毛细管电泳场放大进样化学发光检测10-16mol/L水平钴(Ⅱ)

激光诱导荧光（LIF）以其灵敏度高及应用范围广等特点成为单分子检测的主要手段[1]. 但LIF除了仪器昂贵外, 还需对非荧光物质采用柱前或柱后衍生引入荧光团, 且该法易受拉曼散射、瑞利散射和溶剂噪音的影响, 产生高背景干扰. 化学发光（CL）检测灵敏度高, 光学系统简单、无须外加光源及分光系统, 背景低, 避免了杂散光和光源不稳定性的影响. 这些优异性能使其有可能成为毛细管电泳（CE）中的一种超灵敏检测手段. 关于毛细管电泳在线化学发光检测, 本研究组曾提出一种新的试剂混合模式[2], 并设计了一种新颖的检测接口[3], 降低了噪音, 显著提高了信噪比. 场放大进样在线富集技术可用于提高检测灵敏度, 其对有机化合物[4~6]及金属配合物[7]等方面的研究已有报道. 浓缩因子达到1 500[8]. 我们系统地研究了场放大进样对金属离子的富集作用后发现, 所研究的金属离子如钴（Ⅱ）、铬（Ⅲ）、铜（Ⅱ）及镍（Ⅱ）等的浓缩因子可达到104～106.     

EFFECT OF OVERLAP CONCENTRATION AND PERSISTENCE LENGTH ON DNA SEPARATION IN POLYMER SOLUTIONS BY ELECTROPHORESIS

The persistence length and the overlap concentration(c~*) of poly(ethylene oxide)(PEO) and hydroxyethylcellulose(HEC) with similar molecular weight in 1×TBE buffer were studied by laser light scattering and viscometry.Their effect on DNA separation was investigated by capillary electrophoresis.It was determined that the persistence length of HEC was at least 5 times higher than that of PEO.Therefore,the c~* of HEC was smaller than that of PEO by a factor of ca.2.5.It was also found that the c~* values de...     

Capillary electrophoresis of peptides. Analysis of adrenocorticotropic hormone-related fragments.

Capillary electrophoresis can be used successfully to analyse small peptides to give additional information to that obtained using high-performance liquid chromatography (HPLC). The separation of a modified adrenocorticotropic hormone (4-9) fragment (Org 2766) and several of its fragments was investigated using capillary zone electrophoresis. Prediction of migration in aqueous systems using pKa-related data and the migration behaviour using sodium dodecyl sulphate in the buffer are discussed, as is the choice of buffer systems. The electrophoretic patterns are compared with the HPLC separation.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

High resolution-separation of DNA bands by electrophoresis with a long gel in a fluorescence-detection DNA sequencer.

A high-resolution separation of DNA bands is achieved by electrophoresis with a long gel in DNA base sequencing using fluorescence detection. We separate 760 and 761 base DNA fragments using the 93 cm migration electrophoresis optimized for the separation of DNA bands. A T7 DNA polymerase and an Mn++ buffer are used in sequencing reactions to obtain fluorescence peaks of uniform strength, and the peak areas in the spectrum are used for recognizing the peak number in a cluster of successive peaks. This method is successfully applied to the DNA fragment spectrum obtained by 93 cm migration electrophoresis, which results in a single-band differentiation of bands of 1040 base DNA.     

Thermoresponsive N,N-dialkylacrylamide copolymer blends as DNA sieving matrices with a thermally tunable mesh size

In an earlier study we showed that a blend of thermoresponsive and nonthermoresponsive hydroxyalkylcelluloses could be used to create a thermally tunable polymer network for double-stranded (ds) DNA separation. Here, we show the generality of this approach using a family of polymers suited to a wider range of DNA separations: a blended mixture of N,N-dialkylacrylamide copolymers with different thermoresponsive behaviors. A mixture of 47% w/w N,N-diethylacrylamide (DEA)/53% w/w N,N-dimethylacrylamide (DMA) (DEA47; thermoresponsive, transition temperature = 55 degrees C in water) and 30% w/w DEA/70% w/w DMA (DEA30; nonthermoresponsive, transition temperature > 85 degrees C in water) copolymers in the ratio of 1:5 w/w DEA47:DEA30 was used to separate a dsDNA restriction digest (PhiX174-HaeIII). We investigated the effects of changing mesh size on dsDNA separation, as controlled by temperature. We observed good DNA separation performance with the copolymer blend at temperatures ranging from 25 degrees C to 48 degrees C. The separation selectivity was evaluated quantitatively for certain DNA fragment pairs as a function of temperature. The results were compared with those obtained with a control matrix consisting only of the nonthermoresponsive DEA30. Different DNA fragment pairs of various sizes show distinct temperature-dependent selectivities. Over the same temperature range, no significant temperature dependence of selectivity is observed for these DNA fragment pairs in the nonthermoresponsive control matrix. Overall, the results show similar trends in the temperature dependency of separation selectivity to what was previously observed in hydroxyalkylcellulose blends, for the same DNA fragment pairs. Finally, we showed that a ramped temperature scheme enables improved separation in the blended copolymer matrix for both small and large DNA fragments, simultaneously in a single capillary electrophoresis (CE) run.     

Electroosmotic flow suppression in capillary electrophoresis: chemisorption of trimethoxy silane-modified polydimethylacrylamide

Adsorbed polymers are widely used to suppress electroosmotic flow (EOF) in capillary electrophoresis (CE). Polymeric coatings, physisorbed onto the surface of the capillary wall, are often unstable under harsh conditions. This can be attributed to the reversible nature of the coating which becomes apparent when the adsorbed layer competes with a second species in the electrophoresis buffer solution for attachment/interaction with the capillary surface. In an effort to overcome the problem of coating instability, trimethoxysilane-modified polydimethylacrylamide was synthesized. This copolymer rapidly adsorbs on the wall from ultradilute aqueous solutions. After incubation at a temperature of 60 degrees C silyl groups, which extend from the polymer backbone, form condensation bonds with the silanols on the capillary surface. This enables subsequent formation of strong covalent bonds between the copolymer and the capillary wall. In this research, we establish that physisorption of polymer chains to the surface is essential for close alignment of surface and polymer silane groups which facilitates the formation of covalent bonds.     

Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.     

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range.     

Oligonucleotide trapping method for purification of transcription factors

Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(Hc)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is approximately 3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(Hc) proteins.     

Analysis of diacylglycerols by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry: Double bond location and isomers separation

Diacylglycerols (DAGs) are important lipid intermediates and have been implicated in human diseases. Isomerism complicates their mass spectrometric analysis; in particular, it is difficult to identify fatty acid substituents and locate the double bond positions in unsaturated DAGs. We have developed an analytical strategy using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) in conjunction with dimethyl disulfide (DMDS) derivatization and collision cross-section (CCS) measurement to characterize DAGs in biological samples. The method employs non-aqueous reversed-phase chromatographic separation and profile collision energy (CE) mode for MS^E and MS/MS analyses. Three types of fragment ions were produced simultaneously. Hydrocarbon ions (m/z 50–200) obtained at high CE helped to distinguish unsaturated and saturated DAGs rapidly. Neutral loss ions and acylium ions (m/z 300–400) produced at low CE were used to identify fatty acid substituents. Informative methyl thioalkane fragment ions were used to locate the double bonds of unsaturated DAGs. Mono-methylthio derivatives were formed mainly by the reaction of DAGs with DMDS, where methyl thiol underwent addition to the first double bond farthest from the ester terminus of unsaturated fatty acid chains. The addition of CCS values maximized the separation of isomeric DAG species and improved the confidence of DAG identification. Fourteen DAGs were identified in mouse myotube cells based on accurate masses, characteristic fragment ions, DMDS derivatization, and CCS values.     

High-performance ultrathin layer agarose gel electrophoresis

Large scale, high-resolution DNA fragment analysis, such as genotyping, mapping and genetic profiling requires an affordable, fully automated high-throughput gel electrophoresis based separation device that enables rapid, high-performance analysis in a wide molecular weight range. In this article a novel approach is described that greatly enhances the productivity of DNA fragment analysis by automating the current manual procedure and also reducing the separation time and human intervention from sample loading to data analysis. The ultrathin layer, multilane, high-performance agarose gel electrophoresis system employs integrated scanning laser induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. The separation platform is fabricated in a way that the sieving matrix can be easily replaced in the separation cassette for each run. Visualization of the DNA fragments is accomplished by ‘in migratio' complexation during the electrophoresis process with ultra-sensitive fluorescent agents, also enabling real-time imaging and data analysis.     

Solution-phase parallel synthesis with oligoethylene glycol sorting tags. Preparation of all four stereoisomers of the hydroxybutenolide fragment of murisolin and related acetogenins

The principles of the oligoethylene glycol (OEG) mixture synthesis are illustrated with the synthesis of all four possible stereoisomers of a hydroxybutenolide fragment common to murisolin and many other acetogenins. Modified dimethoxybenzyl groups with varying numbers of OEG units (-CH2CH2O-) are used to protect alcohols and serve as codes for configurations at two stereocenters. The encoded isomers are carried through several steps in a sequence of mixing prior to the reaction and then demixing during the separation to give individual pure products. A new tagging scheme is introduced in which a stereocenter bearing a hydroxy group is given two different tags. These initially redundant tags then serve to encode the configuration of another (untagged) stereocenter by appropriate pairwise reactions of the tagged precursors. The experimental features (reaction, analysis, separation, and characterization) of OEG mixture synthesis are detailed and are compared to and contrasted with those of fluorous mixture synthesis.     

Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.     

Positive secondary-ion mass spectra and thin-layer chromatography/mass spectrometry of phenothiazine drugs

The positive secondary-ion mass spectra of phenothiazine drugs contain abundant protonated molecules and structurally informative fragment ions that are characteristic of the N-substituent. Because the secondary-ion current is carried by a few characteristic ions, selected-ion monitoring is used with chromatographic separation for the determination of these drugs in mixtures. Drugs are extracted in situ from thin-layer chromatograms, and the secondary-ion mass spectra are obtained without destruction of the chromatograva. The spectra are dependent upon the solvent used and not the nature of the underlying chromatographic matrix. Spatially-resolved selected-ion monitoring of the protonated molecule provides the x-y spatial profiles of the sample in the chromatogram. Extended irradiation leads to a beam-induced tailing that alters the profile.     

Separation of the Hageman factor fragment from human serum albumin by chromatography on blue sepharose CL-6B

The separation of the Hageman factor fragment (HFf) activity from human serum albumin by chromatography on Blue Sepharose CL-6B is described. The complete separation cannot be achieved in a single chromatography step due to complex formation between HFf and albumin.     

Procedure for the isolation of a large peptic fragment of human serum albumin

For a thorough investigation of the drug-binding behaviour and other physicochemical properties of human serum albumin, one needs large amounts of specific fragments of albumin. Such fragments were obtained by careful proteolysis of the native protein with pepsin at pH 3.70. The fast protein liquid chromatographic technique was used to find the optimum experimental conditions for the separation of the fragments. By means of anion-exchange chromatography, chromatofocusing and gel permeation, it was possible to obtain a large fragment with a relative molecular mass of 46,000. The fragment could be assigned to segment 1-387 and therefore consists of domains 1 and 2 of the albumin structure. A 1-g amount of albumin produced 50 mg of a fragment that was 98% homogeneous.     

High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.     

Detection of fragment ions produced by collisional activation of multiply charged ions in a floated collision cell

A scan law is derived for the detection of fragment ions formed by collisional activation (CA) of a multiply charged precursor in a floated collision cell of a tandem mass spectrometer. Comparisons of the CA spectra of multiply charged ions obtained in either a floated or a grounded collision cell demonstrate the benefits associated with raising the collision cell above ground potential. In addition to the advantages observed for singly charged ions, floating the collision cell increases the transmission of multiply charged ions through the first mass spectrometer by permitting higher source potentials to be used. This technique also increases the detection efficiency for products of charge separation reactions, which may prove useful in the charge state assignment of the fragment ions.