液质连用分析重组胰高血糖素样肽-1受体激动剂肽图

目的：建立高效液相系统肽图分析法,用于重组胰高血糖素样肽-1受体激动剂（rExendin-4）的质量控制。方法：应用高效液相系统摸索最佳胰蛋白酶切和色谱条件,并采用液质联用系统分析肽段的精确相对分子量和氨基酸序列。结果：根据酶切条件摸索,确定酶切条件为：rExendin-4原液与胰蛋白酶按照质量比为100：1混匀,37℃酶切4小时,根据肽段的色谱保留时间、相对分子质量及对其碰撞诱导解离质谱的解析结果,归属出肽图中各肽段所在的色谱峰,与理论值完全一致。结论：本法精确度高、重复性好、自动化程度高,能够用于rExendin-4原液肽图分析。     

Hochdruck-flüssigkeitschromatographische bestimmungsmethode für freies cortisol im urin

The possibilities were examined for the high-pressure liquid chromatographic analysis of cortisol with methods of adsorption, distribution and reversed-phase chromatography. Free cortisol in urine can be determined by extraction with chloroform and subsequent adsorption chromatography on silica gel with a mobile phase consisting of 1.5% methanol and 0.2% water in chloroform.The time needed for this chromatographic analysis is 10–15 min; the limit of determination is 3 ng of cortisol for one injection.     

Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor 1 from bovine brain tissue

Although Tyr-melanocyte-stimulating hormone release-inhibiting factor 1 (MIF-1) (Tyr-Pro-Leu-Gly-NH2) can exert a number of biological actions in the brain and elsewhere, it has never been isolated from any tissue. Accordingly, we attempted to purify it from acetic acid extracts of bovine brain tissue by gel filtration chromatography and several different high performance liquid chromatographic systems. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against synthetic Tyr-MIF-1. In each of the five applied high performance liquid chromatographic systems, the immunoreactive fractions coincided exactly with the elution time of synthetic Tyr-MIF-1 in the control runnings. The structure of the isolated peptide was identified by microsequence analysis as the tetrapeptide Tyr-Pro-Leu-Gly-NH2 and shown to be biologically active.     

Self-interaction chromatography of proteins on a microfluidic monolith

A novel miniaturized system has been developed for measuring protein-protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column synthesis and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B(22), were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions.     

微波处理对人参皂苷生物转化影响的研究

利用微波技术分别对人参皂苷粗品及人参皂苷转化发酵液进行处理,探讨微波处理对人参皂苷生物转化效果的影响。实验结果通过高效液相色谱分析显示,经微波处理后,人参皂苷峰几乎消失,苷元峰突出,表明微波处理对人参皂苷的转化效果显著。并确定微波的最佳处理条件为微波功率30W,辐射60s。     

重组酶Exo的纯化及多克隆抗体制备

目的:纯化Exo重组酶融合蛋白并制备相应抗体。方法:用阴离子交换柱对蛋白进行初步纯化,然后用Ni-NTA介质填充的层析柱分离纯化含His标签的融合蛋白,用谷胱甘肽琼脂糖4B介质填充的层析柱分离纯化GST融合蛋白;二次纯化的蛋白利用硝酸纤维素膜结合法制备抗原蛋白并免疫实验动物。结果:ELISA结果显示血清抗体效价可达到1∶12 800,说明通过Western免疫印迹自制的多克隆抗体能特异地与Exo重组蛋白相互作用。结论:该蛋白纯化方法操作简单,制备的抗原纯度高,多克隆抗体特异性好。     

A framework for the prediction of scale-up when using compressible chromatographic packings

Experimental data are given for the solid pressure distributions in chromatography columns of various column aspect ratios packed with four types of agarose-based resin. The loss of column wall support at large scales can result in unexpectedly high pressures caused by the compression of the matrix via drag forces exerted by fluid flow through the bed. The need for an accurate model to predict flow conditions at increasing scale is essential for the scaling-up of chromatographic processes and for avoiding bed compression during operation. Several studies have generated correlations that allow for the prediction of column pressure drops, but they either are mathematically complex, which impairs their practical use, or require a large number of experiments to be performed before they can be used. In this study an empirical correlation was developed based on a previously proposed model, which links the critical velocity of operation of a chromatographic system (microcrit), to the gravity-settled bed height (L0), the column diameter (D), the feed viscosity (micro), and the compressibility of the chromatographic media used (micro 10%). The methodology developed in this study is straightforward to use and significantly reduces the burden of preceding laboratory-scale experimentation. The approach can be used to predict the critical velocity of any chromatographic system and will be useful in the development of chromatographic operations and for column sizing.     

Purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase with high specific activity by fast protein liquid chromatography

A rapid procedure for the purification of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rubisco) (EC 4.1.1.39) by fast protein liquid chromatography (FPLC) is described. Chloroplasts isolated mechanically from spinach leaves were used as the source of a stromal extract enriched in rubisco. By subsequent fractionation of this extract on ion-exchange FPLC, highly purified rubisco (sp act 2.10-2.76 mumol/mg protein X min) was obtained in less than 30 min. The high specific activity and excellent stability of the final preparation can be attributed to the use of chloroplasts as a starting material and the short time required for the chromatographic separation, both of which minimize proteolytic activity.     

A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separation

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode--implemented on a quadrupole-TOF instrument--that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain.     

High-performance liquid chromatography using novel square tile-shaped hydroxyapatite crystals as adsorbent

High-performance liquid chromatography using, as adsorbent, novel square tile-shaped hydroxyapatite crystals (with thicknesses of about 2 microns and diameters of 3-7 microns) has been developed. The chromatographic efficiencies of the novel hydroxyapatite packed columns are almost equal to those of the previously developed spherical hydroxyapatite packed columns; high chromatographic resolutions can be obtained by using extremely reduced column lengths of 0.5-3 cm. Since both the square and the spherical hydroxyapatite have roughly the same particle size of some micrometers, the chromatographic efficiency can be deduced to be determined mainly by the particle size rather than the particle shape.     

Data filtration for more robust alignment of chromatograms of complex peptide mixtures

In carrying out proteomic researches using mass-spectrometry there often arises a need to compare experimental data with each other (e.g. control of pathology, the labeled to unlabelled samples). If for peptide identification in different experiments one uses only their exact mass measurements and the retention time in the chromatographic column, difficulties with the identification of chromatographic peaks belonging to the same substances in different chromatograms come up (retention time normalization). Due to inevitable discrepancies in chromatographic conditions of experiments (replacement of chromatographic columns, small changes in mobile phase flow rate or solvent concentration) retention times of the same peptides will diverge from experiment to experiment. In this paper we offer a reliable method for selecting peaks from mass-chromatograms corresponding to the same peptides, which can later be used for retention time normalization (either linear or any other monotone function).     

Comparative analysis of chromatography peaks by computer and disc-integrator

A reliability study was conducted to compare a new, computerized method of measuring the areas under chromatographic curves with the traditional hand method of determining the areas. Three runs of 30 separate injections of standard inorganic phosphate were made using the chromatographic and dry-ashing procedure developed in the laboratory of the Department of Pharmacology of the USC School of Medicine. Results indicate that there is no statistically significant difference between the traditional method and the new computer method for amounts greater than 10 nm, and the computer method is more precise for amounts less than 10 nm. Hence, great savings in time and an increase in precision can be made by using a computer for these analyses.     

A simple procedure for isolation of eukaryotic mRNA polyadenylation factors.

We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.     

A new on-line, in-tube pre-column derivatization technique for high performance liquid chromatographic determination of azithromycin in human serum

Pre-column derivatization methods for high performance liquid chromatographic assay of specific pharmaceutical agents using 9-fluorenylmethyl chloroformate (FMOC-Cl) have received special attention because highly fluorescent and stable adducts are provided by these methods. However, unlike the post-column on-line techniques, long derivatization time is needed and the reaction cannot be well controlled. A new, sensitive and fast pre-column on-line derivatization technique coupled with high-performance liquid chromatography using FMOC-Cl as labeling agent is described and validated for determination of azithromycin in human serum. After extraction of the drug from serum, the residue was reconstituted in mixture of acetonitrile-phosphate buffer (3:1, v/v; pH 8.5) and directly injected onto the chromatographic system. Continuous on-line derivatization and analysis of the compounds were successfully performed using in-tube elution of FMOC-Cl. The total time needed for derivatization and chromatographic analysis of the drug was 13 min. The assay was reliable and reproducible, with limit of quantification of 10 ng/ml. The described technique may offer significant advantages over existing off-line derivatization methods using FMOC-Cl.     

Analysis of bacterial menaquinone mixtures by high performance liquid chromatography

Menaquinone mixtures of microbial origin were separated according to the chain length and the degree of hydrogenation of the polyisoprenyl side-chain by reversephase partition high performance liquid chromatography. Menaquinones can be measured easily and sensitively by the chromatographic system described here.     

Large scale chiral chromatography for the separation of an enantiomer to accelerate drug development

There are several approaches to produce enantiomerically pure drug substances, such as recrystallization, catalytic process (ligand and enzyme), indirect chromatographic resolution, and direct chromatographic resolution. However, the use of preparative chromatography with chiral stationary phases seems to be most effective for early phase projects, where the time and resources on the developments need to be minimized to get the drug candidates into the clinical studies. We showed that by following a well-defined process, chiral chromatography can be easily scaled up from an analytical system to a pilot plant system. We also used the results from a multicolumn continuous chromatography (MCC) study to conclude that MCC can be a cost-effective production method for chiral manufacturing.     

Quantitative enzymatic hydrolysis of tRNAs : Reversed-phase high-performance liquid chromatography of tRNA nucleosides

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme—substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis—HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.     

Hydrolysis of bacterial wall carbohydrates in the microwave using trifluoroacetic acid

By and large, monosaccharide composition and linkage analyses of bacterial cell-surface carbohydrates are achieved by hydrolysis into the corresponding monomeric constituents, and characterization of these, or their derivatives, by chromatographic and spectrometric methods. Normally, these hydrolyses are carried out conveniently with trifluoroacetic acid (TFA) at high temperatures for long periods of time, for example, in 4M TFA at 100 degrees C for 5h in a heating block. In this study, using a closed-vessel system, we investigated the effectiveness and reliability of microwave-assisted TFA hydrolysis of bacterial lipopolysaccharides, capsule, and teichoic-acid polysaccharides that were variably composed of several glycoses. In all cases, we were able to establish that 5min of hydrolysis in the microwave at 120 degrees C with 4M TFA (measured pressure of 90psi) was sufficient time to obtain comparable results to those afforded by conventional hydrolysis. The same observation was made when fully methylated carbohydrates were hydrolyzed. The data obtained with our microwave system (Aurora Instruments MW600) showed that microwave-induced hydrolysis can be used with a high degree of confidence to carry out sugar composition analysis of complex bacterial glycans in markedly shorter periods of time. The results also suggested that non-thermal mechanistic factors must also be involved, at least in part, in accelerating the reaction rate of glycosidic hydrolysis.     

金标法与免疫发光法检测CEA在健康体检中的应用

目的：探讨金标法和免疫发光法检测CEA在健康体检中的应用价值。方法：对728例健康体检的个人同时应用金标法和免疫发光法测定CEA,对结果进行分析。结果：金标法测定的阳性率为0．69％,免疫发光法测定的阳性率为0．41％,两者具有高度一致性。结论：对健康体检人群应先用金标法进行定性,对阳性结果再用免疫发光法进行定量。     

Protein profile study of breast‐tissue homogenates by HPLC‐LIF

Proteomics is a promising approach for molecular understanding of neoplastic processes including response to treatment. Widely used 2D‐gel electrophoresis/Liquid chromatography coupled with mass spectrometry (LC‐MS) are time consuming and not cost effective. We have developed a high‐sensitivity (femto/subfemtomoles of protein/20 μl) High Performance Liquid Chromatography‐Laser Induced Fluorescence HPLC‐LIF instrument for studying protein profiles of biological samples. In this study, we have explored the feasibility of classifying breast tissues by multivariate analysis of chromatographic data. We have analyzed 13 normal, 17 malignant, 5 benign and 4 post‐treatment breast‐tissue homogenates. Data was analyzed by Principal Component Analysis PCA in both unsupervised and supervised modes on derivative and baseline‐corrected chromatograms. Our findings suggest that PCA of derivative chromatograms gives better classification. Thus, the HPLC‐LIF instrument is not only suitable for generation of chromatographic data using femto/subfemto moles of proteins but the data can also be used for objective diagnosis via multivariate analysis. Prospectively, identified fractions can be collected and analyzed by biochemical and/or MS methods. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)