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基于高压制备液相的多维色谱技术在中药分离纯化中的应用 总被引:1,自引:0,他引:1
中药物质基础复杂,对其活性成分的分离一直是中药研究的难题.基于高压制备液相的多维色谱系统在高压制备液相色谱的基础上,结合了多种分离技术,极大地提高了色谱系统的分离性能和分离效率,更有利于对物质基础复杂的中药样品进行分离纯化.本文介绍了基于高压制备液相系统的多维色谱系统的基本原理、分离模式以及关键技术,并综述了其在中药分离纯化中的应用. 相似文献
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蛋白质组学出现之后,多维高效液相色谱(multidimensional HPLC,MD-HPLC)系统以其快速、高效、自动化程度高以及容易与质谱等其他技术联用等优势而成为蛋白质组学相关分析技术中研究应用的热点。本文主要以本实验室在蛋白质组学研究中的技术进展为主线,介绍了多维高效液相色谱技术的发展,包括经典的“bottom-up”技术和“top-down”式的多维高效液相色谱技术路线,以及为了提高系统的分离通量而自行设计搭建的阵列式多维高效液相色谱平台,这些技术路线在蛋白质组学研究中有着极大的潜在应用价值。 相似文献
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多维液相色谱技术的进展 总被引:1,自引:0,他引:1
与一维分离模式相比,多维色谱分离技术的最大特点是可极大地提高峰容量.近几年,随着蛋白质组学的出现尤其是表达谱的开展,对分离技术提出更高的要求.多维高效液相色谱系统以其快速、高效、自动化程度高以及易与质谱等其他技术联用等优势再一次成为研究应用的热点.本文结合本课题组在多维色谱方面的工作介绍了多维色谱技术的发展及应用,重点介绍在蛋白质组学平台中的应用. 相似文献
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1 多维分离技术新进展
一维色谱是目前最常用的分离分析方法,然而对于复杂体系如蛋白质组,采用一维分离模式其分离度远远不能满足要求.Giddings理论告诉我们:对于分离机理相互正交的二维分离系统(如色谱),峰的容量应该为两个色谱柱峰容量的乘积.因此,多维分离系统是解决复杂分离体系的一个最佳选择.在多维色谱中二维气相色谱发展较快,目前全二维气相色谱仪业已商品化,其峰的容量达到104以上.而二维液相色谱,尤其是正相/反相二维液相色谱技术发展较为缓慢,其主要的技术瓶颈在于第一维色谱(正相)分离后的流动相严重干扰第二维色谱(反相)的分离. 相似文献
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《液相色谱法及相关技术杂志》2012,35(12):2771-2787
Abstract The objective of this article is to define, discuss and illustrate the concept of on-line multidimensional liquid chromatography (MDLC) in protein separation. In particular the emphasis of this paper is centered on a special form of on-line MDLC that will be referred to as linear MDLC. Examples of this technique, which involves the coupling of two or more chromatographic columns each employing a different separation mechanism, in both the analytical and preparative mode, are given in order to demonstrate its utility. 相似文献
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Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments. 相似文献
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Simpkins SW Bedard JW Groskreutz SR Swenson MM Liskutin TE Stoll DR 《Journal of chromatography. A》2010,1217(49):7648-7660
Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures. 相似文献
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Flensburg J Tangen A Prieto M Hellman U Wadensten H 《European journal of mass spectrometry (Chichester, England)》2005,11(2):169-179
Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing. 相似文献
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Qing‐Run Li Ke‐Xin Fan Rong‐Xia Li Jie Dai Chao‐Chao Wu Shi‐Lin Zhao Jia‐Rui Wu Chia‐Hui Shieh Rong Zeng 《Rapid communications in mass spectrometry : RCM》2010,24(6):823-832
Increasing attention has been paid to the urinary proteome because it holds the promise of discovering various disease biomarkers. However, most of the urine proteomics studies routinely relied on protein pre‐fractionation and so far did not present characterization on phosphorylation status. Two robust approaches, integrated multidimensional liquid chromatography (IMDL) and Yin‐yang multidimensional liquid chromatography (MDLC) tandem mass spectrometry, were recently developed in our laboratory, with high‐coverage identification of peptide mixtures. In this study, we adopted a strategy without pre‐fractionation on the protein level for urinary proteome identification, using both the IMDL and the Yin‐yang MDLC methods for peptide fractionation followed by identification using a linear ion trap‐orbitrap (LTQ‐Orbitrap) mass spectrometer with high resolution and mass accuracy. A total of 1310 non‐redundant proteins were highly confidently identified from two experiments, significantly including 59 phosphorylation sites. More than half the annotated identifications were membrane‐related proteins. In addition, the lysosomal as well as kidney‐associated proteins were detected. Compared with the six largest datasets of urinary proteins published previously, we found our data included most of the reported proteins. Our study developed a robust approach for exploring the human urinary proteome, which would provide a catalogue of urine proteins on a global scale. It is the first report, to our best knowledge, to profile the urinary phosphoproteome. This work significantly extends current comprehension of urinary protein modification and its potential biological significance. Moreover, the strategy could further serve as a reference for biomarker discovery. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
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Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry 总被引:1,自引:0,他引:1
Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the “drifting peak” phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the “drifting peak” phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures. 相似文献
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Marcus Krapp 《Accreditation and quality assurance》2001,6(7):324-326
Recent developments in international trade will have a significant impact on New Zealand’s measurement infrastructure, especially
for chemical metrology. This article describes the background to these developments and outlines the activities of the Measurement
Standards Laboratory, New Zealand’s National Metrology Institute, in response to these developments. 相似文献
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