Recent developments and contributions from Chinese scientists in multidimensional separations for proteomics and traditional Chinese medicines

The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists in protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Yet the 2-D PAGE-mass spectrometry (MS) approach has its drawbacks with regard to automation, sensitivity, and throughput. Consequently, considerable effort has been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings of 2-D PAGE. In addition, traditional Chinese medicines (TCMs), due to their long period of clinical testing and reliable therapeutic efficacy, are attracting increased global attention. However, hundreds or even thousands of components are usually present in TCMs, which results in great difficulties of separation. As a mainstream separation tool, multidimensional liquid separation systems have shown powerful separation ability, high peak capacity, and excellent detectability in the analysis of complex samples including biological samples and TCMs, etc. Therefore, this review emphasizes the most recent advances in multidimensional liquid chromatography and capillary electrophoresis-based separation techniques, and the corresponding applications in proteomics and TCMs. In view of the significant contributions from Chinese scientists, this review focuses mainly on the work of Chinese scientists in the above fields.     

Novel liquid-chromatography columns for proteomics research

The enormous interest in proteomics research in recent years has inspired many developments in peptide chromatography. Different strategies have been developed to cope with the vast complexity of proteomics samples, trying to provide sufficient degree of separation to be able to exploit fully the potential of protein identification by mass spectrometry (MS). As reversed-phase liquid chromatography (RPLC) coupled to MS is still the method of choice for the analysis of protein digests, many efforts focus on the development of high-efficiency RP methods (e.g., monolithic columns and ultra-high-performance LC). This can also increase the speed and the sensitivity of the analysis of protein digests.As RPLC-MS alone is unlikely to provide sufficient resolution to unravel the composition of highly complex samples comprehensively, multidimensional methods will remain essential in proteome research. In this area, hydrophilic interaction chromatography (HILIC) seems to be a promising alternative to the traditional strong cation-exchange-based methods. Also, HILIC has found application in the analysis of post-translational modifications (e.g., phosphorylation and glycosylation).This review describes recent developments in LC methods for proteomics research, focusing on advances in column technology and the application of novel column materials. Illustrative examples show the possibilities of the new columns in proteomics research.     

Development of a monolithic silica extraction tip for the analysis of proteins

In proteomics, pre-treatment of sample is the most important procedure to remove the matrix for interfacing with mass spectrometry (MS). Additionally, for the samples with low concentration, the process of pre-concentration is required before MS analysis. We have newly developed solid-phase extraction (SPE) tool with pipette-tip shape for purification of bio-samples of various characteristics, utilizing monolithic silica gel as medium. The monolithic silica surface was modified with a C18 phase or coated with titania phase. A C18-bonded tip and a non-modified tip were used for sample concentration, desaltination and removal of detergents from sample. A titania-coated tip was also applied for purification and concentration of phosphorylated peptides. This novel pre-treatment method using monolithic silica extraction tip is much effective and suitable for protein analysis.     

Proteomics as a tool for optimization of human plasma protein separation

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.     

Trends in sample preparation for classical and second generation proteomics

Sample preparation is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extraction and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein separation by 2-DE, protein digestion, mass spectrometry analysis, multidimensional peptide separations and quantification of protein expression level are described.     

Microfluidic systems in proteomics

We present the state-of-the-art in miniaturized sample preparation, immunoassays, one-dimensional and multidimensional analyte separations, and coupling of microdevices with electrospray ionization-mass spectrometry. Hyphenation of these different techniques and their relevance to proteomics will be discussed. In particular, we will show that analytical performances of microfluidic analytical systems are already close to fulfill the requirements for proteomics, and that miniaturization results at the same time in a dramatic increase in analysis throughput. Throughout this review, some examples of analytical operations that cannot be achieved without microfluidics will be emphasized. Finally, conditions for the spreading of microanalytical systems in routine proteomic labs will be discussed.     

复杂生物体系中蛋白质高效分离分析技术的新进展

继人类基因组计划完成之后,作为一种新的研究策略,蛋白质组学在生命科学研究中发挥着愈来愈重要的作用。由于生物体系的复杂性和多样性,使得分离效率高、灵敏度高、通量高和动态范围宽的分离分析技术平台的研究和应用已成为蛋白质组学研究的重点和热点之一。着重介绍了近年来应用日益广泛的多维色谱预分离、毛细管液相色谱-质谱联用、毛细管电泳及其与质谱联用等高效分离分析技术在复杂生物体系的蛋白质分析中的最新进展。引用相关文献40篇。     

鼠肝蛋白质组的纳升级多维液相色谱分离

搭建了一个纳升级的二维液相色谱分离平台(nano-2D-LC),该平台可以自动完成进样、除盐、分离及鉴定。以离子交换色谱(SCX)为第一维,反相液相色谱(RPLC)为第二维,对鼠肝组织的蛋白质组进行了研究。SCX采用阶梯式洗脱,RPLC运用线性梯度洗脱,以200 nL/min的速度进行分离,峰容量可达620。     

多维高效液相色谱技术在蛋白质分离研究中的进展

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。     

Neuropeptide analysis with liquid chromatography-capillary electrophoresis-mass spectrometric imaging

Herein we report the first attempt of coupling multidimensional separations to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging detection. Complex neuropeptide mixtures extracted from crustaceans were first fractionated by reversed-phase liquid chromatography (RPLC), and then subjected to a capillary electrophoresis-mass spectrometric imaging platform. With a specific focus on orcokinin family neuropeptides, we demonstrated that these trace-level analytes from complex neural tissue samples can be fully separated from chemical noise and interfering components and visualized as mass spectrometric imaging signals. A total of 19 putative orcokinins were detected, with highly efficient separations within the family being achieved for the first time. The results indicate that two-dimensional separation coupling to mass spectrometric imaging can serve as a novel and powerful tool in proteomics and peptidomics studies.     

Capillary electrophoresis-based separation techniques for the analysis of proteins

CE offers the advantages of high speed, great efficiency, as well as the requirement of minimum amounts of sample and buffer for the analysis of proteins. In this review, we summarize the CE-based techniques coupled with absorption, LIF, and MS detection systems for the analysis of proteins mostly within the past 5 years. The basic principle of each technique and its advantages and disadvantages for protein analysis are discussed in brief. Advanced CE techniques, including on-column concentration techniques and high-efficiency multidimensional separation techniques, for high-throughput protein profiling of complex biological samples and/or of single cells are emphasized. Although the developed techniques provide improved peak capacity, they have not become practical tools for proteomics, mainly because of poor reproducibility, low-sample lading capacity, and low throughput due to ineffective interfaces between two separation dimensions and that between separation and MS systems. In order to identify the complexities and dynamics of the proteomes expressed by cells, tissues, or organisms, techniques providing improved analytical sensitivity, throughput, and dynamic ranges are still demanded.     

顶空-气相色谱-质谱联用分析桂花和叶中挥发性成分

采用谱库检索结合准确质量测定、保留指数、串联质谱技术的多维定性分析策略鉴定化合物, 能够提高定性分析的效率和准确性. 运用顶空-气相色谱-四极质谱、顶空-气相色谱-飞行时间质谱以及顶空-气相色谱-串联质谱联用技术对桂花样品进行了分析检测, 并采用多维定性分析思路对检出的挥发性成分进行了鉴定. 结果共确认出47种挥发性成分, 其中单萜类和倍半萜类化合物为主要组分. 该定性分析策略准确可靠, 可以广泛应用于复杂样品挥发性成分的定性分析中.     

蛋白质组研究中的多维液相色谱技术

对近年来蛋白质组研究中的多维液相色谱技术进行了系统综述,洋细介绍了在线及离线式阀切换接口的DALPC（direct analysis of large proein complexes）技术及整体式无接口的MudPIT（muhidimensional protein ideutification technology）系统,也系统阐述了其在蛋白质组研究中的应用。引用文献50篇。     

Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. Figure Artistic impression of the HILIC separation mechanism     

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.     

Determination of clofentezine in medical herb extracts by chromatographic methods combined with diode array scanning densitometry

The aim of this study is to report a new procedure for extraction, cleanup and determination of clofentezine in herb extracts by ultrasound‐assisted solvent extraction, SPE and multidimensional planar chromatography with diode array detector (MDPC‐DAD) and/or HPLC‐DAD. The application of various extraction solvents in SPE experiments conducted on octadecyl silane coupled with styrene‐divinylbenzene cartridges for fractionation and purification samples has been described. Normal‐phase systems were used in MDPC experiments on silica layer. The procedure described for the determination of compounds is inexpensive and can be applied to the routine analysis of analytes in plant extracts, after preliminary cleanup and concentration, e.g. by SPE. Application of MDPC‐DAD and HPLC‐DAD is especially useful for correct identification of components of difficult, complicated mixtures, e.g. analytes in medical herbs.     

Multidimensional liquid phase separations for mass spectrometry

Large part of the current research in biology, medicine, and biotechnology depends on the analysis of DNA (genomics), proteins (proteomics), or metabolites (metabolomics). The advances in biotechnology also command development of adequate analytical instrumentation capable to analyze minute amounts of samples. The analysis of the content of single cells may serve as an example of ultimate analytical applications. Most of the separation techniques have been developed in the last three decades and alternative approaches are being investigated. At present, the main protocols for analyses of complex mixtures include 2-DE (IEF) followed by electrophoresis in SDS polyacrylamide gel (SDS-PAGE) and chromatographic techniques. Information-rich techniques such as MS and NMR are essential for the identification and structure analysis of the analyzed compounds. High resolution separation of the individual sample components is often a prerequisite for success. High resolution proteomic analysis in the majority of laboratories still relies on the time consuming and laborious offline methods. This review highlights some of the important aspects of 2-D separations including microfluidics.     

多维液相色谱分离技术及其在蛋白质组研究中的应用

对近年来多维液相色谱技术及其在蛋白质组学研究中的应用进行了系统综述。详细描述了由不同液相色谱模式构建的多维液相色谱系统,并介绍了其在蛋白质组表达谱、翻译后修饰、定量等方面的应用。此外,还对多维液相色谱的发展趋势和前景进行了展望。     

An efficient data format for mass spectrometry-based proteomics

The diverse range of mass spectrometry (MS) instrumentation along with corresponding proprietary and nonproprietary data formats has generated a proteomics community driven call for a standardized format to facilitate management, processing, storing, visualization, and exchange of both experimental and processed data. To date, significant efforts have been extended towards standardizing XML-based formats for mass spectrometry data representation, despite the recognized inefficiencies associated with storing large numeric datasets in XML. The proteomics community has periodically entertained alternate strategies for data exchange, e.g., using a common application programming interface or a database-derived format. However, these efforts have yet to gain significant attention, mostly because they have not demonstrated significant performance benefits over existing standards, but also due to issues such as extensibility to multidimensional separation systems, robustness of operation, and incomplete or mismatched vocabulary. Here, we describe a format based on standard database principles that offers multiple benefits over existing formats in terms of storage size, ease of processing, data retrieval times, and extensibility to accommodate multidimensional separation systems.