A new cyclodipeptide from the cultures of Geotrichum candidum

A new cyclodipeptide,cyclo(phenylalanyl-N-methyltyrosyl)1,was isolated from the mycelial solid cultures of Geotrichum candidum.Its structure was elucidated by spectroscopic methods.The antifungal activity of the new compound against Peronophythora litchii was assessed by microplate Alamar blue assay(MABA).     

Enzyme activity electrophoresis: development and applications

The development of rocket enzyme activity electrophoresis for the detection and quantification of various proteinases, lipases and pectinases is presented. Rocket enzyme activity electrophoresis is more sensitive than the radial diffusion assay and often enables distinction between qualitatively different enzymes present in the same samples, whereas the radial diffusion assay only provides information on the overall enzyme activity. However, calibration and optimization of the enzyme activity electrophoretic assay have to be performed for each new enzyme-substrate system to be analyzed. Some of the common pitfalls in the development of new enzyme activity electrophoretic assays are presented. Enzyme activity electrophoresis can be applied in combination with other electrophoretic assays. Particularly the combination of enzyme activity electrophoresis with various immunoelectrophoretic methods can provide detailed information on the enzymes studies.     

Exploratory data analysis groupware for qualitative and quantitative electrophoretic gel analysis over the Internet-WebGel

Many scientists use quantitative measurements to compare the presence and amount, of various proteins and nucleotides among series of one- and two-dimensional (1-D and 2-D) electrophoretic gels. These gels are often scanned into digital image files. Gel spots are then quantified using stand-alone analysis software. However, as more research collaborations take place over the Internet, it has become useful to share intermediate quantitative data between researchers. This allows research group members to investigate their data and share their work in progress. We developed a World Wide Web group-accessible software system, WebGel, for interactively exploring qualitative and quantitative differences between electrophoretic gels. Such Internet databases are useful for publishing quantitative data and allow other researchers to explore the data with respect to their own research. Because intermediate results of one user may be shared with their collaborators using WebGel, this form of active data-sharing constitutes a groupware method for enhancing collaborative research. Quantitative and image gel data from a stand-alone gel image processing system are copied to a database accessible on the WebGel Web server. These data are then available for analysis by the WebGel database program residing on that server. Visualization is critical for better understanding of the data. WebGel helps organize labeled gel images into montages of corresponding spots as seen in these different gels. Various views of multiple gel images, including sets of spots, normalization spots, labeled spots, segmented gels, etc. may also be displayed. These displays are active and may be used for performing database operations directly on individual protein spots by simply clicking on them. Corresponding regions between sets of gels may be visually analyzed using Flicker-comparison (Electrophoresis 1997, 18, 122-140) as one of the WebGel methods for qualitative analysis. Quantitative exploratory data analysis can be performed by comparing protein concentration values between corresponding spots for multiple samples run in separate gels. These data are then used to generate reports on statistical differences between sets of gels (e.g., between different disease states such as benign or metastatic cancers, etc.). Using combined visual and quantitative methods, WebGel can help bridge the analysis of dissimilar gels which are difficult to analyze with stand-alone systems and can serve as a collaborative Internet tool in a groupware setting.     

Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities

The analysis of microbial communities is of increasing importance in life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods suffer from many disadvantages. They are unable to give a complete qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Obviously, the determination of static or dynamic balances among microorganisms is of fast growing interest. The generation of species specific and fluorescently labeled 16S ribosomal DNA (rDNA) fragments by the terminal restriction fragment length polymorphism (T-RFLP) technique is a suitable tool to overcome the problems other methods have. For the separation of these fragments polyacrylamide gel sequencers are preferred as compared to capillary sequencers using linear polymers until now because of their higher electrophoretic resolution and therefore sizing accuracy. But modern capillary sequencers, especially multicapillary sequencers, offer an advanced grade of automation and an increased throughput necessary for the investigation of complex communities in long-time studies. Therefore, we adapted a T-RFLP technique to an automated high-throughput multicapillary electrophoresis device (ABI 3100 Genetic Analysis) with regard to a precise qualitative and quantitative characterization of microbial communities.     

Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin

The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms.     

Enzyme enhanced quantitative determination of multiple DNA targets based on capillary electrophoresis

In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targets based on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, which reacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled probes, to hybridize with three corresponding targets. The resulting mixture containing double-strand DNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated by capillary electrophoresis, and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The catalytic product was detected with electrochemical detection. With this protocol, the limits of quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 × 10⁻¹¹, 2.4 × 10⁻¹¹ and 3.0 × 10⁻¹¹ M, respectively. The multiplex assay also provided good specificity without any cross-reaction.     

Microchip electrophoresis with hydrodynamic injection and waste-removing function for quantitative analysis

Quantitative analysis is problematic for microchip electrophoresis for several reasons including chip-to-chip variation, discontinuous sample re-loading, channel reconditioning, and electrokinetic injection bias. In this study, the capability for quantitative analysis on a flow-through based microchip electrophoresis, which provides continuous sample re-loading, channel washing, reconditioning and hydrodynamic injection as well as waste removing is demonstrated to be more quantifiable and more reproducible compared to manual electrokinetic injection method. Using the flow-through microchip with waste-removing function, FITC-labeled estrogen or Rhodamine B could be continuously analyzed without significant changes (R.S.D. < 6.6%) in signal intensity for over 3 h, which is sufficient for a complete set of quantitative analysis. With the use of a phosphorylated kinase substrate as the model, a calibration curve for quantitative analysis of phosphopeptides were constructed and results indicate that both R2 value of the linearity and R.S.D. values of the peak intensity were around 0.9961 and 3.16%, respectively, without the use of an internal standard. These values were slightly improved to be around 0.9986 and 2.27%, respectively, with the use of a non-phosphopeptide counterpart as the internal standard. The potential of this flow-through device for the development of a kinase phosphorylation assay based on the quantitative method was also briefly discussed.     

Computer-assisted liquid chromatography for automated qualitative and quantitative analysis of toxic drugs

A computer-assisted liquid chromatographic system (MCASYST) is evaluated for automatic qualitative and quantitative analysis of toxic drugs in poisoned human fluids. The system performance is confirmed by the actual analyses of several cases of poisoning. The results indicate that the accuracy of identification by retention prediction and UV spectral search is very high, and quantitation by the memorized calibration curves can be performed very conveniently. However, it is difficult at present to identify all peaks that appear in chromatograms for human urine and serum because the drugs may be changed to their metabolites in those fluids. Metabolite identification will be the next step to improve the performance of this system.     

A review of the CLIP system for the quantitative analysis of two-dimensional electrophoresis gels

This paper reviews the CLIP image processing system for the complete analysis of two-dimensional electrophoresis images. The analysis problem for two-dimensional gel images can be broken down into three issues: segmentation of individual gel images, alignment and comparison of pairs of gel images, and information storage and retrieval. This paper describes these problems and reviews how the CLIP system handles each of them. Segmentation is the location and isolation of each protein spot on an individual gel image and also the extraction of individual spot data such as position, area and volume. There are three basic stages: background field correction, noise filtering, spot detection and information extraction. Alignment and comparison of gel images involves matching protein spots between two gels. This can be quite difficult because there is not a simple relationship which can transform one gel image onto another. The database issues concern storing all the information which has been obtained from the above operations such that retrieval of this information can be readily performed. The advantage of the CLIP system over others is speed of processing. CLIP series computers use one processor for every pixel of the camera image such that image processing algorithms run in parallel. The main disadvantage is in the cost of these machines. With the declining trend in the cost of parallel processors, these machines will become more and more viable alternatives. This papers reviews the algorithms for the analysis of two-dimensional gels. It is shown that CLIP is flexible enough to perform more than one type of algorithm for a particular operation.     

Enzyme separation and isozyme heterogeneity analysis using non-denaturing two-dimensional electrophoresis

Carboxylesterase and sorbitol dehydrogenase are separated by non-denaturing two-dimensional electrophoresis (2-DE) of isoelectric focusing separation using 5% carrier ampholyte (pH 6-8) and 1.25% carrier ampholyte (pH 3-10) and size separation. Furthermore, activities of sorbitol, malate and lactate dehydrogenases are sequentially examined when the enzymes are separated by 2-DE and are sequentially reacted to sorbitol, malic and lactic acid, respectively, in the presence of nicotinamide adenine dinucleotide, nitro blue tetrazolium and phenazine methosulphate. Several kinds of enzymes including lactate dehydrogenize isozymes can be simultaneously separated using 2-DE. Furthermore, the binding differences between lactate dehydrogenase isozymes and concanavalin A (con A) can be examined using a combination of 2-DE and non-denaturing stacking gel electrophoresis. The results of this study indicate that non-denaturing 2-DE can be applied to both enzyme separation and isozyme heterogeneity analysis.     

Immunostaining of rocket immunoelectrophoresis precipitates too weak for identification following staining with Coomassie brilliant blue

Low enzyme concentrations can be determined quantitatively using rocket immunoelectrophoresis if the resolved peaks are transferred electrophoretically to a nitrocellulose membrane and immunostained.     

Guanidination chemistry for qualitative and quantitative proteomics

The application of guanidination chemistry, the conversion of lysine into homoarginine residues, is used to illustrate several important general considerations relating to the use of differential isotope labelling for relative quantification in proteomics. The derivatisation procedure has been optimised for automation using a liquid handling station designed for proteomics. Automated application of the procedure to the analysis of in-gel tryptic digests of multiple spots from the two-dimensional gel electrophoretic (2DE) analysis of proteins from the FDCP-mix cell line shows near-universal improvement in protein identification as a result of derivatisation. This chemistry has been extended for relative quantification, applicable to matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and also tandem mass spectrometry (MS/MS). It provides a robust method for the quantitative comparison of two samples that have been separated by 2DE. A peptide pair may display poor detection during MS analysis, causing their reliable relative quantification to be difficult. In such circumstances, the additional selectivity of detection provided by MS/MS can substantiate identification and allow relative quantification of these species via product ion signal ratios.     

Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure       

High-resolution mass spectrometry for integrated qualitative and quantitative analysis of pharmaceuticals in biological matrices

Quantitative and qualitative high-resolution (HR) dependent and independent acquisition schemes on a QqTOF MS (with resolving power 20,000–40,000) were investigated for the analysis of pharmaceutical compounds in biological fluids. High-resolution selected reaction monitoring (HR-SRM) was found to be linear over three orders of magnitude for quantitative analysis of paracetamol in human plasma, offering a real alternative to triple quadrupole LC–SRM/MS. Metabolic stability of talinolol in microsomes was characterized by use of three different acquisition schemes: (i) information-dependent acquisition (IDA) with a TOF MS experiment as survey scan and product-ion scan as dependent scan; (ii) MS^ALL by collecting TOF mass spectra with and without fragmentation by alternating the collision energy of the collision cell between a low (i.e., 10 eV) and high setting (i.e., 40 eV); and (iii) a novel independent acquisition mode referred to as “sequential window acquisition of all theoretical fragment-ion spectra” (SWATH) or “global precursor ions scan mode” (GPS) in which sequential precursor ions windows (typically 20 u) are used to collect the same spectrum precursor and fragment ions using a collision energy range. SWATH or GPS was found to be superior to IDA or MS^ALL in combination with UHPLC for qualitative analysis but requires a rapidly acquiring mass spectrometer. Finally, the GPS concept was used for QUAL/QUAN analysis (i.e. integration of qualitative and quantitative analysis) of bosentan and its metabolites in urine over a concentration range from 5 to 2,500 ng mL⁻¹.     

High-energy collisional spectroscopy in qualitative and quantitative analysis of diesel particulates

High-energy collisional spectroscopy provides a powerful analytical method for the characterization of polynuclear aromatic hydrocarbons present in diesel particulates. The absence of any need for extraction procedures and the large amount of information available from collisional spectra makes this technique of great interest. In particular, 3-methylcholanthrene can be determined from the daughter spectra of its molecular ion, with methylene iodide as internal standard.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Use of a discrete diode array spectrophotometer for the quantitative and qualitative analysis of cytochrome P-450

Cytochrome P-450, the hemoprotein located in the endoplasmic reticulum of mammalian cells and responsible for the metabolism of xenobiotics, was qualitatively and quantitatively analyzed using the HP-8450A diode array spectrophotometer. The diode array instrument was compared to a conventional spectrophotometer and the advantages of the diode array instrument over conventional spectrophotometry with respect to the analysis of cytrochrome P-450 were discussed.     

酶切过程中肽段过烷基化对蛋白质定性和定量分析的影响

蛋白质的还原-烷基化是蛋白质酶切中的重要步骤,常用的烷基化试剂是碘乙酰胺(IAA),但是IAA除了和半胱氨酸发生反应,也可能和其他多种氨基酸发生副反应。我们模拟常规的酶切条件,系统地研究了蛋白质真实酶切时所有酶切肽段发生烷基化的情况。结果表明,多种氨基酸可以发生烷基化,其趋势为:半胱氨酸>肽段N端氨基酸>天冬氨酸>谷氨酸>组氨酸>天冬酰胺>赖氨酸>酪氨酸,同时也发现同一肽段上的氨基酸烷基化具有排他性和聚集性。根据定性结果,采用质谱多反应监测(MRM)技术对多个肽段进行了定量分析,评估了过烷基化对蛋白质定量分析的影响。该研究结果表明,过量的烷基化修饰对蛋白质的定性与定量分析都可能产生较大影响。在蛋白质组学研究的样本处理流程中,应避免样本的过烷基化。