Screening method for inherited disorders of purine and pyrimidine metabolism by capillary electrophoresis with reversed electroosmotic flow

Capillary electrophoresis with electroosmotic flow reversed by cationic surfactant for diagnosis of purine and pyrimidine inherited enzyme deficiencies is reported. Final separation conditions consist of 45 mM borate, 55 mM N-tris[hydroxymethyl]methylglycine, 10 mM tartrate, 1 mM cetyltrimethylammonium bromide and 0.44% tetrabutylammonium hydroxide-2-amino-2-methyl-1,3-propanediol (pH 8.6). Average sensitivity (2.51 microM), reproducibility of migration times (run-to-run C.V. < or = 0.6%, day-to-day C.V. < or = 2.5%), linearity (R2>0.994) and imprecision (mean intra-assay RSD 4.7% and inter-assay RSD 6.6%) of the method are acceptable for diagnostic purposes. Applicability of the method is demonstrated on urine samples from patients with enzymatically proven enzyme deficiencies.     

Optimization of capillary coating by hydroxyethyl methacrylate for capillary zone electrophoresis of proteins

This work describes further improvements of coating fused silica capillaries with 2-hydroxyethyl methacrylate (HEMA) by atom transfer radical polymerization (ATRP). First, endcapping with a sterically less bulky silanyl reagent reduces the electrosmotic flow (EOF) by 25% in addition to the 40% EOF reduction caused by HEMA coating compared to a bare fused silica capillary. An additional hydrolysis step was introduced into the preparation of HEMA coated capillaries and leads to better reproducible migration times. The influence of the solvent during ATRP and the resulting polymer coating was investigated by replacement of DMF with water or water-methanol mixtures. The quality of the optimized coating was characterized by protein separations at pH 3. HEMA coated capillaries reveal up to 746000 plates. The polyvinyl alcohol (PVA) coated capillary provides only half of this efficiency. A long-term test at pH 9 shows good stability of the HEMA coated capillaries in basic medium. Also the numbers of plates in this medium was about 30% higher than for separations with the PVA capillary. In addition, the phosphate buffer was replaced by a volatile ammonium acetate buffer for later use with mass spectrometry (MS).     

Analysis of recombinant human growth hormone by capillary electrophoresis with bilayer-coated capillaries using UV and MS detection

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.     

Development of a capillary electrophoresis method for the screening of human urine for multiple drugs of abuse

A new capillary electrophoresis (CE) method was developed and validated for the screening of human urine for nineteen drugs of abuse. In order to achieve sufficient separation, the electrolyte composition was modified using beta-cyclodextrin (beta-CD) and organic solvents. To process each sample, a sequential injection-solid-phase extraction (SI-SPE) system was constructed. Using this device, matrix clean-up, extraction, and preconcentration of analytes were performed onto a C(18) cartridge. Optimal separation and detection were obtained using a background electrolyte consisting of 100mM phosphate adjusted to pH 6.0, with 20 mM beta-CD, 5% acetonitrile and 20% isopropanol. Electrokinetic injection was performed at 5 kV for 10s, separation voltage was 25 kV and column temperature was set to 25 degrees C. The separation was carried out in a 67.0 cm x 50 microm fused-silica capillary with UV detection at 214 nm. The combination of SI-SPE and sample stacking showed significant sensitivity enhancement with limits of detection in the range of 5-30 ng ml(-1). A validation study showed good reproducibility of both migration time (RSD=0.003-0.088%) and peak area (RSD=0.54-4.8%). Overall, this automated and miniaturized SI-SPE system provides a rapid, sensitive, and robust procedure for analysis; as well as minimizes sample and solvent consumption.     

Plastic substrates based separation channels in electromigration techniques

Three types of plastic materials (polyester, polyurethane and polymethylmethacrylate) were tested as materials for manufacturing separation columns (polyester and polyurethane capillaries were used) or separation channels (polymethylmethacrylate) in the chip format. A set of 11 fluorescein isothiocyanate amino acid derivatives was used as the test mixture. Using alpha-cyclodextrin additive to the background electrolyte in the case of the chip separation was also tested. The main problem with all plastic separation media was the selectivity of the separation. The best results, practically identical with bare fused silica capillary, were obtained with the polymethylmethacrylate chip, provided that alpha-cyclodextrin in a concentration 40 mmol/l was added to the background electrolyte. An important observation was that in SDS containing background electrolyte all the plastic materials used exhibited a distinct electroosmotic flow, which was ascribe to the sorption of the negatively charged constituents of the background electrolyte to the capillary wall. Regarding the order in which the individual components of the test mixture were brought to the detector only a single change was observed. Histidine migrated in the polystyrene and polymethylmethacrylate separation channels more slowly than in the bare silica or polyurethane based capillaries.     

Restriction fragment length polymorphism analysis of ERBB2 oncogene by capillary electrophoresis

Capillary electrophoresis (CE) with a sieving buffer containing ethidium bromide was applied to the detection of PCR-amplified RFLP samples. With CE, in contrast to agarose gel electrophoresis, run times are short, i.e., typically less than 30 min, the capillary can be re-used, and full automation is feasible. The addition of ethidium bromide to the buffer system in conjunction with a field amplification injection technique led to increased sample detectability and resolution. Migration time precision was better than 0.2% RSD with a approximately 12-bp resolution for the DNA fragment sizes of interest. RFLP samples were analyzed for homo- or heterozygosity based on the presence of 500- and/or 520-bp DNA fragments. Special software was used to correct for run-to-run migration time variations, thus facilitating genotype assignment.     

Applications of monolithic silica capillary columns in proteomics

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research.     

Acyl migration in 1,2-dipalmitoyl-sn-glycerol

The acyl migration of 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) to 1,3-dipalmitoylglycerol (1,3-DPG) in different states, neat, in the presence of egg yolk lecithin (sonicated and unsonicated) and on silica gel was studied. The isomerization was quantitated by scanning densitometry of charred TLC plates, at different temperatures and for varying periods of time. At equilibrium the amount of 1,3-DPG was found to be 56%. The rates of initial isomerization, and the time required to isomerize to half the equilibrium quantity (i.e., t1/2 eq. = 1,3-DPG 28%) under the above conditions was estimated. In the case of neat melt at 74 degrees C and in an organic solvent the time required to t1/2 eq. is 18 h and a few days, respectively. However, at 62 degrees C in the presence of a polar solvent (sodium phosphate buffer at pH 7.0) the t1/2 eq. is 1-2 h. On dry silica gel (TLC plate) at 24 degrees C the t1/2 eq. is reached in less than 1 h.     

The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns

Amino acids were reacted with o-phthalaldehyde and 2-mercaptoethanol and were separated using a simple linear gradient from 10 to 65% methanol over 15 min on an octyl silica (C8) column by reversed-phase chromatography. The separation obtained was found to be sensitive to the pH, ionic strength, and tetrahydrofuran concentration of aqueous solvent A [THF: sodium acetate (45 mM), pH 5.7, (4:96)]. These effects were characterized and used to design a rapid (17 min) separation of the amino acids commonly found in acid hydrolysates of proteins. A more involved procedure was used to separate the more complex mixture of amino acids that are found in enzymatic hydrolysates of proteins or in physiological fluids. The simplicity of the methods allows their use on different chromatographic systems with little or no alteration.     

Preparation and evaluation of bovine serum albumin immobilized chiral monolithic column for affinity capillary electrochromatography

A system of capillary silica monolith with bovine serum albumin (BSA) functionalized through two approaches for affinity monolithic capillary electrochromatography (AMCEC) was developed. Covalent immobilization conditions for two different Schiff base methods, which employed 3-glycidopropyl trimethoxysilane (GPTS) and 3-aminopropyl trimethoxysilane (APTS) as starting materials, respectively, were investigated to obtain good and stable chiral separation. The BSA immobilized silica monoliths were evaluated in terms of morphology, electroosmotic flow, retention time, column efficiency and resolution of model compound (±)-tryptophan. The columns exhibited satisfactory run-to-run, column-to-column repeatability and maintained their enantioselectivity for more than 3 months. Both developed methods can baseline separate tryptophan enantiomers, whereas shorter retention time, better column efficiency, and enantiomeric recognition between two pairs of drug enantiomers (pantoprazole and atenolol) were obtained by the GPTS method.     

Array based capillary IEF with a whole column image of laser-induced fluorescence in coupling to capillary RPLC as a comprehensive 2-D separation system for proteome analysis

Based on array CIEF (ACIEF) and a novel whole column imaging detection (WCID), a comprehensive 2-D system with laser-induced fluorescence was developed for protein mapping. By coupling capillary RPLC (CRPLC) as the first dimension and ACIEF as the second dimension, a high-throughput and high-resolution proteomic expression profiling was obtained. An array of up to 60 capillaries was assembled, with electrical connections made through filling small breaks, created on each capillary at positions of buffer reservoirs, with a porous polymer. A whole column image system with laser-induced fluorescence (LIF) was devised. Spot excitation was performed with a laser converted to produce linear light, and a CCD camera was employed to take images of the protein fluorescence during line laser scanning of the capillary array. Quantitative detection of thousands of focusing protein bands in the capillary array was achieved. Details on the capillary array fabrication and scanning LIF detection system devices are discussed. The efficiency of this CRPLC-ACIEF-LIF-WCID system was further demonstrated using samples of soluble proteins extracted from liver cancer tissue. The overall peak capacity was estimated to be around 18 000 in an analysis time of less than 3 h. The reproducibility of consecutive runs and different columns were assessed as having an RSD of 1.5% and 2.2% in focusing positions, respectively.     

Synthesis and application of [18F]FDG-maleimidehexyloxime ([18F]FDG-MHO): a [18F]FDG-based prosthetic group for the chemoselective 18F-labeling of peptides and proteins

2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG) as the most important PET radiotracer is available in almost every PET center. However, there are only very few examples using [(18)F]FDG as a building block for the synthesis of (18)F-labeled compounds. The present study describes the use of [(18)F]FDG as a building block for the synthesis of (18)F-labeled peptides and proteins. [(18)F]FDG was converted into [(18)F]FDG-maleimidehexyloxime ([(18)F]FDG-MHO), a novel [(18)F]FDG-based prosthetic group for the mild and thiol group-specific (18)F labeling of peptides and proteins. The reaction was performed at 100 degrees C for 15 min in a sealed vial containing [(18)F]FDG and N-(6-aminoxy-hexyl)maleimide in 80% ethanol. [(18)F]FDG-MHO was obtained in 45-69% radiochemical yield (based upon [(18)F]FDG) after HPLC purification in a total synthesis time of 45 min. Chemoselecetive conjugation of [(18)F]FDG-MHO to thiol groups was investigated by the reaction with the tripeptide glutathione (GSH) and the single cysteine containing protein annexin A5 (anxA5). Radiolabeled annexin A5 ([(18)F]FDG-MHO-anxA5) was obtained in 43-58% radiochemical yield (based upon [(18)F]FDG-MHO, n = 6), and [(18)F]FDG-MHO-anxA5 was used for a pilot small animal PET study to assess in vivo biodistribution and kinetics in a HT-29 murine xenograft model.     

Entamoeba histolytica: cDNAs cloned as 30kDa collagen-binding proteins (CBP) belong to an antioxidant molecule family. Protection of hamsters from amoebic liver abscess by immunization with recombinant CBP

A cDNA expression library of Entamoeba histolytica was screened with antiserum to native amoebic collagen binding proteins (CBPs), and two clones C13 and C7 which partially encode for the 30 kDa CBP were obtained. The sequenced clones were 90% homologous. C7 had a 69 bp deletion at the 5' end that is present in C13 and encodes for a Glu-Cys-Lys rich region and a four amino acids repeat (Glu-Lys-Glu-Cys). Purified fusion proteins from these cDNA clones were able to bind native type I collagen gels in a pH, calcium, ionic strength, and temperature dependent way. The binding of pgtC13 to collagen gel was time and temperature stable, while pgtC7 binding was not, suggesting that the deleted region in C7 is important for the binding. The clones reported here partially encode a 30 kDa CBP that also belong to an antioxidant molecule family. We demonstrated that the fusion protein pgtC13 is immunogenic and partially protective as a subunit vaccine in the hamster model of amoebic liver abscess.     

The interaction of the factor VIII/von Willebrand factor complex (VIII/vWf), with guanidinium-derivatized matrices.

Five different guanidinium (Gu)-derivatized agarose matrices were investigated for their potential in chromatographically resolving the Factor VIII/von Willebrand complex, VIII/vWf, fibrinogen, Fg, and fibronectin, Fn, from cryoprecipitate. Using conventional NaCl gradient methodology it was found that the order of elution of specific plasma proteins, and the yield of VIII/vWf, varied with the methods used to derivatize the agarose beads. Good yields of VIII:C (generally 30-45%) were obtained with Gu-matrices prepared by bis-oxirane coupling procedures. Cryoprecipitate binding studies showed that the capacity of Gu-Sepharose 4B, prepared by isourea modification of amino-Sepharose 4B, was 36 units VIII/vWf per ml matrix. The product, depleted of both Fg and Fn, had a specific activity of 2 units VIII:C per mg total protein, (yield 100% vWf:Ag and 47% VIII:C).     

HPLC法测定头孢克肟的含量

采用高效液相色谱法测定头孢克肟的含量 ,HPL C法 C1 8为填充柱 (6 .0× 15 0 mm ) ,以氢氧化四丁铵 -乙腈 (34 0 :16 0 )为流动相 ;检测波长为 2 5 4nm。结果 :线性范围为 0 .144 mg/ ml～ 0 .2 80 mg/ ml(r=0 .9999) ,平均回收率为 99.80 %,RSD=0 .2 0 %(n=3)。本法具有柱效高、经济等优点 ,优于 USP法。     

Preparative protein purification on underivatized silica

This article describes the use of underivatized silica gel as a preparative stationary phase for process purification of proteins. Although silica has been frequently used as a stationary phase backbone matrix, direct adsorption of proteins on underivatized silica has not been widely exploited for industrial applications. In this study an effort was made to fundamentally understand the interaction mechanisms between a protein and silica surface by using several proteins with a wide range of isoelectric points (pIs) and surface hydrophobicity. Interactions in silica were found to be largely dominated by a combination of ionic and hydrophobic forces. Accordingly, a predictive model was derived for describing linear retention of proteins on silica. Finally, a case study is described investigating the role of silica in an industrial purification process. It was found that the integration of the two modes of interaction confers silica with a unique selectivity that can be very effectively utilized in downstream bioprocessing.     

Predominant Low-Molecular-Weight Proteins in Isolated Brain Capillaries Are Histones

Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.     

A method for the quantitation of tryptophan in Escherichia coli fermentation broth by isocratic high-performance liquid chromatography with ultraviolet detection.

A rapid, isocratic method for the determination of tryptophan in Escherichia coli fermentation broths by reversed-phase HPLC is described. Tryptophan can be measured in fermentations containing either chemically defined media or media with hydrolyzed protein supplements. The procedure was rugged and rereproducible (RSD = 1.7%). The sample response was found to be linear up to 10 mcg of tryptophan/ml. Two different columns--Vydac C18 30 mm and "deactivated" SupelcoSil LC-18-DB--were compared and evaluated for use in the analysis. The deactivated columns had the residual silanols on the silica gel chemically inactivated to reduce the interaction with basic groups or analytes. The deactivated column was found to provide better peak shape (peak assymetry factor less than 1.1) and superior efficiency (plate count greater than 40000/m) and durability (greater than 3000 injections per column) than the non-deactivated column. The procedure described was found to be more selective than a fluorometric procedure.     

Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system

Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.     

高效毛细管电泳法直接测定红豆杉悬浮培养细胞中游离芳香族氨基酸

建立了红豆杉悬浮培养细胞中游离芳香族氨基酸的高效毛细管电泳的分离直接紫外吸收测定方法。在优化的实验条件下 ,以间苯二酚为内标 ,未涂层融硅毛细管 (75 μmi.d .,总长 37cm ,有效分离长度 30cm)为分离通道 ,40mmol/L磷酸缓冲液 (pH10 .46 )为电泳介质 ,3sec(0 .5 psi)压力进样 ,15kV恒压电泳 ,2 0 0nm检测 ,在 9min内三种氨基酸得到分离测定。色氨酸、苯丙氨酸和酪氨酸的线性范围分别为 2 .5 0～ 2 5 0 μmol/L(r =0 .996 2 ,RSD =2 .5 2 % )、1.2 5～ 2 5 0 μmol/L(r =0 .996 6 ,RSD =2 .12 % )和 2 .5 0～ 2 5 0 μmol/L(r=0 .9940 ,RSD =2 .93% ) ,苯丙氨酸回收率为 96 .8%± 1.37% ,酪氨酸回收率为 99.2 %± 3.0 4%。