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Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses.

A simple multidimensional liquid chromatography system utilizing an isocratic pump and a HPLC system is described for the comprehensive proteomic analysis of complex peptide digest mixtures by coupled LC-LC-MS-MS techniques. A binary ion-exchange separation was achieved through the use of a strong cation-exchange column followed by a reversed-phase column for data-dependent LC-MS-MS analysis of the unbound analytes, and following salt elution (and concomitant column reequilibration), the bound analytes. Off-line validation of the platform showed near quantitative recovery of fractionated peptides and essentially complete ion-exchange partitioning. In comparative analyses of a highly complex peptide digest mixture a >40% increase in the number of peptide and protein identifications was achieved using this multidimensional platform compared to an unfractionated control.

Amino Acid Sequence↗

Nuclear magnetic resonance chemical shifts of potassium ions (39K) on ion exchange resins and in muscle.

Chemical shifts of potassium (39K) were calculated from frequencies of beat patterns measured by a pulsed NMR method. This method indicates large chemical shifts for 3 molar KNO3 and 6 molal KI in good quantitative agreement with the steady state NMR measurements of previous investigators. 39K on two wet ion exchange resins and 39K in fresh muscle show insignificant chemical shifts relative to 0.1 M KCl solution. Previous studies showed marked shortening of NMR relaxation times (T1 and T2) for 39K in ion exchange resins and in muscle compared to free solution. These results seem to indicate that even though potassium on resins and in muscle experiences high electric field gradients, these have relatively little effect on the chemical shift of potassium, which may be correlated with the low pK values of the anionic groups in the resins and muscle.

Animals↗

Column ion exchange chromatographic production of human immune serum globulin for intravenous use.

Column ion exchange chromatographic processes were developed for the production of immune serum globulin (ISG) for intravenous use. Sequential chromatography on two cross-linked agarose gel anion exchangers yielded the same immunoelectrophoretic pure ISG as a single column DEAE-Sephadex procedure with the advantage of repeated 'in column' cycling. ISG with a low content of aggregated IgG and low anticomplementary activity was prepared from plasma with 53-69% efficiency. The ISG prepared by these processes was characterized with respect to the content of IgA, prekallikrein activator (PKA), plasminogen, fragmentation, and heavy chain subclass distribution.

Chromatography, Ion Exchange↗

Removal of THM precursors by coagulation or ion exchange.

The removal of natural organic matter (NOM) from drinking water supplies can be achieved by different processes, among them coagulation and adsorption. Synthetic waters made from concentrates of humic substances from reservoir and river waters were tested in the laboratory for ease of removAl of NOM by coagulation with cationic organic polymers and with alum, and by adsorption on anion exchangers. For polymers such as high molecular weight polydiallyldimethylammonium chloride (polyDADMAC) and cationic polyacrylamides of high charge, performance was nearly as effective as alum, with colour removals 86-100% of those obtained for alum. Ion exchange using the best commercially available resins designed for this purpose, a gel polystyrene and a macroporous acrylic resin, was more effective than alum treatment for two of the natural waters studied, but inferior for a third. The resins were overall superior to cationic polymers. The NOM was separated into four fractions based on hydrophobic and hydrophilic properties. Alum was not as effective as ion exchange for the elimination of individual ionic NOM fractions. It was better than cationic polymers for removal of humic and fulvic acids, although polyDADMAC was as good for one water. For the removal of charged compounds alum then polyDADMAC were the best performers for that water. Unequivocal evidence was obtained that coagulants remove material that is not adsorbed by resins, and vice versa. A combination of coagulation with a cationic polymer and adsorption by an anion exchanger removed essentially all of the NOM. The preference of the coagulants was for the larger, more hydrophobic molecules, and of resins for smaller highly charged hydrophilic molecules. Each fraction had trihalomethane formation potentials in the range 11-24 microg/mg, except for one water that was more reactive. Hence, the actual amount of each fraction in the original water becomes a crucial factor.

Alum Compounds↗

Models for biological ion exchangers. II. Solvent selectivity in DMSO-water-Dowex 50W.

Dimethylsulfoxide-water-Dowex 50W-X8 systems are characterized by measurements of solvent selectively and proton magnetic resonance spectra. The H+-, Li+-, NH4+-, NHe4+-, NBu4+-, Mg2+-, Zn2+-, and La3+- forms are studied over a wide range of binary-solvent mole fractions. The relative selectivities for water by the ion exchanger, based on an integrated Kipling parameter, are Zn2+-form (reference) --1.00, Mg2+ --0.43, La3+ --0.38, Li+ +0.17, NMe4+ 0.20, NH4+ 0.31, NBU4+ 0.33, and H+ 0.68, the polyvalent counterions preferring DMSO. All of the ionic forms except the NH4+-form exhibit over much of the mole fraction range separations between the external water and the internal water peaks exceeding 50 Hz, the magnitude of the separation varying with the counter-ion. Comparison of results is facilitated by maintaining a constant ratio between the total number of moles of solvent and the number of equivalents of ions exchanger.

Cation Exchange Resins↗

Products for the treatment of hemophilia A and B prepared by ion exchange chromatography.

In France, since 1988, factor VIII concentrate are prepared by ion exchanged chromatography from plasma cryoprecipitate. The final product is a very high purity factor VIII with a specific activity (SA) between 150 and 250 IU/mg of proteins. Viral inactivation is obtained after solvent detergent method with TNBP and Tween 80. Since 1989 hemophilia B patients are treated with a factor IX preparation purified from a prothrombin complex concentrate and after solvent detergent inactivation. Factor IX concentrate is a high purity product with a SA of 50 to 100 U/mg of protein.

Chromatography, Ion Exchange↗

Ion exchange tumor targeting: a new approach.

Connective tissues are distinguished by the types, concentrations, and organizations of material in the extracellular matrix. Many physiological functions are determined largely by the nature and organization of the extracellular components. The components are characterized by their content and distribution of charged, mostly anionic groups. The distinct roles played by the charges are sometimes modeled by analogy to the transport theory of ion exchange resins. The intent of this study was to investigate whether the properties of the tumor matrix could be used for selective, charge-dependent accumulation of charge-modified dextran. Ten patients with diagnosed superficial urinary bladder carcinoma were included in the study. They received intravesical instillations of technetium-99m-labeled charge-modified dextran derivatives (approximately 0.1-1 mg; approximately 50 MBq in saline; 30-min incubation). After treatment and resection, samples were taken from normal and diseased tissue. The result clearly demonstrated a charge-dependent difference in the quotient of radioactive uptake in tumor tissue: normal tissue. Instillations of cationic dextran yielded a high quotient, up to 3000. Normal tissue had background activity. Anionic dextran yielded a low quotient, 1.8-2, with increased background (i.e. uptake in normal tissue). Neutral dextran gave a quotient of up to 90. No radioactivity could be detected in blood. The tumors in this study apparently displayed cation-exchanging properties. We will continue this investigation and determine whether this is a general property of bladder carcinomas and whether other carcinomas display ion exchange properties. If this is the case, the finding could have important implications for the local treatment of several cancers.

Dextrans↗

Effect of charge regulation on steric mass-action equilibrium for the ion-exchange adsorption of proteins.

A thermodynamic formalism is developed for incorporating the effects of charge regulation on the ion-exchange adsorption of proteins under mass-overloaded conditions as described by the steric mass-action (SMA) isotherm. To accomplish this, the pH titration behavior of a protein and the associated adsorption equilibrium of the various charged forms of a protein are incorporated into a model which also accounts for the steric hindrance of salt counterions caused by protein adsorption. For the case where the protein is dilute, the new model reduces to the protein adsorption model described recently by the authors which accounts for charge regulation. Similarly, the new model reduces to the steric mass-action isotherm developed by Brooks and Cramer which applies to mass-overloaded conditions for the case where charge regulation is ignored so that the protein has a fixed charge. Calculations using the new model were found to agree with experimental data for the adsorption of bovine serum albumin (BSA) on an anion-exchange column packing when using reasonable physical properties. The new model was also used to develop an improved theoretical criterion for determining the conditions required for an adsorbed species to displace a protein in displacement chromatography when the pH is near the protein pI.

Adsorption↗

[Clinical study on unmodified intravenous dried ion-exchange resin treated human normal immunoglobulin, SM-4300].

Unmodified intravenous dried ion-exchange resin treated human normal immunoglobulin, SM-4300, was administered to 4 patients with severely intractable infectious diseases. Obvious antipyretic effect, opsonic effect and and investigational findings on healing are not observed. However, in both subjective and objective clinical effects, we obtained the following test results, that is, 1 case of effective and 2 cases of rather effective. No side effects by SM-4300 were observed.

Adult↗

[A study of DEAE ion exchange chromatography for the purification of hen's yolk IgG].

A DEAE ion exchange chromatography procedure performed by using either gradient or stepwise elution was developed for the purification of chicken IgG antibodies from the egg yolk of immunized hens. Following this procedure, highly purified yolk IgG preparations were obtained as identified by Sephadex G 200 gel filtration as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, this procedure guaranteed the recovery of antibodies of undiminished activity. The results demonstrate that the procedure is simple, reliable and practical for the yolk IgG purification. The establishment of such a method is beneficial to the production and application of egg yolk antibodies.

Animals↗

Preparation and characterization of polyalkene membranes modified with four different ion-exchange groups by radiation-induced graft polymerization.

Three different cation-exchange membranes with carboxylic acid, phosphoric acid, and sulfonic acid groups, and one anion-exchange membrane with a triethylamine group were prepared by radiation-induced graft polymerization. The maximum capacities of the carboxylic acid, phosphoric acid, sulfonic acid, and triethylamine groups were 3.0, 2.30, 2.43, and 1.42 mmol/g, respectively. The physico-chemical properties of the polyalkene non-woven fabric membrane with four different ion-exchange groups were examined by thermogravimetric analysis, X-ray photoelectron spectrometry (XPS), and scanning electron microscopy. The prepared ion-exchange membrane was applied to the removal of terephthalic acid in dyeing wastewater.

Carboxylic Acids↗

Isolation of glycosylated hemoglobins by a combination of ion-exchange and gel-filtration chromatographies: a pilot study.

The techniques of ion exchange and gel filtration have been combined in a single chromatographic column which allows the simultaneous isolation of hemoglobins glycosylated at their beta-amino termini from other hemoglobin species as well as from molecules differing in size from the hemoglobins. This method is unique because it makes possible isolation of preparative quantities of glycosylated hemoglobins within approximately 15 min. The method works most efficiently with a dry weight-to-weight ratio of Biorex 70 to Sephadex G25 of 1.4 to 1.0. The technique was applied to the determination of the apparent first-order rate constant for the deglycosylation of the labile form of hemoglobin AIc.

Chromatography, Gel↗

Ion-exchange chromatographic determination of hypoglycin A in canned ackee fruit.

An ion-exchange chromatographic method was developed to determine hypoglycin A (HG-A) levels in canned ackee fruit by using an amino acid analyzer. HG-A was extracted by homogenizing the sample in 80% alcohol. An isocratic buffer system, consisting of 30% sodium citrate buffer (pH 3.15) and 70% sodium chloride-sodium acetate buffer (pH 7.40) was used to obtain baseline separation between HG-A and the other amino acids. The system can detect HG-A levels as low as 4.8 micrograms/mL. HG-A levels in the edible portion of fruit in 6 cans ranged from 11.0 to 66.5 mg HG-A/can. Recoveries by standard addition averaged 102.5%.

Amino Acids↗

Drug loading onto ion-exchange microspheres: modeling study and experimental verification.

A new mathematical model was developed and an exact analytical solution without approximations of previous work was derived for the description of the kinetics and equilibrium characteristics of drug loading from a finite external solution onto ion-exchange microspheres. The influence of important parameters pertinent to material properties and loading conditions on the kinetics, efficiency, and equilibrium of drug loading was analyzed using the developed model and equations. The numerical results showed that the rate of drug loading increased with increasing initial drug concentration in the solution or with the relative volume of the external solution and the microsphere. The maximum binding capacity of the micrsophere and the association rate constant had positive effects on the loading rate and the equilibrium loading. A decrease in microsphere radius or an increase in drug diffusion coefficient accelerated the loading process but did not influence the equilibrium drug loading. The model prediction agreed with experimental results of verapamil hydrochloride loading onto sulfopropyl dextran microspheres. The usefulness of the model in the design of loading experiments for desired drug loading efficiency and equilibrium loading was demonstrated by numerical analysis.

Biocompatible Materials↗

Analysis of cyanobacterial-derived saxitoxins using high-performance ion exchange chromatography with chemical oxidation/fluorescence detection.

A single run HPLC method utilizing ion exchange as the separation mode with a novel mobile phase system coupled to chemical postcolumn oxidation and fluorescence detection has been developed and demonstrated to be applicable to the quantitative analysis of paralytic shellfish poisons (PSPs) produced by Australian cyanobacteria (Anabaena circinalis) and other cyanobacteria. Both the cyanobacterial matrix and natural water constituents did not significantly affect the performance of this method. The daily precision of this method was adequate for it to be considered as a routine analytical tool for direct PSP analysis (prePSP concentration is not required) of cyanobacterial extracts and water bodies containing PSPs (C1, C2, GTX2, GTX3, NEO, STX) in the low parts per billion concentration range (10-70 ppb).

Anabaena↗

Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography.

A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab')(2) antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab')(2) from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab')(2) antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab')(2) product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab')(2) antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab')(2) product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.

Animals↗

Breakthrough performance of linear-DNA on ion-exchange membrane columns.

Breakthrough performance of linear-DNA adsorption on ion-exchange membrane columns was theoretically and experimentally investigated using batch and fixed-bed systems. System dispersion curves showed the absence of flow non-idealities in the experimental arrangement. Breakthrough curves were not significantly affected by flow-rate or inlet solution concentration. In the theoretical analysis a model was integrated by the serial coupling of the membrane transport model and the system dispersion model. A transport model that considers finite kinetic rate and column dispersed flow was used in the study. A simplex optimization routine coupled to the solution of the partial differential model equations was employed to estimate the maximum adsorption capacity constant, the equilibrium desorption constant and the forward interaction rate-constant, which are the parameters of the membrane transport model. Through this approach a good prediction of the adsorption phenomena is obtained for inlet concentrations and flow rates greater than 0.2 mg/ml and 0.16 ml/min.

Chromatography, Ion Exchange↗