and Affinity for Computers

DTIC Science & Technology

1991-03-01

differences on personality dimensions between the respondents, and to explore the relationship between these differences and computer affinity. The results...between the respondents, and to explore the relationship between these differences and computer affinity. The results revealed no significant differences...type to this measure of computer affinity. 2 II. LITERATURZ REVIEW The interest of this study was the relationship between a person’s psychological

Smooth affine shear tight frames: digitization and applications

NASA Astrophysics Data System (ADS)

Zhuang, Xiaosheng

2015-08-01

In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

Analysis of chemical signals in red fire ants by gas chromatography and pattern recognition techniques

USDA-ARS?s Scientific Manuscript database

The combination of gas chromatography and pattern recognition (GC/PR) analysis is a powerful tool for investigating complicated biological problems. Clustering, mapping, discriminant development, etc. are necessary to analyze realistically large chromatographic data sets and to seek meaningful relat...

Segregation of human peripheral blood lymphocytes according to their affinity for insolubilized histamine. Principal differences between males and females.

PubMed Central

Tartakovsky, B; Segal, S; Shani, A; Hellerstein, S; Weinstein, Y; Bentwich, Z

1979-01-01

An attempt was made to investigate the possible existence of differences in the composition of peripheral blood lymphocytes between males and females. Using affinity chromatography of human peripheral mononuclear cells on insolubilized histamine together with staining by fluoresceinated histamine-rabbit serum albumin (HRSA) we revealed that males possess a significantly higher proportion of mononuclear cells which bind to HRSA. These results are also reflected in sex-related differences in proliferative responses of the HRSA-non-adherent mononuclear cell population to T cell-dependent mitogens antigens and allogeneic mononuclear cells. PMID:160849

Protein A affinity chromatography of Chinese hamster ovary (CHO) cell culture broths containing biopharmaceutical monoclonal antibody (mAb): Experiments and mechanistic transport, binding and equilibrium modeling.

PubMed

Grom, Matic; Kozorog, Mirijam; Caserman, Simon; Pohar, Andrej; Likozar, Blaž

2018-04-15

Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins. Packing breakthrough curves were recorded at an array of prolonged residence times. A mathematical simulation model was developed, applied and validated in combination with non-linear regression algorithms on bed effluent concentrations to determine the previously-unknown binding properties of stationary phase materials. Apart from the columns' differential partitioning, the whole external system was also integrated. It was confirmed that internal pore diffusion is the global rate-limiting resistance of the compound retention process. Immobilizing substrate characteristics, obtained in this engineering study, are indispensable for the scale-up of the periodic counter-current control with mechanistic load, elution and wash reduction. Furthermore, unit's volumetric flow screening measurements revealed dynamic effect correlation to eluate quality parameters, like the presence of aggregates, the host cell-related impurities at supernatant's extended feeding, and titre. Numerical sensitivity outputs demonstrated the impacts of fluidics (e.g. axial dispersion coefficient), thermodynamics (Langmuir adsorption) and mass transfer fluxes. Copyright © 2018 Elsevier B.V. All rights reserved.

Preparation of a silica stationary phase co-functionalized with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography.

PubMed

Li, Hengye; Zhang, Xuemeng; Zhang, Lin; Wang, Xiaojin; Kong, Fenying; Fan, Dahe; Li, Lei; Wang, Wei

2017-04-15

A silica stationary phase was designed and synthesized through the co-functionalization of silica with Wulff-type phenylboronate and C12 for mixed-mode liquid chromatography applications. The as-synthesized stationary phase was characterized by elemental analysis and Fourier Transform-InfraRed Spectroscopy (FT-IR). Retention mechanisms, including boronate affinity (BA), reversed-phase (RP) and anion-exchange (AE), were involved. Retention mechanism switching was easily realized by adjustment of the mobile phase constitution. Cis-diol compounds could be selectively captured under neutral conditions in BA mode and off-line separated in RP mode. Neutral, basic, acidic and amphiprotic compounds were chromatographed on the column in RP chromatography, while inorganic anions were chromatographed in AE chromatography to characterize the mixed-mode nature of the prepared stationary phase. In addition, the RP performance was compared with an octadecyl silica column in terms of column efficiency (N/m), asymmetry factor (A f ), retention factor (k) and resolution (Rs). The prepared stationary phase offered multiple interactions with analytes in addition to hydrophobic interactions under RP elution conditions. Based on the mixed-mode properties, off-line 2D-LC, for selective capture and separation of urinary nucleosides, was successfully realized on a single column, demonstrating its powerful application potential for complex samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Thin layer chromatography coupled to paper spray ionization mass spectrometry for cocaine and its adulterants analysis.

PubMed

De Carvalho, Thays C; Tosato, Flavia; Souza, Lindamara M; Santos, Heloa; Merlo, Bianca B; Ortiz, Rafael S; Rodrigues, Rayza R T; Filgueiras, Paulo R; França, Hildegardo S; Augusti, Rodinei; Romão, Wanderson; Vaz, Boniek G

2016-05-01

Thin layer chromatography (TLC) is a simple and inexpensive type of chromatography that is extensively used in forensic laboratories for drugs of abuse analysis. In this work, TLC is optimized to analyze cocaine and its adulterants (caffeine, benzocaine, lidocaine and phenacetin) in which the sensitivity (visual determination of LOD from 0.5 to 14mgmL(-1)) and the selectivity (from the study of three different eluents: CHCl3:CH3OH:HCOOHglacial (75:20:5v%), (C2H5)2O:CHCl3 (50:50v%) and CH3OH:NH4OH (100:1.5v%)) were evaluated. Aiming to improve these figures of merit, the TLC spots were identified and quantified (linearity with R(2)>0.98) by the paper spray ionization mass spectrometry (PS-MS), reaching now lower LOD values (>1.0μgmL(-1)). The method developed in this work open up perspective of enhancing the reliability of traditional and routine TLC analysis employed in the criminal expertise units. Higher sensitivity, selectivity and rapidity can be provided in forensic reports, besides the possibility of quantitative analysis. Due to the great simplicity, the PS(+)-MS technique can also be coupled directly to other separation techniques such as the paper chromatography and can still be used in analyses of LSD blotter, documents and synthetic drugs. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

HPTLC-aptastaining - Innovative protein detection system for high-performance thin-layer chromatography

NASA Astrophysics Data System (ADS)

Morschheuser, Lena; Wessels, Hauke; Pille, Christina; Fischer, Judith; Hünniger, Tim; Fischer, Markus; Paschke-Kratzin, Angelika; Rohn, Sascha

2016-05-01

Protein analysis using high-performance thin-layer chromatography (HPTLC) is not commonly used but can complement traditional electrophoretic and mass spectrometric approaches in a unique way. Due to various detection protocols and possibilities for hyphenation, HPTLC protein analysis is a promising alternative for e.g., investigating posttranslational modifications. This study exemplarily focused on the investigation of lysozyme, an enzyme which is occurring in eggs and technologically added to foods and beverages such as wine. The detection of lysozyme is mandatory, as it might trigger allergenic reactions in sensitive individuals. To underline the advantages of HPTLC in protein analysis, the development of innovative, highly specific staining protocols leads to improved sensitivity for protein detection on HPTLC plates in comparison to universal protein derivatization reagents. This study aimed at developing a detection methodology for HPTLC separated proteins using aptamers. Due to their affinity and specificity towards a wide range of targets, an aptamer based staining procedure on HPTLC (HPTLC-aptastaining) will enable manifold analytical possibilities. Besides the proof of its applicability for the very first time, (i) aptamer-based staining of proteins is applicable on different stationary phase materials and (ii) furthermore, it can be used as an approach for a semi-quantitative estimation of protein concentrations.

Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

PubMed

Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

2016-05-04

The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

Alkali Metal Cation versus Proton and Methyl Cation Affinities: Structure and Bonding Mechanism

PubMed Central

Boughlala, Zakaria; Fonseca Guerra, Célia

2016-01-01

Abstract We have analyzed the structure and bonding of gas‐phase Cl−X and [HCl−X]+ complexes for X+= H+, CH3 +, Li+, and Na+, using relativistic density functional theory (DFT). We wish to establish a quantitative trend in affinities of the anionic and neutral Lewis bases Cl− and HCl for the various cations. The Cl−X bond becomes longer and weaker along X+ = H+, CH3 +, Li+, and Na+. Our main purpose is to understand the heterolytic bonding mechanism behind the intrinsic (i.e., in the absence of solvent) alkali metal cation affinities (AMCA) and how this compares with and differs from those of the proton affinity (PA) and methyl cation affinity (MCA). Our analyses are based on Kohn–Sham molecular orbital (KS‐MO) theory in combination with a quantitative energy decomposition analysis (EDA) that pinpoints the importance of the different features in the bonding mechanism. Orbital overlap appears to play an important role in determining the trend in cation affinities. PMID:27551660

Affinity Chart Analysis: A Method for Structured Collection, Aggregation, and Response to Customer Needs in Radiology.

PubMed

Boll, Daniel T; Rubin, Geoffrey D; Heye, Tobias; Pierce, Laura J

2017-04-01

The objective of this study is to analyze implementation of the voice-of-the-customer method to assess the current state of image postprocessing and reporting delivered by a radiology department and to plan improvements on the basis of referring physicians' preferences. The voice-of-the-customer method consisted of discovery, analysis, and optimization phases. Fifty referring physicians were invited to be interviewed. Interviews addressed the topics of structure, process, outcome, and support. Interviews were dissected into individual statements categorized as fact or feeling. Statements were grouped to find collective voices. Improvements were compiled from affinity charts and were processed by identifying insights. Ninety-four percent (47/50) of physicians participated, generating 352 statements (81 facts and 271 feelings) that subsequently underwent affinity chart clustering. The resultant affinity charts covered distinct themes: "we need you to know us better," "we need you to consider our workflow," "we need more from your services," "we want to review your data in certain ways," and "we want to do more with you." As a result of the insights gained, the following optimizations were implemented: a software application that improves study requesting, performance tracking, study prioritization, and longitudinal data archiving; six prototype reports containing tabulated data and annotated images; two prototype longitudinal reporting templates assessing aneurysm evolution and treatment-induced changes in organ size over time; and a teaching curriculum for trainees. This study has shown the clinical feasibility to assess the current state of image postprocessing and reporting and to implement improvements of and investments in image postprocessing and reporting infrastructure on the basis of referring physicians' preferences using the voice-of-the-customer method.

Affinity extraction of emerging contaminants from water based on bovine serum albumin as a binding agent.

PubMed

Papastavros, Efthimia; Remmers, Rachael A; Snow, Daniel D; Cassada, David A; Hage, David S

2018-03-01

Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides, and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on-line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions than a traditional C 18 support for most of the tested emerging contaminants. Potential advantages of using these protein-based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Litopenaeus vannamei BiP as a novel cellular attachment protein for white spot syndrome virus by using a biotinylation based affinity chromatography method.

PubMed

Yuan, Zengzhi; Chen, Meng; Wang, Jingting; Li, Zhuoyu; Geng, Xuyun; Sun, Jinsheng

2018-05-05

White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target. Copyright © 2018. Published by Elsevier Ltd.

Single-step affinity purification for fungal proteomics.

PubMed

Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

2010-05-01

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

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)

Micellar liquid chromatography

NASA Astrophysics Data System (ADS)

Basova, Elena M.; Ivanov, Vadim M.; Shpigun, Oleg A.

1999-12-01

Background and possibilities of practical applications of micellar liquid chromatography (MLC) are considered. Various retention models in MLC, the effects of the nature and concentration of surfactants and organic modifiers, pH, temperature and ionic strength on the MLC efficiency and selectivity are discussed. The advantages and limitations of MLC are demonstrated. The performance of MLC is critically evaluated in relationship to the reversed-phase HPLC and ion-pair chromatography. The potential of application of MLC for the analysis of pharmaceuticals including that in biological fluids and separation of inorganic anions, transition metal cations, metal chelates and heteropoly compounds is described. The bibliography includes 146 references.

Hb San Cataldo [β144(HC1)Lys→Thr; HBB: C.434A > C]: A New Hemoglobin Variant with Increased Affinity for Oxygen.

PubMed

Vinciguerra, Margherita; Passarello, Cristina; Cassarà, Filippo; Leto, Filippo; Cannata, Monica; Crivello, Anna; Di Salvo, Veronica; Maggio, Aurelio; Giambona, Antonino

2016-08-01

A 59-year-old Italian woman came to our center for revaluation of a previous diagnosis of polycythemia vera. The patient presented with a lifelong history of polycythemia, no increase in white blood cells (WBCs) and platelets, and a negative bone marrow biopsy. Analysis of hemoglobin (Hb) fractions showed an abnormal fast moving Hb component. We aimed to determine if this variant was the cause of polycythemia in this patient. A complete blood count (CBC) was performed by an automated cell counter and Hb fractions were determined by high performance liquid chromatography (HPLC). Standard stability tests and oxygen affinity evaluation were also performed. Genomic DNA was extracted from peripheral blood leukocytes using the phenol chloroform method and the entire β-globin gene was analyzed by direct sequencing. At the hematological level, no anemia or hemolysis was observed but an abnormal Hb fraction was detected using cation exchange HPLC. Molecular analysis of the β-globin gene showed heterozygosity for an AAG > ACG substitution at codon 144, resulting in a Lys→Thr amino acid replacement. We demonstrated that this is a new Hb variant with increased oxygen affinity. Its altered physiology is caused by the reduction of 2,3-diphosphoglycerate (2,3-DPG) effects, due to an amino acid substitution in the central pocket near the C-terminal of the β chain. We called this new variant Hb San Cataldo for the native city of proband.

Considerations on comprehensive and off-line supercritical fluid chromatography x reversed-phase liquid chromatography for the analysis of triacylglycerols in fish oil.

PubMed

François, Isabelle; Pereira, Alberto dos Santos; Sandra, Pat

2010-06-01

The separation of the triacylglycerols in fish oil was performed by comprehensive and off-line supercritical fluid chromatography combined with RP-LC. The first dimension consisted of two serially coupled silver-ion (SI)-loaded columns operated with a supercritical mobile phase (supercritical fluid chromatography, SFC) in both the cases, whereas the second dimension was performed in non-aqueous RP mode (NARP-LC) on a 10-cm monolithic octadecyl silica (ODS) or a 45-cm long ODS column packed with 1.8 microm particles for the comprehensive and off-line separations, respectively. Despite the outstanding performance of the SI-SFC x NARP-LC interface, the high complexity of the sample rendered the online separation far from complete. The off-line approach gave much better separation mainly because of the higher peak capacity of the second-dimension column, but even in this case, the use of MS was mandatory to elucidate the different triacylglycerols in fish oil. The disadvantage of the off-line procedure was the long analysis time.

Mobile Technology Affinity in Renal Transplant Recipients.

PubMed

Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

Solid-phase microextraction coupled with high performance liquid chromatography: a complementary technique to solid-phase microextraction-gas chromatography for the analysis of pesticide residues in strawberries.

PubMed

Wang, Z; Hennion, B; Urruty, L; Montury, M

2000-11-01

Solid-phase microextraction coupled with high performance liquid chromatography has been studied for the analysis of methiocarb, napropamide, fenoxycarb and bupirimate in strawberries. The strawberries were blended and centrifuged. Then, an aliquot of the resulting extracting solution was subjected to solid-phase microextraction (SPME) on a 60 microns polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre for 45 min at room temperature. The extracted pesticides on the SPME fibre were desorbed into SPME/high performance liquid chromatography (HPLC) interface for HPLC analysis with diode-array detection (DAD). The method is organic solvent-free for the whole extraction process and is simple and easy to manipulate. The detection limits were shown to be at low microgram kg-1 level and the linear response covered the range from 0.05 to 2 mg kg-1 of pesticides in strawberries with a regression coefficient larger than 0.99. A good repeatability with RSDs between 2.92 and 9.25% was obtained, depending on compounds.

A safe, effective, and facility compatible cleaning in place procedure for affinity resin in large-scale monoclonal antibody purification.

PubMed

Wang, Lu; Dembecki, Jill; Jaffe, Neil E; O'Mara, Brian W; Cai, Hui; Sparks, Colleen N; Zhang, Jian; Laino, Sarah G; Russell, Reb J; Wang, Michelle

2013-09-20

Cleaning-in-place (CIP) for column chromatography plays an important role in therapeutic protein production. A robust and efficient CIP procedure ensures product quality, improves column life time and reduces the cost of the purification processes, particularly for those using expensive affinity resins, such as MabSelect protein A resin. Cleaning efficiency, resin compatibility, and facility compatibility are the three major aspects to consider in CIP process design. Cleaning MabSelect resin with 50mM sodium hydroxide (NaOH) along with 1M sodium chloride is one of the most popular cleaning procedures used in biopharmaceutical industries. However, high concentration sodium chloride is a leading cause of corrosion in the stainless steel containers used in large scale manufacture. Corroded containers may potentially introduce metal contaminants into purified drug products. Therefore, it is challenging to apply this cleaning procedure into commercial manufacturing due to facility compatibility and drug safety concerns. This paper reports a safe, effective and environmental and facility-friendly cleaning procedure that is suitable for large scale affinity chromatography. An alternative salt (sodium sulfate) is used to prevent the stainless steel corrosion caused by sodium chloride. Sodium hydroxide and salt concentrations were optimized using a high throughput screening approach to achieve the best combination of facility compatibility, cleaning efficiency and resin stability. Additionally, benzyl alcohol is applied to achieve more effective microbial control. Based on the findings, the recommended optimum cleaning strategy is cleaning MabSelect resin with 25 mM NaOH, 0.25 M Na2SO4 and 1% benzyl alcohol solution every cycle, followed by a more stringent cleaning using 50 mM NaOH with 0.25 M Na2SO4 and 1% benzyl alcohol at the end of each manufacturing campaign. A resin life cycle study using the MabSelect affinity resin demonstrates that the new cleaning strategy

Determination of alkylphenols by gas chromatography, elution liquid chromatography, and gel permeation chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wittmann, S.; Decsy, Z.; Regensperger, S.

1984-01-01

The separation and determination of groups of alkylphenols with C/sub 15/-C/sub 33/ isoalkane chains by gas chromatography, elution liquid chromatography, and gel permeation chromatography are described. Paraffinic hydrocarbons, monoalkylphenols, dialkylphenols, and bis(hydroxyphenyl)alkanes were identified in industrial alkylphenols by mass spectrometry. 7 references, 3 figures, 2 tables.

Application of thin-layer chromatography/infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry to structural analysis of bacteria-binding glycosphingolipids selected by affinity detection.

PubMed

Müsken, Anne; Souady, Jamal; Dreisewerd, Klaus; Zhang, Wenlan; Distler, Ute; Peter-Katalinić, Jasna; Miller-Podraza, Halina; Karch, Helge; Müthing, Johannes

2010-04-15

Glycosphingolipids (GSLs) play key roles in the manifestation of infectious diseases as attachment sites for pathogens. The thin-layer chromatography (TLC) overlay assay represents one of the most powerful approaches for the detection of GSL receptors of microorganisms. Here we report on the direct structural characterization of microbial GSL receptors by employment of the TLC overlay assay combined with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF-MS). The procedure includes TLC separation of GSL mixtures, overlay of the chromatogram with GSL-specific bacteria, detection of bound microbes with primary antibodies against bacterial surface proteins and appropriate alkaline phosphatase labeled secondary antibodies, and in situ MS analysis of bacteria-specific GSL receptors. The combined method works on microgram scale of GSL mixtures and is advantageous in that it omits laborious and time-consuming GSL extraction from the silica gel layer. This technique was successfully applied to the compositional analysis of globo-series neutral GSLs recognized by P-fimbriated Escherichia coli bacteria, which were used as model microorganisms for infection of the human urinary tract. Thus, direct TLC/IR-MALDI-o-TOF-MS adds a novel facet to this fast and sensitive method offering a wide range of applications for the investigation of carbohydrate-specific pathogens involved in human infectious diseases. 2010 John Wiley & Sons, Ltd.

Application of enhanced electronegative multimodal chromatography as the primary capture step for immunoglobulin G purification.

PubMed

Wang, Yanli; Chen, Quan; Xian, Mo; Nian, Rui; Xu, Fei

2018-06-01

In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.

Supercritical fluid chromatography with photodiode array detection for pesticide analysis in papaya and avocado samples.

PubMed

Pano-Farias, Norma S; Ceballos-Magaña, Silvia G; Gonzalez, Jorge; Jurado, José M; Muñiz-Valencia, Roberto

2015-04-01

To improve the analysis of pesticides in complex food matrices with economic importance, alternative chromatographic techniques, such as supercritical fluid chromatography, can be used. Supercritical fluid chromatography has barely been applied for pesticide analysis in food matrices. In this paper, an analytical method using supercritical fluid chromatography coupled to a photodiode array detection has been established for the first time for the quantification of pesticides in papaya and avocado. The extraction of methyl parathion, atrazine, ametryn, carbofuran, and carbaryl was performed through the quick, easy, cheap, effective, rugged, and safe methodology. The method was validated using papaya and avocado samples. For papaya, the correlation coefficient values were higher than 0.99; limits of detection and quantification ranged from 130-380 and 220-640 μg/kg, respectively; recovery values ranged from 72.8-94.6%; precision was lower than 3%. For avocado, limit of detection values were ˂450 μg/kg; precision was lower than 11%; recoveries ranged from 50.0-94.2%. Method feasibility was tested for lime, banana, mango, and melon samples. Our results demonstrate that the proposed method is applicable to methyl parathion, atrazine, ametryn, and carbaryl, toxics pesticides used worldwide. The methodology presented in this work could be applicable to other fruits. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of protein prenylation and S-acylation using gas chromatography-coupled mass spectrometry.

PubMed

Sorek, Nadav; Akerman, Amir; Yalovsky, Shaul

2013-01-01

Lipid modifications play a key role in protein targeting and function. The two Arabidopsis Gγ subunits, AGG1 and AGG2, have been shown to undergo prenylation (AGG1) and S-acylation (AGG2). Prenylation involves covalent nonreversible attachment of either farnesyl (15 carbons) or geranylgeranyl (20 carbons) isoprenoids to conserved cysteine residues at or near the C-terminus of proteins. S-acylation, frequently referred to as palmitoylation, involves the attachment of acyl fatty acids to thiol groups of cysteine residues through a reversible thioester bond. The procedures described below allow direct analysis of the prenyl and acyl moieties using gas chromatography-coupled mass spectrometry (GC-MS). These methods are based on (1) cleavage of prenyl groups with the Raney nickel catalyst and (2) analysis of protein S-acylation following cleavage of the acyl fatty acids from proteins by hydrogenation with platinum (IV) oxide. The hydrogenation under these conditions causes an acid transesterification of the acyl moieties, adding an ethyl group to the carboxyl head of the fatty acid. The addition of the ethyl group reduces the polarity of the fatty acids, allowing their efficient separation by gas chromatography.

Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

PubMed

Kanomata, Naoki; Matsuura, Shiro; Nomura, Tsunehisa; Kurebayashi, Junichi; Mori, Taisuke; Kitawaki, Jo; Moriya, Takuya

2017-01-01

Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated

Study of Separation and Identification of the Active Ingredients in Gardenia jasminoides Ellis Based on a Two-Dimensional Liquid Chromatography by Coupling Reversed Phase Liquid Chromatography and Hydrophilic Interaction Liquid Chromatography.

PubMed

Zhou, Xuan; Chen, Cen; Ye, Xiaolan; Song, Fenyun; Fan, Guorong; Wu, Fuhai

2017-01-01

In this paper, by coupling reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), a two-dimensional liquid chromatography system was developed for separation and identification of the active ingredients in Gardenia jasminoides Ellis (GJE). By applying the semi-preparative C18 column as the first dimension and the core-shell column as the second dimension, a total of 896 peaks of GJE were separated. Among the 896 peaks, 16 active ingredients including geniposide, gardenoside, gardoside, etc. were identified by mass spectrometry analysis. The results indicated that the proposed two-dimensional RPLC/HILIC system was an effective method for the analysis of GJE and might hold a high potential to become a useful tool for analysis of other complex mixtures. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

Quantum properties of affine-metric gravity with the cosmological term

NASA Astrophysics Data System (ADS)

Baurov, A. Yu; Pronin, P. I.; Stepanyantz, K. V.

2018-04-01

The paper contains analysis of the one-loop effective action for affine-metric gravity of the Hilbert–Einstein type with the cosmological term. We discuss different approaches to the calculation of the effective action, which depends on two independent variables, namely, the metric tensor and the affine connection. In the one-loop approximation we explain how the effective action can be obtained, if, at the first step of the calculation, the metric tensor is integrated out. It is demonstrated that the result is the same as in the case when one starts by integrating out the connection.

[Identification of the interacting proteins with S100A8 or S100A9 by affinity purification and mass spectrometry].

PubMed

Wang, Jing; Zhang, Xuemei; Li, Zheng; Li, Xiayu; Ma, Jian; Shen, Shourong

2017-04-28

To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS).
 Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins. Co-immunoprecipitation (Co-IP) was applied to confirm the proteins interacted with S100A8 or S100A9.
 Results: Fourteen proteins including pyruvate kinase, muscle (PKM), nucleophosmin (NPM1) and eukaryotic translation initiation factor 5A (EIF5A), which potentially interacted with S100A8, were successfully identified by Flag-tag affinity purification followed by LC-MS/MS analysis. Six proteins, such as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (14-3-3ε) and PKM, which potentially interacted with S100A9, were successfully identified. Gene ontology analysis of the identified proteins suggested that proteins interacted with S100A8 or S100A9 were involved in several biological pathways, including canonical glycolysis, positive regulation of NF-κB transcription factor activity, negative regulation of apoptotic process, cell-cell adhesion, etc. Co-IP experiment confirmed that PKM2 can interact with both S100A8 and S100A9, and 14-3-3ε can interact with S100A8.
 Conclusion: PKM2 is identified to interact with both S100A8 and S100A9, while 14-3-3ε can interact with S100A9. These results may provide a new clue for the role of S100A8 or S100A9 in the progression of colitis-associated colorectal cancer.

Affinity Spaces and 21st Century Learning

ERIC Educational Resources Information Center

Gee, James Paul

2017-01-01

This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

PubMed

Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

2016-02-01

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multimodal charge-induction chromatography for antibody purification.

PubMed

Tong, Hong-Fei; Lin, Dong-Qiang; Chu, Wen-Ning; Zhang, Qi-Lei; Gao, Dong; Wang, Rong-Zhu; Yao, Shan-Jing

2016-01-15

Hydrophobic charge-induction chromatography (HCIC) has advantages of high capacity, salt-tolerance and convenient pH-controlled elution. However, the binding specificity might be improved with multimodal molecular interactions. New ligand W-ABI that combining tryptophan and 5-amino-benzimidazole was designed with the concept of mutimodal charge-induction chromatography (MCIC). The indole and benzimidazole groups of the ligand could provide orientated mutimodal binding to target IgG under neutral pH, while the imidazole groups could induce the electrostatic repulsion forces for efficient elution under acidic pH. W-ABI ligand was coupled successfully onto agarose gel, and IgG adsorption behaviors were investigated. High affinity to IgG was found with the saturated adsorption capacity of 70.4 mg/ml at pH 7, and the flow rate of mobile phase showed little impact on the dynamic binding capacity. In addition, efficient elution could be achieved at mild acidic pH with high recovery. Two separation cases (IgG separation from albumin containing feedstock and monoclonal antibody purification from cell culture supernatant) were verified with high purity and recovery. In general, MCIC with the specially-designed ligand is an expanding of HCIC with improved adsorption selectivity, which would be a potential alternative to Protein A-based capture for the cost-effective purification of antibodies. Copyright © 2015 Elsevier B.V. All rights reserved.

ANALYSIS OF TRACE-LEVEL ORGANIC COMBUSTION PROCESS EMISSIONS USING NOVEL MULTIDIMENSIONAL GAS CHROMATOGRAPHY-MASS SPECTROMETRY PROCEDURES

EPA Science Inventory

The paper discusses the analysis of trace-level organic combustion process emissions using novel multidimensional gas chromatography-mass spectrometry (MDGC-MS) procedures. It outlines the application of the technique through the analyses of various incinerator effluent and produ...

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.

Taste characteristics of Chinese bayberry juice characterized by sensory evaluation, chromatography analysis, and an electronic tongue.

PubMed

Yu, Haiyan; Zhang, Yan; Zhao, Jie; Tian, Huaixiang

2018-05-01

To evaluate the taste characteristics of Chinese bayberry juice, four types of bayberry juice sourced from different origins and varieties were analysed using sensory evaluation, chromatography, spectroscopy analysis and an electronic tongue (E-tongue). Nine organic acids and three sugars were assessed using high performance liquid chromatography. Total polyphenols were measured by spectrophotometry. The overall taste profile was collected using the E-tongue. The four types of bayberry juice differed in the sensory attributes of sour, sweet, bitter, and astringent. The E-tongue responses combined with discriminant analysis were able to characterise the taste profiles of the juices. The relationships between the taste compounds and the sensory panel scores established by partial least squares showed that total polyphenols, quininic acid, maleic acid, fructose, citric acid, lactic acid, succinic acid and sucrose made significant contributions to the taste characteristics of the Chinese bayberry juice.

Neutral monosaccharide composition analysis of plant-derived oligo- and polysaccharides by high performance liquid chromatography.

PubMed

Yan, Jun; Shi, Songshan; Wang, Hongwei; Liu, Ruimin; Li, Ning; Chen, Yonglin; Wang, Shunchun

2016-01-20

A novel analytical method for neutral monosaccharide composition analysis of plant-derived oligo- and polysaccharides was developed using hydrophilic interaction liquid chromatography coupled to a charged aerosol detector. The effects of column type, additives, pH and column temperature on retention and separation were evaluated. Additionally, the method could distinguish potential impurities in samples, including chloride, sulfate and sodium, from sugars. The results of validation demonstrated that this method had good linearity (R(2) ≥ 0.9981), high precision (relative standard deviation ≤ 4.43%), and adequate accuracy (94.02-103.37% recovery) and sensitivity (detection limit: 15-40 ng). Finally, the monosaccharide compositions of the polysaccharide from Eclipta prostrasta L. and stachyose were successfully profiled through this method. This report represents the first time that all of these common monosaccharides could be well-separated and determined simultaneously by high performance liquid chromatography without additional derivatization. This newly developed method is convenient, efficient and reliable for monosaccharide analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

On the structure of self-affine convex bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voynov, A S

2013-08-31

We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

Identification and Affinity-Quantification of ß-Amyloid and α-Synuclein Polypeptides Using On-Line SAW-Biosensor-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Slamnoiu, Stefan; Vlad, Camelia; Stumbaum, Mihaela; Moise, Adrian; Lindner, Kathrin; Engel, Nicole; Vilanova, Mar; Diaz, Mireia; Karreman, Christiaan; Leist, Marcel; Ciossek, Thomas; Hengerer, Bastian; Vilaseca, Marta; Przybylski, Michael

2014-08-01

Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS- acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (KD) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

QUANTITATIVE ANALYSIS OF 68 POLAR COMPOUNDS FROM TEN CHEMICAL CLASSES BY DIRECT AQUEOUS INJECTION GAS CHROMATOGRAPHY

EPA Science Inventory

Porous polymer packings have been used successfully in many applications of direct aqueous injection gas chromatography. The authors have expanded the use of aqueous injection to the quantitative analysis of 68 alcohols, acetates, ketones, ethers, sulfides, aldehydes, diols, dion...

Development of a sensitive and rapid method for rifampicin impurity analysis using supercritical fluid chromatography.

PubMed

Li, Wei; Wang, Jun; Yan, Zheng-Yu

2015-10-10

A novel simple, fast and efficient supercritical fluid chromatography (SFC) method was developed and compared with RPLC method for the separation and determination of impurities in rifampicin. The separation was performed using a packed diol column and a mobile phase B (modifier) consisting of methanol with 0.1% ammonium formate (w/v) and 2% water (v/v). Overall satisfactory resolutions and peak shapes for rifampicin quinone (RQ), rifampicin (RF), rifamycin SV (RSV), rifampicin N-oxide (RNO) and 3-formylrifamycinSV (3-FR) were obtained by optimization of the chromatography system. With gradient elution of mobile phase, all of the impurities and the active were separated within 4 min. Taking full advantage of features of SFC (such as particular selectivity, non-sloping baseline in gradient elution, and without injection solvent effects), the method was successfully used for determination of impurities in rifampicin, with more impurity peaks detected, better resolution achieved and much less analysis time needed compared with conventional reversed-phase liquid chromatography (RPLC) methods. Copyright © 2015 Elsevier B.V. All rights reserved.

The Use of Gas Chromatography for Biogas Analysis

NASA Astrophysics Data System (ADS)

Andersen, Amanda; Seeley, John; Aurandt, Jennifer

2010-04-01

Energy from natural gas accounts for 24 percent of energy consumed in the US. Natural gas is a robust form of energy which is rich in methane content and is low in impurities. This quality suggests that it is a very clean and safe gas; it can be used in providing heat, a source for cooking, and in powering vehicles. The downside is that it is a non-renewable resource. On the contrary, methane rich gas that is produced by the breakdown of organic material in an anaerobic environment, called biogas, is a renewable energy source. This research focuses on the gas analysis portion of the creation of the anaerobic digestion and verification laboratory where content and forensic analysis of biogas is performed. Gas Chromatography is implemented as the optimal analytical tool for quantifying the components of the biogas including methane, carbon dioxide, hydrogen sulfide and siloxanes. In addition, the problems associated with the undesirable components are discussed. Anaerobic digestion of primary sludge has consistently produced about 55 percent methane; future goals of this research include studying different substrates to increase the methane yield and decrease levels of impurities in the gas.

Analysis of D-penicillamine by gas chromatography utilizing nitrogen--phosphorus detection.

PubMed

Rushing, L G; Hansen, E B; Thompson, H C

1985-01-11

A method is presented for the analysis of the "orphan" drug D-penicillamine (D-Pa), which is used for the treatment of the inherited rare copper metabolism dysfunction known as Wilson's disease, by assaying a derivative of the compound by gas chromatography employing a rubidium sensitized nitrogen--phosphorus detector. Analytical procedures are described for the analyses of residues of D-Pa X HCl salt in animal feed and for the analyses of the salt or free base from aqueous solutions by utilizing a single-step double derivatization with diazomethane--acetone. Stability data for D-Pa X HCl in animal feed and for the free base in water are presented. An ancillary fluorescence derivatization procedure for the analysis of D-Pa in water is also reported.

Core-Shell Columns in High-Performance Liquid Chromatography: Food Analysis Applications

PubMed Central

Preti, Raffaella

2016-01-01

The increased separation efficiency provided by the new technology of column packed with core-shell particles in high-performance liquid chromatography (HPLC) has resulted in their widespread diffusion in several analytical fields: from pharmaceutical, biological, environmental, and toxicological. The present paper presents their most recent applications in food analysis. Their use has proved to be particularly advantageous for the determination of compounds at trace levels or when a large amount of samples must be analyzed fast using reliable and solvent-saving apparatus. The literature hereby described shows how the outstanding performances provided by core-shell particles column on a traditional HPLC instruments are comparable to those obtained with a costly UHPLC instrumentation, making this novel column a promising key tool in food analysis. PMID:27143972

Multivariate analysis of progressive thermal desorption coupled gas chromatography-mass spectrometry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Benthem, Mark Hilary; Mowry, Curtis Dale; Kotula, Paul Gabriel

Thermal decomposition of poly dimethyl siloxane compounds, Sylgard{reg_sign} 184 and 186, were examined using thermal desorption coupled gas chromatography-mass spectrometry (TD/GC-MS) and multivariate analysis. This work describes a method of producing multiway data using a stepped thermal desorption. The technique involves sequentially heating a sample of the material of interest with subsequent analysis in a commercial GC/MS system. The decomposition chromatograms were analyzed using multivariate analysis tools including principal component analysis (PCA), factor rotation employing the varimax criterion, and multivariate curve resolution. The results of the analysis show seven components related to offgassing of various fractions of siloxanes that varymore » as a function of temperature. Thermal desorption coupled with gas chromatography-mass spectrometry (TD/GC-MS) is a powerful analytical technique for analyzing chemical mixtures. It has great potential in numerous analytic areas including materials analysis, sports medicine, in the detection of designer drugs; and biological research for metabolomics. Data analysis is complicated, far from automated and can result in high false positive or false negative rates. We have demonstrated a step-wise TD/GC-MS technique that removes more volatile compounds from a sample before extracting the less volatile compounds. This creates an additional dimension of separation before the GC column, while simultaneously generating three-way data. Sandia's proven multivariate analysis methods, when applied to these data, have several advantages over current commercial options. It also has demonstrated potential for success in finding and enabling identification of trace compounds. Several challenges remain, however, including understanding the sources of noise in the data, outlier detection, improving the data pretreatment and analysis methods, developing a software tool for ease of use by the chemist, and demonstrating our

Comprehensive lipidomic analysis of human plasma using multidimensional liquid- and gas-phase separations: Two-dimensional liquid chromatography-mass spectrometry vs. liquid chromatography-trapped-ion-mobility-mass spectrometry.

PubMed

Baglai, Anna; Gargano, Andrea F G; Jordens, Jan; Mengerink, Ynze; Honing, Maarten; van der Wal, Sjoerd; Schoenmakers, Peter J

2017-12-29

Recent advancements in separation science have resulted in the commercialization of multidimensional separation systems that provide higher peak capacities and, hence, enable a more-detailed characterization of complex mixtures. In particular, two powerful analytical tools are increasingly used by analytical scientists, namely online comprehensive two-dimensional liquid chromatography (LC×LC, having a second-dimension separation in the liquid phase) and liquid chromatography-ion mobility-spectrometry (LC-IMS, second dimension separation in the gas phase). The goal of the current study was a general assessment of the liquid-chromatography-trapped-ion-mobility-mass spectrometry (LC-TIMS-MS) and comprehensive two-dimensional liquid chromatography-mass spectrometry (LC×LC-MS) platforms for untargeted lipid mapping in human plasma. For the first time trapped-ion-mobility spectrometry (TIMS) was employed for the separation of the major lipid classes and ion-mobility-derived collision-cross-section values were determined for a number of lipid standards. The general effects of a number of influencing parameters have been inspected and possible directions for improvements are discussed. We aimed to provide a general indication and practical guidelines for the analyst to choose an efficient multidimensional separation platform according to the particular requirements of the application. Analysis time, orthogonality, peak capacity, and an indicative measure for the resolving power are discussed as main characteristics for multidimensional separation systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

PubMed

Hentz, N G; Daunert, S

1996-11-15

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Classical affine W-algebras associated to Lie superalgebras

NASA Astrophysics Data System (ADS)

Suh, Uhi Rinn

2016-02-01

In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalization of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.

PubMed

Dooley, Helen; Stanfield, Robyn L; Brady, Rebecca A; Flajnik, Martin F

2006-02-07

The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

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. © 2016 American Institute of Chemical Engineers.

Classical affine W-algebras associated to Lie superalgebras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suh, Uhi Rinn, E-mail: uhrisu1@math.snu.ac.kr

2016-02-15

In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalizationmore » of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.« less

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

PubMed

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Analysis of Drought Stress-Responsive Raffinose Family Oligosaccharides in Plant Tissues.

PubMed

Jorge, Tiago F; Florêncio, Maria H; António, Carla

2017-01-01

Drought is a major limiting factor in agriculture and responsible for dramatic crop yield losses worldwide. The adjustment of the metabolic status via accumulation of drought stress-responsive osmolytes is one of the many strategies that some plants have developed to cope with water deficit conditions. Osmolytes are highly polar compounds, analysis of whcih is difficult with typical reversed-phase chromatography. Porous graphitic carbon (PGC) has shown to be a suitable alternative to reversed-phase stationary phases for the analysis of highly polar compounds typically found in the plant metabolome. In this chapter, we describe the development and validation of a PGC-based liquid chromatography tandem mass spectrometry (LC-MS n ) method suitable for the target analysis of water-soluble carbohydrates, such as raffinose family oligosaccharides (RFOs). We present detailed information regarding PGC column equilibration, LC-MS n system operation, data analysis, and important notes to be considered during the steps of method development and validation.

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

PubMed Central

Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

2016-01-01

Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

Vaccine Efficacy and Affinity Maturation

NASA Astrophysics Data System (ADS)

Lee, Hayoun; Deem, Michael W.

2002-03-01

We propose macroscopic equations to describe variable vaccine efficacy between repeated vaccinee and first time vaccinee. The main ingredients are antigenic distance between epidemic strain and vaccne strain, and affinity maturation dynamics which differs in primary and second response. Increase of affinity by repeated vaccine leads to localization in immune space. This localization decreases the ability of the immune system to response to distant, but related epidemic strains.

Trace analysis in the food and beverage industry by capillary gas chromatography: system performance and maintenance.

PubMed

Hayes, M A

1988-04-01

Gas chromatography (GC) is the most widely used analytical technique in the food and beverage industry. This paper addresses the problems of sample preparation and system maintenance to ensure the most sensitive, durable, and efficient results for trace analysis by GC in this industry.

Characterization of synthetic dyes by comprehensive two-dimensional liquid chromatography combining ion-exchange chromatography and fast ion-pair reversed-phase chromatography.

PubMed

Pirok, Bob W J; Knip, Jitske; van Bommel, Maarten R; Schoenmakers, Peter J

2016-03-04

In the late 19th century, newly invented synthetic dyes rapidly replaced the natural dyes on the market. The characterization of mixtures of these so-called early synthetic dyes is complicated through the occurrence of many impurities and degradation products. Conventional one-dimensional liquid chromatography does not suffice to obtain fingerprints with sufficient resolution and baseline integrity. Comprehensive two-dimensional liquid chromatography (LC×LC) is employed in this study, with ion-exchange chromatography in the first dimension and fast ion-pair liquid chromatography in the second. Retention in the first dimension is largely determined by the number of charges, while the selection of a small ion-pair reagent (tetramethylammonium hydroxide) in the second dimension causes retention to be largely determined by the molecular structure of the dye. As a result, there is a high degree of orthogonality of the two dimensions, similar to the values typically encountered in GC×GC. The proposed LC×LC method shows a theroretical peak capacity of about 2000 in an analysis time of about three hours. Clear, informative fingerprints are obtained that open a way to a more efficient characterization of dyes used in objects of cultural heritage. Copyright © 2016 Elsevier B.V. All rights reserved.

Multiplex gas chromatography for use in space craft

NASA Technical Reports Server (NTRS)

Valentin, J. R.

1985-01-01

Gas chromatography is a powerful technique for the analysis of gaseous mixtures. Some limitations in this technique still exist which can be alleviated with multiplex gas chromatography (MGC). In MGC, rapid multiple sample injections are made into the column without having to wait for one determination to be finished before taking a new sample. The resulting data must then be reduced using computational methods such as cross correlation. In order to efficiently perform multiplexgas chromatography, experiments in the laboratory and on board future space craft, skills, equipment, and computer software were developed. Three new techniques for modulating, i.e., changing, sample concentrations were demonstrated by using desorption, decomposition, and catalytic modulators. In all of them, the need for a separate gas stream as the carrier was avoided by placing the modulator at the head of the column to directly modulate a sample stream. Finally, the analysis of an environmental sample by multiplex chromatography was accomplished by employing silver oxide to catalytically modulate methane in ambient air.

Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution.

PubMed

Yodoshi, Masahiro; Oyama, Takehiro; Masaki, Ken; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

2011-01-01

Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry.

Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts.

PubMed

Vanzolini, K L; da F Sprenger, R; Leme, G M; de S Moraes, V R; Vilela, A F L; Cardoso, C L; Cass, Q B

2018-05-10

The use of affinity-based protein assay produced by covalently linking acetylcholinesterase to magnetic beads, followed by chemical characterization of the selective binders using Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC-HRMS) is herein described for profiling crude aqueous natural product extracts. The fishing assay was first modulated using galanthamine as a reference ligand and then, the assay condition was adjusted for the aqueous leaves extracts obtained from Lippia gracilis Schauer (genotype 201) that was used as the natural combinatory library. From the experiments, a selective binder has been undisclosed with an accurate mass of 449.1131 m/z and identified as eriodictyol 2'-O-glucoside or eriodictyol 3'-O-glucoside. The selectivity of the binding assay was demonstrated, as much as, that erydictiol 7-O-glucoside was not fished, although it was present in the crude aqueous extract. The binding assay platform exhibited high specificity and did not require any sample pretreatment, making it appropriate for profiling binders at natural libraries. Copyright © 2018 Elsevier B.V. All rights reserved.

Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

PubMed

Bian, Liujiao; Ji, Xu; Hu, Wei

2014-07-01

In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. Copyright © 2013 John Wiley & Sons, Ltd.

[Preparation of flavonoid reference standards from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry analysis].

PubMed

Guo, Henan; Yang, Xuedong; Liu, Jun; Zheng, Wenfeng

2012-07-01

Flavonoid reference standards were targeted-prepared from Scutellariae Radix under the guidance of high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. With HPLC-MS analysis of Scutellariae Radix, 19 flavonoid components were identified by analyzing and comparing their retention times, ultraviolet spectra, and mass spectrometry data with literature. The separation and purification protocols of all targeted flavonoid reference standards were optimally designed according to the results of HPLC-MS analysis and related literature. The ethanol extract of Scutellariae Radix was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol successively. The ethyl acetate extract and n-butanol extract were separately subjected to primary separation by low pressure reverse phase preparative chromatography. Then the fractions containing targeted compounds were further purified by low pressure reverse and normal phases preparative chromatography. Finally, baicalin and wogonoside reference standards were obtained from n-butanol extract; baicaelin, wogonin, and oroxylin A reference standards were obtained from ethyl acetate extract. The structures of the 5 reference standards were identified by mass spectrometry (MS) and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The HPLC analytical results showed that the purities of the 5 reference standards were all above 98%. It is demonstrated that the rapid targeted-preparation method under the guidance of the HPLC-MS analysis is applicable for the isolation and preparation of chemical components in traditional Chinese medicines.

Laboratory and field based evaluation of chromatography ...

EPA Pesticide Factsheets

The Monitor for AeRosols and GAses in ambient air (MARGA) is an on-line ion-chromatography-based instrument designed for speciation of the inorganic gas and aerosol ammonium-nitrate-sulfate system. Previous work to characterize the performance of the MARGA has been primarily based on field comparison to other measurement methods to evaluate accuracy. While such studies are useful, the underlying reasons for disagreement among methods are not always clear. This study examines aspects of MARGA accuracy and precision specifically related to automated chromatography analysis. Using laboratory standards, analytical accuracy, precision, and method detection limits derived from the MARGA chromatography software are compared to an alternative software package (Chromeleon, Thermo Scientific Dionex). Field measurements are used to further evaluate instrument performance, including the MARGA’s use of an internal LiBr standard to control accuracy. Using gas/aerosol ratios and aerosol neutralization state as a case study, the impact of chromatography on measurement error is assessed. The new generation of on-line chromatography-based gas and particle measurement systems have many advantages, including simultaneous analysis of multiple pollutants. The Monitor for Aerosols and Gases in Ambient Air (MARGA) is such an instrument that is used in North America, Europe, and Asia for atmospheric process studies as well as routine monitoring. While the instrument has been evaluat

Analysis of polymeric phenolics in red wines using different techniques combined with gel permeation chromatography fractionation.

PubMed

Guadalupe, Zenaida; Soldevilla, Alberto; Sáenz-Navajas, María-Pilar; Ayestarán, Belén

2006-04-21

A multiple-step analytical method was developed to improve the analysis of polymeric phenolics in red wines. With a common initial step based on the fractionation of wine phenolics by gel permeation chromatography (GPC), different analytical techniques were used: high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-mass spectrometry (MS), capillary zone electrophoresis (CZE) and spectrophotometry. This method proved to be valid for analyzing different families of phenolic compounds, such as monomeric phenolics and their derivatives, polymeric pigments and proanthocyanidins. The analytical characteristics of fractionation by GPC were studied and the method was fully validated, yielding satisfactory statistical results. GPC fractionation substantially improved the analysis of polymeric pigments by CZE, in terms of response, repeatability and reproducibility. It also represented an improvement in the traditional vanillin assay used for proanthocyanidin (PA) quantification. Astringent proanthocyanidins were also analyzed using a simple combined method that allowed these compounds, for which only general indexes were available, to be quantified.

Analysis of phospholipids in bio-oils and fats by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

PubMed

Viidanoja, Jyrki

2015-09-15

A new, sensitive and selective liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method was developed for the analysis of Phospholipids (PLs) in bio-oils and fats. This analysis employs hydrophilic interaction liquid chromatography-scheduled multiple reaction monitoring (HILIC-sMRM) with a ZIC-cHILIC column. Eight PL class selective internal standards (homologs) were used for the semi-quantification of 14 PL classes for the first time. More than 400 scheduled MRMs were used for the measurement of PLs with a run time of 34min. The method's performance was evaluated for vegetable oil, animal fat and algae oil. The averaged within-run precision and between-run precision were ≤10% for all of the PL classes that had a direct homologue as an internal standard. The method accuracy was generally within 80-120% for the tested PL analytes in all three sample matrices. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiresidue analysis of pesticides in traditional Chinese medicines using gas chromatography - negative chemical ionization tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

In this study, a residue analysis method for the simultaneous determination of 107 pesticides in the traditional Chinese medicines (TCMs), Angelica sinensis, Angelica dahurica, Leonurus heterophyllus Sweet, Pogostemon cablin, and Lonicera japonica Thunb, was developed using gas chromatography couple...

[Analysis of phthalates in plastic food-packaging bags by thin layer chromatography].

PubMed

Chen, Hui; Wang, Yuan; Zhu, Ruohua

2006-01-01

The method for simultaneous determination of four phthalates, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) in plastic food-packaging bags by thin layer chromatography (TLC) was developed. The plastic food-packaging bags were extracted with ethanol by ultrasonication, then the mixture was filtrated through membrane (0.45 microm). The mixture of ethyl acetate-anhydrous ether-isooctane (1 : 4 : 15, v/v) was used as developing agent on the TLC silica gel plate for development. The filtered liquid was spotted on the TLC plate dealt by acetone, and detected with scanning wavelength of 275 nm and reference wavelength of 340 nm. The qualitative analysis of the phthalates was performed using the R(f) values of the chromatogram. The quantitative analysis was performed with external standard method. Good linearities were obtained for DMP, DEP, DBP and DEHP. The detection limits were 2.1 ng for DMP, 2.4 ng for DEP, 3.4 ng for DBP and 4.0 ng for DEHP. The relative standard deviations (RSDs) of the four phthalates were 2.8% - 3.5%. The recoveries of the four phthalate standards in real sample were 78.58% - 111.04%. The method presented has the advantages of high precision, high sensitivity, small sample size, and simple pretreatment . The method was used to detect the four phthalates in the food-packaging bags. The contents in real samples were close to the results by gas chromatography.

Quantitative analysis of benzodiazepines in vitreous humor by high-performance liquid chromatography

PubMed Central

Bazmi, Elham; Behnoush, Behnam; Akhgari, Maryam; Bahmanabadi, Leila

2016-01-01

Objective: Benzodiazepines are frequently screened drugs in emergency toxicology, drugs of abuse testing, and in forensic cases. As the variations of benzodiazepines concentrations in biological samples during bleeding, postmortem changes, and redistribution could be biasing forensic medicine examinations, hence selecting a suitable sample and a validated accurate method is essential for the quantitative analysis of these main drug categories. The aim of this study was to develop a valid method for the determination of four benzodiazepines (flurazepam, lorazepam, alprazolam, and diazepam) in vitreous humor using liquid–liquid extraction and high-performance liquid chromatography. Methods: Sample preparation was carried out using liquid–liquid extraction with n-hexane: ethyl acetate and subsequent detection by high-performance liquid chromatography method coupled to diode array detector. This method was applied to quantify benzodiazepines in 21 authentic vitreous humor samples. Linear curve for each drug was obtained within the range of 30–3000 ng/mL with coefficient of correlation higher than 0.99. Results: The limit of detection and quantitation were 30 and 100 ng/mL respectively for four drugs. The method showed an appropriate intra- and inter-day precision (coefficient of variation < 10%). Benzodiazepines recoveries were estimated to be over 80%. The method showed high selectivity; no additional peak due to interfering substances in samples was observed. Conclusion: The present method was selective, sensitive, accurate, and precise for the quantitative analysis of benzodiazepines in vitreous humor samples in forensic toxicology laboratory. PMID:27635251

[Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

PubMed

Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

2017-03-01

Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography.

PubMed

Bergmann, Marianne L; Sadjadi, Seyed; Schmedes, Anne

2017-07-01

The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns. Copyright © 2017 Elsevier B.V. All rights reserved.

Instrumentation: Ion Chromatography.

ERIC Educational Resources Information Center

Fritz, James S.

1987-01-01

Discusses the importance of ion chromatography in separating and measuring anions. The principles of ion exchange are presented, along with some applications of ion chromatography in industry. Ion chromatography systems are described, as well as ion pair and ion exclusion chromatography, column packings, detectors, and programming. (TW)

Atomic Force Microscope Mediated Chromatography

NASA Technical Reports Server (NTRS)

Anderson, Mark S.

2013-01-01

The atomic force microscope (AFM) is used to inject a sample, provide shear-driven liquid flow over a functionalized substrate, and detect separated components. This is demonstrated using lipophilic dyes and normal phase chromatography. A significant reduction in both size and separation time scales is achieved with a 25-micron-length column scale, and one-second separation times. The approach has general applications to trace chemical and microfluidic analysis. The AFM is now a common tool for ultra-microscopy and nanotechnology. It has also been demonstrated to provide a number of microfluidic functions necessary for miniaturized chromatography. These include injection of sub-femtoliter samples, fluidic switching, and sheardriven pumping. The AFM probe tip can be used to selectively remove surface layers for subsequent microchemical analysis using infrared and tip-enhanced Raman spectroscopy. With its ability to image individual atoms, the AFM is a remarkably sensitive detector that can be used to detect separated components. These diverse functional components of microfluidic manipulation have been combined in this work to demonstrate AFM mediated chromatography. AFM mediated chromatography uses channel-less, shear-driven pumping. This is demonstrated with a thin, aluminum oxide substrate and a non-polar solvent system to separate a mixture of lipophilic dyes. In conventional chromatographic terms, this is analogous to thin-layer chromatography using normal phase alumina substrate with sheardriven pumping provided by the AFM tip-cantilever mechanism. The AFM detection of separated components is accomplished by exploiting the variation in the localized friction of the separated components. The AFM tip-cantilever provides the mechanism for producing shear-induced flows and rapid pumping. Shear-driven chromatography (SDC) is a relatively new concept that overcomes the speed and miniaturization limitations of conventional liquid chromatography. SDC is based on a

Gene expression analysis of microtubule affinity-regulating kinase 2 in non-small cell lung cancer.

PubMed

Marshall, Erin A; Ng, Kevin W; Anderson, Christine; Hubaux, Roland; Thu, Kelsie L; Lam, Wan L; Martinez, Victor D

2015-12-01

Lung cancer is the leading cause of cancer death worldwide, and has a five-year survival rate of 18% [1]. MARK2 is a serine/threonine-protein kinase, and is a key component in the phosphorylation of microtubule-associated proteins [2], [3]. A recent study published by Hubaux et al. found that microtubule affinity-regulating kinase 2 (MARK2) showed highly frequent DNA and RNA level disruption in lung cancer cell lines and independent non-small cell lung cancer (NSCLC) cohorts [4]. These alterations result in the acquisition of oncogenic properties in cell lines, such as increased viability and anchorage-independent growth. Furthermore, a microarray-based transcriptome analysis of three short hairpin RNA (shRNA)-mediated MARK2 knockdown lung adenocarcinoma cell lines (GEO#: GSE57966) revealed an association between MARK2 gene expression and cell cycle activation and DNA damage response. Here, we present a detailed description of transcriptome analysis to support the described role of MARK2 in promoting a malignant phenotype.

Affinity-based biosensors in sport medicine and doping control analysis.

PubMed

Mazzei, Franco; Antiochia, Riccarda; Botrè, Francesco; Favero, Gabriele; Tortolini, Cristina

2014-01-01

Affinity-based biosensors (ABBs) have started to be considered in sport medicine and doping control analysis because they are cheap, easy to use and sufficiently selective analytical devices, characterized by a reversible interaction with the analyte under investigation allowing the use of the same sensor for multiple analyses. In this review we describe the main categories of substances reported in the World Anti-Doping Agency Prohibited List and how ABBs may contribute to their detection. Although several ABBs proposed in the last few years display limit of detections that are in principle matching the World Anti-Doping Agency requirements, their application in the framework of 'traditional' antidoping tests seems quite unlikely, mainly because of the still insufficient selectivity especially in the case of 'pseudo-endogenous' compounds, and on the lack of complete information regarding potential matrix effects in real samples and following their routine use. At the same time, ABBs could contribute to fill a significant information gap concerning complementary evidence that can be obtained from their use 'on the spot', as well as to preselect a risk population of individuals to be targeted for a full antidoping test; while in sport medicine they could contribute to obtaining analytical information of physiological relevance from the measurement of specific parameters or markers before, during and after physical exercise.

The analysis of clingfilms by infrared spectroscopy and thermal desorption capillary gas chromatography.

PubMed

Gilburt, J; Ingram, J M; Scott, M P; Underhill, M

1991-01-01

An automated thermal desorption gas chromatography technique has been adapted to analyse traces of volatile compounds in proprietary food-wrapping films. Fourteen brands of polyvinylchloride film, seven brands of polyethylene film and one polyvinylidene chloride film were discriminated. Prior infrared analysis was used to identify the polymer type. The chromatograms showed minor changes in volatiles along the length of a roll of film and major changes in films exposed to daylight or in contact with cannabis resin.

Tetracycline residues in royal jelly and honey by liquid chromatography tandem mass spectrometry: validation study according to Commission Decision 2002/657/EC.

PubMed

Giannetti, L; Longo, F; Buiarelli, F; Russo, M V; Neri, B

2010-09-01

A specific, sensitive and robust liquid chromatography tandem mass spectrometry method for determining oxytetracycline, tetracycline, chlortetracycline and doxycycline in royal jelly and honey samples is presented. Extraction of drug residues was performed by ammonium acetate buffer as extractant followed by a clean-up with metal chelate affinity chromatography and solid-phase extraction. Tetracycline analysis was performed using liquid chromatography-electrospray ionisation-tandem mass spectrometry. The presented method is the first validated for royal jelly and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples at 5.0, 10.0, 20.0 and 30.0 μg kg(-1), were 79% to 90% for honey and 77% to 90% for royal jelly. The intra-day precision (RSD) ranged between 8.1% and 15.0% for honey and from 9.1% to 16.3% for royal jelly, while inter-day precision values were from 10.2% to 17.6% and from 10.6% to 18.4% respectively for honey and royal jelly. Linearity for the four analytes was calculated from 5.0 to 50.0 μg kg(-1). The decision limits (CCα) ranged from 6.2 to 6.4 μg kg(-1) and from 6.1 to 6.5 μg kg(-1) for honey and royal jelly, respectively. Detection capabilities values (CCβ) ranged between 7.2 and 7.7 μg kg(-1) and from 7.3 to 7.9 μg kg(-1) respectively for honey and royal jelly. The developed method is currently in use for confirmation of the official control analysis of honey and royal jelly samples.

Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

PubMed

Periat, Aurélie; Krull, Ira S; Guillarme, Davy

2015-02-01

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Notch and affinity boundaries in Drosophila.

PubMed

Herranz, Héctor; Milán, Marco

2006-02-01

Cells in multicellular organisms often do not intermingle freely with each other. Differential cell affinities can contribute to organizing cells into different tissues. Drosophila limbs and the vertebrate central nervous system are subdivided into compartments. Cells in adjacent compartments do not mix. Cell interactions mediated by Notch-family receptors have been implicated in the specification of these compartment boundaries. Two recent reports analyze the role of the Notch signaling pathway in the generation of an affinity boundary in the Drosophila wing. The first report analyzes the connection between Notch and the actin cytoskeleton. The second report analyzes the differential requirements of Notch and the transcription factor Suppressor of Hairless in generating the affinity boundary.

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

PubMed

Ferguson, A D; Breed, J; Diederichs, K; Welte, W; Coulton, J W

1998-07-01

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Size-exclusion chromatography using core-shell particles.

PubMed

Pirok, Bob W J; Breuer, Pascal; Hoppe, Serafine J M; Chitty, Mike; Welch, Emmet; Farkas, Tivadar; van der Wal, Sjoerd; Peters, Ron; Schoenmakers, Peter J

2017-02-24

Size-exclusion chromatography (SEC) is an indispensable technique for the separation of high-molecular-weight analytes and for determining molar-mass distributions. The potential application of SEC as second-dimension separation in comprehensive two-dimensional liquid chromatography demands very short analysis times. Liquid chromatography benefits from the advent of highly efficient core-shell packing materials, but because of the reduced total pore volume these materials have so far not been explored in SEC. The feasibility of using core-shell particles in SEC has been investigated and contemporary core-shell materials were compared with conventional packing materials for SEC. Columns packed with very small core-shell particles showed excellent resolution in specific molar-mass ranges, depending on the pore size. The analysis times were about an order of magnitude shorter than what could be achieved using conventional SEC columns. Copyright © 2016 Elsevier B.V. All rights reserved.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

New insights into liquid chromatography for more eco-friendly analysis of pharmaceuticals.

PubMed

Shaaban, Heba

2016-10-01

Greening the analytical methods used for analysis of pharmaceuticals has been receiving great interest aimed at eliminating or minimizing the amount of organic solvents consumed daily worldwide without loss in chromatographic performance. Traditional analytical LC techniques employed in pharmaceutical analysis consume tremendous amounts of hazardous solvents and consequently generate large amounts of waste. The monetary and ecological impact of using large amounts of solvents and waste disposal motivated the analytical community to search for alternatives to replace polluting analytical methodologies with clean ones. In this context, implementing the principles of green analytical chemistry (GAC) in analytical laboratories is highly desired. This review gives a comprehensive overview on different green LC pathways for implementing GAC principles in analytical laboratories and focuses on evaluating the greenness of LC analytical procedures. This review presents green LC approaches for eco-friendly analysis of pharmaceuticals in industrial, biological, and environmental matrices. Graphical Abstract Green pathways of liquid chromatography for more eco-friendly analysis of pharmaceuticals.

Determination of mycotoxins produced by Fusarium isolates from banana fruits by capillary gas chromatography and high-performance liquid chromatography.

PubMed

Jiménez, M; Mateo, R

1997-08-22

A method of analysis for trichothecenes (nivalenol, deoxynivalenol, 3- and 15-acetyldeoxynivalenol, diacetoxyscirpenol, neosolaniol, T-2 tetraol, T-2 and HT-2 toxins), zearalenone and zearalenols, and another method for determination of fumonisin B1 are described and applied to cultures of Fusarium isolated from bananas. Both methods were adapted from different techniques of extraction, clean-up and determination of these mycotoxins. The first method involves extraction with methanol-1% aqueous sodium chloride, clean-up of extracts by partition with hexane and dichloromethane, additional solid reversed-phase clean-up and analysis of two eluates by both high-performance liquid chromatography with ultraviolet detection and capillary gas chromatography. The method for fumonisin B1 implies extraction with aqueous methanol, concentration, clean-up with water and methanol on Amberlite XAD-2 column, formation of a fluorescent 4-fluoro-7-nitrobenzofurazan derivative and analysis by high-performance liquid chromatography with fluorescence detection. Both procedures give good limits of detection and recoveries, and are considered suitable for the detection and quantification of the studied toxins in corn and rice cultures of Fusarium spp. isolated from banana fruits.

High performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC) for the qualitative and quantitative analysis of Calendula officinalis-advantages and limitations.

PubMed

Loescher, Christine M; Morton, David W; Razic, Slavica; Agatonovic-Kustrin, Snezana

2014-09-01

Chromatography techniques such as HPTLC and HPLC are commonly used to produce a chemical fingerprint of a plant to allow identification and quantify the main constituents within the plant. The aims of this study were to compare HPTLC and HPLC, for qualitative and quantitative analysis of the major constituents of Calendula officinalis and to investigate the effect of different extraction techniques on the C. officinalis extract composition from different parts of the plant. The results found HPTLC to be effective for qualitative analysis, however, HPLC was found to be more accurate for quantitative analysis. A combination of the two methods may be useful in a quality control setting as it would allow rapid qualitative analysis of herbal material while maintaining accurate quantification of extract composition. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

High performance liquid chromatography of substituted aromatics with the metal-organic framework MIL-100(Fe): Mechanism analysis and model-based prediction.

PubMed

Qin, Weiwei; Silvestre, Martin Eduardo; Li, Yongli; Franzreb, Matthias

2016-02-05

Metal-organic framework (MOF) MIL-100(Fe) with well-defined thickness was homogenously coated onto the outer surface of magnetic microparticles via a liquid-phase epitaxy method. The as-synthesized MIL-100(Fe) was used as stationary phase for high-performance liquid chromatography (HPLC) and separations of two groups of mixed aromatic hydrocarbons (toluene, styrene and p-xylene; acetanilide, 2-nirtoaniline and 1-naphthylamine) using methanol/water as mobile phase were performed to evaluate its performance. Increasing water content of the mobile phase composition can greatly improve the separations on the expense of a longer elution time. Stepwise elution significantly shortens the elution time of acetanilide, 2-nirtoaniline and 1-naphthylamine mixtures, while still achieving a baseline separation. Combining the experimental results and in-depth modeling using a recently developed chromatographic software (ChromX), adsorption equilibrium parameters, including the affinities and maximum capacities, for each analyte toward the MIL-100(Fe) are obtained. In addition, the pore diffusivity of aromatic hydrocarbons within MIL-100(Fe) was determined to be 5×10(-12)m(2)s(-1). While the affinities of MIL-100(Fe) toward the analyte molecules differs much, the maximum capacities of the analytes are in a narrow range with q*MOFmax,toluene=3.55molL(-1), q*MOFmax,styrene or p-xylene=3.53molL(-1), and q*MOFmax,anilines=3.12molL(-1) corresponding to approximately 842 toluene and 838 styrene or p-xylene, and 740 aniline molecules per MIL-100(Fe) unit cell, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Comprehensive two-dimensional gas chromatography for biogas and biomethane analysis.

PubMed

Hilaire, F; Basset, E; Bayard, R; Gallardo, M; Thiebaut, D; Vial, J

2017-11-17

The gas industry is going to be revolutionized by being able to generate bioenergy from biomass. The production of biomethane - a green substitute of natural gas - is growing in Europe and the United-States of America. Biomethane can be injected into the gas grid or used as fuel for vehicles after compression. Due to various biomass inputs (e.g. agricultural wastes, sludges from sewage treatment plants, etc.), production processes (e.g. anaerobic digestion, municipal solid waste (MSW) landfills), seasonal effects and purification processes (e.g. gas scrubbers, pressure swing adsorption, membranes for biogas upgrading), the composition and quality of biogas and biomethane produced is difficult to assess. All previous publications dealing with biogas analysis reported that hundreds of chemicals from ten chemical families do exist in trace amounts in biogas. However, to the best of our knowledge, no study reported a detailed analysis or the implementation of comprehensive two-dimensional gas chromatography (GC×GC) for biogas matrices. This is the reason why the benefit of implementing two-dimensional gas chromatography for the characterization of biogas and biomethane samples was evaluated. In a first step, a standard mixture of 89 compounds belonging to 10 chemical families, representative of those likely to be found, was used to optimize the analytical method. A set consisting of a non-polar and a polar columns, respectively in the first and the second dimension, was used with a modulation period of six seconds. Applied to ten samples of raw biogas, treated biogas and biomethane collected on 4 industrial sites (two MSW landfills, one anaerobic digester on a wastewater treatment plant and one agricultural biogas plant), this analytical method provided a "fingerprint" of the gases composition at the molecular level in all biogas and biomethane samples. Estimated limits of detection (far below the μgNm -3 ) coupled with the resolution of GC×GC allowed the comparison

High-affinity K+ uptake in pepper plants.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2005-06-01

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

Electron affinity of liquid water

DOE PAGES

Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; ...

2018-01-16

Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

Electron affinity of liquid water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco

Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

Column Chromatography To Obtain Organic Cation Sorption Isotherms.

PubMed

Jolin, William C; Sullivan, James; Vasudevan, Dharni; MacKay, Allison A

2016-08-02

Column chromatography was evaluated as a method to obtain organic cation sorption isotherms for environmental solids while using the peak skewness to identify the linear range of the sorption isotherm. Custom packed HPLC columns and standard batch sorption techniques were used to intercompare sorption isotherms and solid-water sorption coefficients (Kd) for four organic cations (benzylamine, 2,4-dichlorobenzylamine, phenyltrimethylammonium, oxytetracycline) with two aluminosilicate clay minerals and one soil. A comparison of Freundlich isotherm parameters revealed isotherm linearity or nonlinearity was not significantly different between column chromatography and traditional batch experiments. Importantly, skewness (a metric of eluting peak symmetry) analysis of eluting peaks can establish isotherm linearity, thereby enabling a less labor intensive means to generate the extensive data sets of linear Kd values required for the development of predictive sorption models. Our findings clearly show that column chromatography can reproduce sorption measures from conventional batch experiments with the benefit of lower labor-intensity, faster analysis times, and allow for consistent sorption measures across laboratories with distinct chromatography instrumentation.

[Analysis of pigments from Rhodotorula glutinis by Raman spectroscopy and thin layer chromatography].

PubMed

Yuan, Yu-feng; Tao, Zhan-hua; Wang, Xue; Li, Yong-qing; Liu, Jun-xian

2012-03-01

The pigments from Rhodotorula glutinis were separated by using thin layer chromatography, and the result showed that Rhodotorula glutinis cells could synthesize at least three kinds of pigments, which were beta-carotene, torulene, and torularhodin. The Raman spectra based on the three pigments were acquired, and original spectra were preprocessed by background elimination, baseline correction, and three-point-smoothing, then the averaged spectra from different pigments were investigated, and the result indicated that Raman shift which represents C-C bond was different, and the wave number of beta-carotene demonstrated the largest deviation, finally torulene and torularhodin in Rhodotorula glutinis had more content than beta-carotene. Quantitative analysis of Raman peak height ratio revealed that peak height ratio of pigments showed little difference, which could be used as parameters for further research on living cells, providing reference content of pigments. The above results suggest that Raman spectroscopy combined with thin layer chromatography can be applied to analyze pigments from Rhodotorula glutinis, provides abundant information about pigments, and serves as an effective method to study pigments.

Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

PubMed

Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

2013-07-02

Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

High Performance Liquid Chromatography

NASA Astrophysics Data System (ADS)

Talcott, Stephen

High performance liquid chromatography (HPLC) has many applications in food chemistry. Food components that have been analyzed with HPLC include organic acids, vitamins, amino acids, sugars, nitrosamines, certain pesticides, metabolites, fatty acids, aflatoxins, pigments, and certain food additives. Unlike gas chromatography, it is not necessary for the compound being analyzed to be volatile. It is necessary, however, for the compounds to have some solubility in the mobile phase. It is important that the solubilized samples for injection be free from all particulate matter, so centrifugation and filtration are common procedures. Also, solid-phase extraction is used commonly in sample preparation to remove interfering compounds from the sample matrix prior to HPLC analysis.

ANALYSIS OF ELECTROLESS NICKEL SOLUTIONS BY ANION CHROMATOGRAPHY

EPA Science Inventory

The principal appeal of ion chromatography (IC) as analytical technique lies in the ability to rapidly analyze a mixture of ions of widely varying concentrations and properties in a single elution. It is therefore not surprising that IC has been hampered by the similar ion exchan...

Quantitative determination of triterpenes from Amphiptherygium adstringens by liquid chromatography and thin-layer chromatography and morphological analysis of cuachalalate preparations.

PubMed

Navarrete, Andres; Avula, Bharathi; Joshi, Vaishali C; Ji, Xiuhong; Hersh, Paul; Khan, Ikhlas A

2006-01-01

Amphiptherygium adstringens (Anacardiaceae/Julianaceae), local name "cuachalalate," is used in folk medicine for the treatment of cholelithiasis, fevers, fresh wounds, hypercholesterolemia, gastritis, gastric ulcers, and cancer of the gastrointestinal tract. The development of column high-performance liquid chromatography-photodiode array detector (LC-PDA) and high-performance thin-layer chromatography (HPTLC)-densitometry methods for the determination of masticadienonic acid and 3-hydroxymasticadienonic acid in cuachalalate preparations is described in this paper. Good separation of the compounds could be achieved by both methods. Either might be preparable depending on the requirements. The LC separation was performed on a Phenomenex Synergi MAX-RP 80A reversed-phase column operated at 40 degrees C with detection at 215 nm. The plant materials were extracted with methanol by sonication. The triterpenes present in the plant material and commercial extracts were separated with an acetonitrile-water reagent alcohol isocratic system. The limit of detection was 0.1-0.2 microg/mL. The relative standard deviation values for the determination of triterpenes in plant extracts were less than 1.00%. This is the first report of an analytical method developed for the quantitative analysis of triterpenes from Amphiptherygium adstringens by LC-PDA and HPTLC. The stem bark showed higher amounts of triterpenes, and low amounts in root and stem root. The microscopic description of the crude drug of cuachalalate was also provided.

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Chromatography resin support

DOEpatents

Dobos, James G.

2002-01-01

An apparatus and method of using an improved chromatography resin support is disclosed. The chromatography support platform is provided by a stainless steel hollow cylinder adapted for being inserted into a chromatography column. An exterior wall of the stainless steel cylinder defines a groove for carrying therein an "O"-ring. The upper surface of the stainless steel column is covered by a fine stainless steel mesh welded to the edges of the stainless steel cylinder. When placed upon a receiving ledge defined within a chromatography column, the "O"-ring provides a fluid tight seal with the inner edge wall of the chromatography cylinder. The stainless steel mesh supports the chromatography matrix and provides a back flushable support which is economical and simple to construct.

Ionic liquids as silica deactivating agents in gas chromatography for direct analysis of primary amines in water.

PubMed

Krzyżaniak, Agnieszka; Weggemans, Wilko; Schuur, Boelo; de Haan, André B

2011-12-16

Analysis of primary amines in aqueous samples remains a challenging analytical issue. The preferred approach by gas chromatography is hampered by interactions of free silanol groups with the highly reactive amine groups, resulting in inconsistent measurements. Here, we report a method for direct analysis of aliphatic amines and diamines in aqueous samples by gas chromatography (GC) with silanol deactivation using ionic liquids (ILs). ILs including trihexyl(tetradecyl)phosphonium bis 2,4,4-(trimethylpentyl)phosphinate (Cyphos IL-104), 1-methyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide [pmim][Tf(2)N] and N″-ethyl-N,N,N',N'-tetramethylguanidinium tris(pentafluoroethyl)trifluorophosphate [etmg][FAP] were tested as deactivating media for the GC liner. Solutions of these ILs in methanol were injected in the system prior to the analysis of primary amines. Butane-1,4-diamine (putrescine, BDA) was used as a reference amine. The best results were obtained using the imidazolium IL [pmim][Tf(2)N]. With this deactivator, excellent reproducibility of the analysis was achieved, and the detection limit of BDA was as low as 1mM. The applicability of the method was proven for the analysis of two different primary amines (C4-C5) and pentane-1,5-diamine. Copyright © 2011 Elsevier B.V. All rights reserved.

Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies.

PubMed

Nelson, Bryce; Adams, Jarrett; Kuglstatter, Andreas; Li, Zhijian; Harris, Seth F; Liu, Yang; Bohini, Sandya; Ma, Han; Klumpp, Klaus; Gao, Junjun; Sidhu, Sachdev S

2018-07-06

Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Thin-Layer Chromatography Experiments That Illustrate General Problems in Chromatography.

ERIC Educational Resources Information Center

Lederer, M.; Leipzig-Pagani, E.

1996-01-01

Describes experiments that illustrate a number of general principles such as pattern identification, displacement chromatography, and salting-out adsorption, plus an experiment that demonstrates that identification by chromatography alone is impossible. Illustrates that chromatography is still possible with quite simple means, notwithstanding the…

Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates.

PubMed

Moerdijk-Poortvliet, Tanja C W; Schierbeek, Henk; Houtekamer, Marco; van Engeland, Tom; Derrien, Delphine; Stal, Lucas J; Boschker, Henricus T S

2015-07-15

We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ(13)C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although LC/IRMS is expected to be more accurate and precise, no direct comparison has been reported. GC/IRMS with the aldonitrile penta-acetate (ANPA) derivatisation method was compared with LC/IRMS without derivatisation. A large number of glucose standards and a variety of natural samples were analysed for five neutral carbohydrates at natural abundance as well as at (13)C-enriched levels. Gas chromatography/chemical ionisation mass spectrometry (GC/CIMS) was applied to check for incomplete derivatisation of the carbohydrate, which would impair the accuracy of the GC/IRMS method. The LC/IRMS technique provided excellent precision (±0.08‰ and ±3.1‰ at natural abundance and enrichment levels, respectively) for the glucose standards and this technique proved to be superior to GC/IRMS (±0.62‰ and ±19.8‰ at natural abundance and enrichment levels, respectively). For GC/IRMS measurements the derivatisation correction and the conversion of carbohydrates into CO2 had a considerable effect on the measured δ(13)C values. However, we did not find any significant differences in the accuracy of the two techniques over the full range of natural δ(13)C abundances and (13)C-labelled glucose. The difference in the performance of GC/IRMS and LC/IRMS diminished when the δ(13)C values were measured in natural samples, because the chromatographic performance and background correction became critical factors, particularly for LC/IRMS. The derivatisation of carbohydrates for the GC/IRMS method was complete. Although both LC/IRMS and GC/IRMS are reliable techniques for compound-specific stable carbon isotope analysis of carbohydrates (provided that derivatisation is complete and the

A Figment of Their Imagination: Adolescent Poetic Literacy in an Online Affinity Space

ERIC Educational Resources Information Center

Padgett, Elizabeth R.; Curwood, Jen Scott

2016-01-01

Drawing on sociocultural perspectives, this case study investigated ways that youths write, read, and critique poetry in an online affinity space. Specifically, it used a thematic analysis of interviews, a linguistic analysis of online feedback mechanisms, and poetic analysis to gain insight into how four teens engaged with poetry on the Figment…

Rapid direct analysis to discriminate geographic origin of extra virgin olive oils by flash gas chromatography electronic nose and chemometrics.

PubMed

Melucci, Dora; Bendini, Alessandra; Tesini, Federica; Barbieri, Sara; Zappi, Alessandro; Vichi, Stefania; Conte, Lanfranco; Gallina Toschi, Tullia

2016-08-01

At present, the geographical origin of extra virgin olive oils can be ensured by documented traceability, although chemical analysis may add information that is useful for possible confirmation. This preliminary study investigated the effectiveness of flash gas chromatography electronic nose and multivariate data analysis to perform rapid screening of commercial extra virgin olive oils characterized by a different geographical origin declared in the label. A comparison with solid phase micro extraction coupled to gas chromatography mass spectrometry was also performed. The new method is suitable to verify the geographic origin of extra virgin olive oils based on principal components analysis and discriminant analysis applied to the volatile profile of the headspace as a fingerprint. The selected variables were suitable in discriminating between "100% Italian" and "non-100% Italian" oils. Partial least squares discriminant analysis also allowed prediction of the degree of membership of unknown samples to the classes examined. Copyright © 2016. Published by Elsevier Ltd.

Update of on-line coupled liquid chromatography - gas chromatography for the analysis of mineral oil hydrocarbons in foods and cosmetics.

PubMed

Biedermann, Maurus; Munoz, Celine; Grob, Koni

2017-10-27

On-line coupled high performance liquid chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID) is the most widely used method for the analysis of mineral oil hydrocarbons in food, food contact materials, tissues and cosmetics. With comprehensive two-dimensional gas chromatography (GCxGC), a tool became available for better establishing the elution sequence of the various types of hydrocarbons from the HPLC column used for isolating the mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). The performance of a heavily used HPLC column with reduced retention for MOAH was investigated to improve the robustness of the method. Updates are recommended that render the MOSH/MOAH separation less dependent of the state of the HPLC column and more correct in cases of highly refined mineral oil products of high molecular mass. Cyclohexyl cyclohexane (Cycy), used as internal standard, turned out to be eluted slightly after cholestane (Cho); apparently the size exclusion effect predominates the extra retention by ring number on the 60Å pore size silica gel. Hence, Cycy can be used to determine the end of the MOSH fraction. Long chain alkyl benzenes were eluted earlier than tri-tert. butyl benzene (Tbb). It is proposed to start the MOAH transfer immediately after the MOSH fraction and use a gradient causing breakthrough of dichloromethane (visible in the UV chromatogram) at a time suitable to elute perylene (Per) at the end of the fraction. In this way, a decrease in retention power of the HPLC column can be tolerated without adjustment of the MOAH fraction until some MOAH start being eluted into the MOSH fraction. This critical point can be checked either with di(2-ethylhexyl) benzene (DEHB) as a marker or the HPLC-UV chromatogram. Finally, based on new findings in rats and human tissues, it is recommended to integrate the MOSH and MOAH up to the retention time of the n-alkane C40. Copyright © 2017 Elsevier B.V. All rights

Extreme disorder in an ultrahigh-affinity protein complex

NASA Astrophysics Data System (ADS)

Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

2018-03-01

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

Centrifugal precipitation chromatography

PubMed Central

Ito, Yoichiro; Lin, Qi

2009-01-01

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. The 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. PMID:19541553

Data analysis and calibration for a bulk-refractive-index-compensated surface plasmon resonance affinity sensor

NASA Astrophysics Data System (ADS)

Chinowsky, Timothy M.; Yee, Sinclair S.

2002-02-01

Surface plasmon resonance (SPR) affinity sensing, the problem of bulk refractive index (RI) interference in SPR sensing, and a sensor developed to overcome this problem are briefly reviewed. The sensor uses a design based on Texas Instruments' Spreeta SPR sensor to simultaneously measure both bulk and surface RI. The bulk RI measurement is then used to compensate the surface measurement and remove the effects of bulk RI interference. To achieve accurate compensation, robust data analysis and calibration techniques are necessary. Simple linear data analysis techniques derived from measurements of the sensor response were found to provide a versatile, low noise method for extracting measurements of bulk and surface refractive index from the raw sensor data. Automatic calibration using RI gradients was used to correct the linear estimates, enabling the sensor to produce accurate data even when the sensor has a complicated nonlinear response which varies with time. The calibration procedure is described, and the factors influencing calibration accuracy are discussed. Data analysis and calibration principles are illustrated with an experiment in which sucrose and detergent solutions are used to produce changes in bulk and surface RI, respectively.

Structure of Greyhound hemoglobin: origin of high oxygen affinity.

PubMed

Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

2011-05-01

This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

Single-cell measurement of red blood cell oxygen affinity.

PubMed

Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M; Schonbrun, Ethan

2015-08-11

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

Single-cell measurement of red blood cell oxygen affinity

PubMed Central

Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

2015-01-01

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen–Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2–3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability. PMID:26216973

EXTRACTION AND QUANTITATIVE ANALYSIS OF ELEMENTAL SULFUR FROM SULFIDE MINERAL SURFACES BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY. (R826189)

EPA Science Inventory

A simple method for the quantitative determination of elemental sulfur on oxidized sulfide minerals is described. Extraction of elemental sulfur in perchloroethylene and subsequent analysis with high-performance liquid chromatography were used to ascertain the total elemental ...

Determination of dasatinib in the tablet dosage form by ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis.

PubMed

Gonzalez, Aroa Garcia; Taraba, Lukáš; Hraníček, Jakub; Kozlík, Petr; Coufal, Pavel

2017-01-01

Dasatinib is a novel oral prescription drug proposed for treating adult patients with chronic myeloid leukemia. Three analytical methods, namely ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis, were developed, validated, and compared for determination of the drug in the tablet dosage form. The total analysis time of optimized ultra high performance liquid chromatography and capillary zone electrophoresis methods was 2.0 and 2.2 min, respectively. Direct ultraviolet detection with detection wavelength of 322 nm was employed in both cases. The optimized sequential injection analysis method was based on spectrophotometric detection of dasatinib after a simple colorimetric reaction with folin ciocalteau reagent forming a blue-colored complex with an absorbance maximum at 745 nm. The total analysis time was 2.5 min. The ultra high performance liquid chromatography method provided the lowest detection and quantitation limits and the most precise and accurate results. All three newly developed methods were demonstrated to be specific, linear, sensitive, precise, and accurate, providing results satisfactorily meeting the requirements of the pharmaceutical industry, and can be employed for the routine determination of the active pharmaceutical ingredient in the tablet dosage form. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative analysis of psilocybin and psilocin in psilocybe baeocystis (Singer and Smith) by high-performance liquid chromatography and by thin-layer chromatography.

PubMed

Beug, M W; Bigwood, J

1981-03-27

Rapid quantification of psilocybin and psilocin in extracts of wild mushrooms is accomplished by reversed-phase high-performance liquid chromatography with paired-ion reagents. Nine solvent systems and three solid supports are evaluated for their efficiency in separating psilocybin, psilocin and other components of crude mushroom extracts by thin-layer chromatography.

New exopolysaccharides produced by Aureobasidium pullulans grown on glucosamine.

PubMed

Cescutti, Paola; Pupulin, Raffaella; Delben, Franco; Abbate, Maria; Dentini, Mariella; Sparapano, Lorenzo; Rizzo, Roberto; Crescenzi, Vittorio

2002-07-16

The polysaccharides produced by Aureobasidium pullulans, grown using glucosamine as the carbon source, were investigated by means of methylation analysis, affinity chromatography and NMR spectroscopy. The results indicated that, besides a small amount of pullulan, this micro-organism was capable of producing-in low yields-mixtures of at least two different complex polysaccharides containing mainly mannose and galactose. (1)H NMR spectra of two fractions obtained by lectin affinity chromatography indicated that one polymer was constituted exclusively of mannose residues while the other contained both galactofuranosyl and mannopyranosyl residues.

Analysis of trace dicyandiamide in stream water using solid phase extraction and liquid chromatography UV spectrometry.

PubMed

Qiu, Huidong; Sun, Dongdi; Gunatilake, Sameera R; She, Jinyan; Mlsna, Todd E

2015-09-01

An improved method for trace level quantification of dicyandiamide in stream water has been developed. This method includes sample pretreatment using solid phase extraction. The extraction procedure (including loading, washing, and eluting) used a flow rate of 1.0mL/min, and dicyandiamide was eluted with 20mL of a methanol/acetonitrile mixture (V/V=2:3), followed by pre-concentration using nitrogen evaporation and analysis with high performance liquid chromatography-ultraviolet spectroscopy (HPLC-UV). Sample extraction was carried out using a Waters Sep-Pak AC-2 Cartridge (with activated carbon). Separation was achieved on a ZIC(®)-Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) (50mm×2.1mm, 3.5μm) chromatography column and quantification was accomplished based on UV absorbance. A reliable linear relationship was obtained for the calibration curve using standard solutions (R(2)>0.999). Recoveries for dicyandiamide ranged from 84.6% to 96.8%, and the relative standard deviations (RSDs, n=3) were below 6.1% with a detection limit of 5.0ng/mL for stream water samples. Copyright © 2015. Published by Elsevier B.V.

High-affinity cannabinoid binding site in brain: A possible marijuana receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nye, J.S.

The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one classmore » of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.« less

Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

PubMed

Jiang, Han-Peng; Chu, Jie-Mei; Lan, Meng-Dan; Liu, Ping; Yang, Na; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

2016-09-02

More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two

Employees' Perceptions of Female Affinity Groups

ERIC Educational Resources Information Center

Gabriel, Peter J.

2017-01-01

Female affinity groups offered by corporations are formalized corporate initiatives focused on networking and professional development opportunities for women (Welbourne & McLaughlin, 2013). The scholarly literature focused on female affinity groups was limited. The purpose of this grounded theory Dissertation in Practice study was to…

Qualitative and quantitative temporal analysis of licit and illicit drugs in wastewater in Australia using liquid chromatography coupled to mass spectrometry.

PubMed

Bade, Richard; White, Jason M; Gerber, Cobus

2018-01-01

The combination of qualitative and quantitative bimonthly analysis of pharmaceuticals and illicit drugs using liquid chromatography coupled to mass spectrometry is presented. A liquid chromatography-quadrupole time of flight instrument equipped with Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) was used to qualitatively screen 346 compounds in influent wastewater from two wastewater treatment plants in South Australia over a 14-month period. A total of 100 compounds were confirmed and/or detected using this strategy, with 61 confirmed in all samples including antidepressants (amitriptyline, dothiepin, doxepin), antipsychotics (amisulpride, clozapine), illicit drugs (cocaine, methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA)), and known drug adulterants (lidocaine and tetramisole). A subset of these compounds was also included in a quantitative method, analyzed on a liquid chromatography-triple quadrupole mass spectrometer. The use of illicit stimulants (methamphetamine) showed a clear decrease, levels of opioid analgesics (morphine and methadone) remained relatively stable, while the use of new psychoactive substances (methylenedioxypyrovalerone (MDPV) and Alpha PVP) varied with no visible trend. This work demonstrates the value that high-frequency sampling combined with quantitative and qualitative analysis can deliver. Graphical abstract Temporal analysis of licit and illicit drugs in South Australia.

Affinity+: Semi-Structured Brainstorming on Large Displays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.

2013-04-27

Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

High performance thin layer chromatography fingerprint analysis of guava (Psidium guajava) leaves

NASA Astrophysics Data System (ADS)

Astuti, M.; Darusman, L. K.; Rafi, M.