Journal of Chromatography B (v.849, #1-2)

Methods for samples preparation in proteomic research by Anna Bodzon-Kulakowska; Anna Bierczynska-Krzysik; Tomasz Dylag; Anna Drabik; Piotr Suder; Marek Noga; Justyna Jarzebinska; Jerzy Silberring (1-31).
Sample preparation is one of the most crucial processes in proteomics research. The results of the experiment depend on the condition of the starting material. Therefore, the proper experimental model and careful sample preparation is vital to obtain significant and trustworthy results, particularly in comparative proteomics, where we are usually looking for minor differences between experimental-, and control samples. In this review we discuss problems associated with general strategies of samples preparation, and experimental demands for these processes.
Keywords: Sample; Proteomics; Analysis; Proteins; Peptides; Identification; Sequence;

Leveraging protein purification strategies in proteomics by Ipsita Roy; Kalyani Mondal; Munishwar N. Gupta (32-42).
The proteomic studies, although, tend to be analytical in nature, yet many strategies of preparative protein purification can be usefully employed in such studies. This review points out the importance of purification techniques which are capable of dealing with samples which are suspensions rather than clear solution, e.g. aqueous two phase partitioning, three phase partitioning, expanded bed chromatography, etc. The review also outlines the potential of non-chromatographic techniques in dealing with fractionation of proteomes. Separation protocols which can deal with post-translationally modified (PTM) proteins are also considered.
Keywords: Post-translational modification; Protein arrays; Affinity precipitation; Three phase partitioning; Aqueous two phase system;

The plasma proteome has a wide dynamic range of protein concentrations and is dominated by a few highly abundant proteins. Discovery of novel cancer biomarkers using proteomics is particularly challenging because specific biomarkers are expected to be low abundance proteins with normal blood concentrations of low nanograms per milliliter or less. Conventional, one- and two-dimensional proteomic methods including 2D PAGE, 2D DIGE, LC–MS/MS, and LC/LC–MS/MS do not have the capacity to consistently detect many proteins in this range. In contrast, new higher dimensional (Hi-D) separation strategies, utilizing more than two dimensions of fractionation, can profile the low abundance proteome.
Keywords: Cancer; Proteomics; Plasma proteome; Serum proteome; Blood proteome; Multidimensional separation; Biomarkers; Protein separation; Peptide separation;

Current insights on protein behaviour in hydrophobic interaction chromatography by M. Elena Lienqueo; Andrea Mahn; J. Cristian Salgado; Juan A. Asenjo (53-68).
This paper gives a summary of different aspects for predicting protein behaviour in hydrophobic interaction chromatography (HIC). First, a brief description of HIC, hydrophobic interactions, amino acid and protein hydrophobicity is presented. After that, several factors affecting protein chromatographic behaviour in HIC are described. Finally, different approaches for predicting protein retention time in HIC are shown. Using all this information, it could be possible to carry out computational experiments by varying the different operating conditions for the purification of a target protein; and then selecting the best conditions in silico and designing a rational protein purification process involving an HIC step.
Keywords: Hydrophobic interaction chromatography; Retention time prediction; Retention mechanism; Hydrophobicity;

The ultimate goal of proteomics is to identify biologically active proteins and to produce them using biotechnology tools such as bacterial hosts. However, proteins produced by Escherichia coli must be refolded to their native state. Protein folding liquid chromatography (PFLC) is a new method developed in recent years, and it is widely used in molecular biology and biotechnology. In this paper, the new method, PFLC is introduced and its recent development is reviewed. In addition the paper includes definitions, advantages, principles, applications for both laboratory and large scales, apparatus, and effecting factors of PFLC. In addition, the role of this method in the future is examined.
Keywords: Recombinant proteins; Proteomics; Protein folding liquid chromatography; Industrialization;

Affinity chromatography: A useful tool in proteomics studies by Mohamed Azarkan; Joëlle Huet; Danielle Baeyens-Volant; Yvan Looze; Guy Vandenbussche (81-90).
Separation or fractionation of a biological sample in order to reduce its complexity is often a prerequisite to qualitative or quantitative proteomic approaches. Affinity chromatography is an efficient protein separation method based on the interaction between target proteins and specific immobilized ligands. The large range of available ligands allows to separate a complex biological extract in different protein classes or to isolate the low abundance species such as post-translationally modified proteins. This method plays an essential role in the isolation of protein complexes and in the identification of protein–protein interaction networks. Affinity chromatography is also required for quantification of protein expression by using isotope-coded affinity tags.
Keywords: Proteomics; Affinity chromatography; Mass spectrometry; Phosphoproteome; Glycoproteome; Disulfide proteome; Isotope-coded affinity tags;

Enhanced detectability in proteome studies by Supawadee Sriyam; Supachok Sinchaikul; Payungsak Tantipaiboonwong; Ching Tzao; Suree Phutrakul; Shui-Tein Chen (91-104).
The discovery of candidate biomarkers from biological materials coupled with the development of detection methods holds both incredible clinical potential as well as significant challenges. However, the proteomic techniques still provide the low dynamic range of protein detection at lower abundances. This review describes the current development of potential methods to enhance the detection and quantification in proteome studies. It also includes the bioinformatics tools that are helpfully used for data mining of protein ontology. Therefore, we believe that this review provided many proteomic approaches, which would be very potent and useful for proteome studies and for further diagnostic and therapeutic applications.
Keywords: Proteome; Proteomic analysis; Detection; Limitation; Sample preparation;

Biomarker discovery from body fluids using mass spectrometry by Ana Villar-Garea; Matthias Griese; Axel Imhof (105-114).
Systems analysis of body fluids by mass spectrometry (MS) is an upcoming field of biomarker research. This approach is extremely attractive because it does not require biomarker candidates and the sample preparation is simple. However, during the development of the technique strong critical comments were made on the poor reproducibility, the special characteristics of blood as a source of peptides and on the frequent non-adequate statistical analysis of the data. Here we discuss the efforts made in the last few years to develop suitable protocols, which could lead to biomarker discovery from body fluids by mass spectrometry. Our review focuses on the systems analysis of non-digested blood serum or plasma samples by MALDI-TOF and SELDI-TOF.
Keywords: Biomarker; Mass spectrometry; Serum analysis; High throughput;

Glycoproteomics based on tandem mass spectrometry of glycopeptides by Manfred Wuhrer; M. Isabel Catalina; André M. Deelder; Cornelis H. Hokke (115-128).
Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.
Keywords: Collision-induced dissociation; Electron-capture dissociation; Electron-transfer-dissociation; Glycopeptide; Glycosylation;

Towards the entire proteome of the model bacterium Bacillus subtilis by gel-based and gel-free approaches by Susanne Wolff; Haike Antelmann; Dirk Albrecht; Dörte Becher; Jörg Bernhardt; Sierd Bron; Knut Büttner; Jan Maarten van Dijl; Christine Eymann; Andreas Otto; Le Thi Tam; Michael Hecker (129-140).
With the emergence of mass spectrometry in protein science and the availability of complete genome sequences, proteomics has gone through a rapid development. The soil bacterium Bacillus subtilis, as one of the first DNA sequenced species, represents a model for Gram-positive bacteria and its proteome was extensively studied throughout the years. Having the final goal to elucidate how life really functions, one basic requirement is to know the entirety of cellular proteins. This review presents how far we have got in unraveling the proteome of B. subtilis. The application of gel-based and gel-free technologies, the analyses of different subcellular proteome fractions, and the pursuance of various physiological strategies resulted in a coverage of more than one-third of B. subtilis theoretical proteome.
Keywords: Proteome coverage; Stress and starvation signatures; Subproteomic fractions; Gel-based and gel-free proteomics;

Prokaryotic elongation factors EF-Tu form a family of homologous, three-domain molecular switches catalyzing the binding of aminoacyl-tRNAs to ribosomes during the process of mRNA translation. They are GTP-binding proteins, or GTPases. Binding of GTP or GDP regulates their conformation and thus their activity. Because of their particular structure and regulation, various activities (also outside of the translation system) and a relative abundance they represent attractive tools for studies of many basic but still not fully understood mechanisms both of the translation process, the structure–function relationships in EF-Tu molecules themselves and proteins and energy transduction mechanisms in general. The review critically summarizes procedures for the isolation and purification of native and engineered eubacterial elongation factors EF-Tu and their mutants on a large as well as small scale. Current protocols for the purification of both native and polyHis-tagged or glutathione-S-transferase (GST)-tagged EF-Tu proteins and their variants using conventional procedures and the Ni-NTA-Agarose or Glutathione Sepharose are presented.
Keywords: Bacterial elongation factors EF-Tu; Detection methods; Separation methods; Purification methods; EF-Tu.GDP; EF-Tu.EF-Ts; EF-Tu mutants; G-domains; Protein synthesis; Escherichia coli; Bacillus stearothermophilus; Bacillus subtilis; Streptomyces aureofaciens; Streptomyces coelicolor; Thermus aquaticus; Thermus thermophilus;

Current methods for phosphoprotein isolation and enrichment by Stefan R. Schmidt; Fritz Schweikart; Martin E. Andersson (154-162).
The phosphorylation of proteins is a central paradigm of signal transduction. The substitution of neutral hydroxyl groups of serine, threonine and tyrosine with a negatively charged phosphate group alters the physicochemical and immunogenic properties of the protein, which then can be used to isolate these isoforms. In the last decades several different techniques were applied, attempting to selectively enrich protein populations with this post-translational modification. This review aims to give an overview on the arsenal of available methods to extract phosphoproteins focusing on chromatographic approaches.
Keywords: Phosphoprotein; Chromatography; IMAC; Chromatofocusing; Immunoprecipitation;

Analytical methodologies for the detection and structural characterization of phosphorylated proteins by Chiara D’Ambrosio; Anna Maria Salzano; Simona Arena; Giovanni Renzone; Andrea Scaloni (163-180).
Phosphorylation of proteins is a frequent post-translational modification affecting a great number of fundamental cellular functions in living organisms. Because of its key role in many biological processes, much effort has been spent over the time on the development of analytical methodologies for characterizing phosphoproteins. In the past decade, mass spectrometry-based techniques have emerged as a viable alternative to more traditional methods of phosphorylation analysis, providing accurate information for a purified protein on the number of the occurring phosphate groups and their exact localization on the polypeptide sequence. This review summarizes the analytical methodologies currently available for the analysis of protein phosphorylation, emphasizing novel mass spectrometry (MS) technologies and dedicated biochemical procedures that have been recently introduced in this field. A formidable armamentarium is now available for selective enrichment, exaustive structural characterization and quantitative determination of the modification degree for phosphopeptides/phosphoproteins. These methodologies are now successfully applied to the global analysis of cellular proteome repertoire according a holistic approach, allowing the quantitative study of phosphoproteomes on a dynamic time-course basis. The enormous complexity of the protein phosphorylation pattern inside the cell and its dynamic modification will grant important challenges to future scientists, contributing significantly to deeper insights into cellular processes and cell regulation.
Keywords: Phosphorylation; Proteomics; Electrophoresis; Mass spectrometry;

The proteomics of keratin proteins by Jeffrey E. Plowman (181-189).
Keratin proteins are widespread in nature, being found in the nuclei and cytoplasm of almost all differentiated eukaryote cells. However, they are best known as the principal structural proteins in hair, wool and skin. Because of difficulties associated with their extraction from biological samples, high sequence homology and the presence of numerous post-translational modifications, they have been less well studied than other protein families. Thanks to the advent of modern proteomic techniques we now have available a good suite of tools to study this neglected family of proteins.
Keywords: Cytokeratins; Hair; Wool; Fish; Cancer; Cystic fibrosis; Ocular keratins;

Cell proteomes are complex, given they consist of several thousand proteins. Two-dimensional electrophoresis (2DE) is unique not only for its ability to simultaneously separate thousands of proteins but also for detecting post- and co-translational modifications, which cannot be predicted from genome sequences.This review will describe the protocols applied to prepare 2D gels properly, and analyse and summarise the major challenges for successful proteome analysis using 2DE, i.e. the ability to analyse very alkaline, hydrophobic and/or low or high M r proteins with high resolution and the ability to detect minor components. Challenges involving sample preparation and solubilisation prior to the first dimension IEF/IPG step will be studied in depth. Sample preparation is crucial in 2DE studies and greatly influences other stages of the technique.It is the aim of this review not only to describe the challenges and limitations of 2DE but also to suggest the avenues, the evolution, the potential and the future of 2DE in proteomics.
Keywords: Two-dimensional electrophoresis; Sample preparation; Expression proteomics;

Human plasma consists of mainly large proteins, which vary in terms of both composition and concentration with the physiological state of the individual. Alterations in protein concentrations reflect the current state of the individual's health and thus may be utilized as valuable biomarkers for a specific biological process or disease. Two-dimensional gel electrophoresis (2-DE) has proven to be a valuable method for the separation and comparison of complex protein mixtures, for example, from disease and healthy states, as this method provides information regarding the variation, relative quantities, and structures of the intact proteins. The procedures utilized for the preparation of samples for 2-DE are critical to the acquisition of high-quality results for the discovery of biomarkers. The objective of this study was to review the preparation methods of plasma for 2-DE, particularly those designed to improve the detection of proteins in low abundance in plasma on 2-DE. The use of anticoagulants and protease inhibitors during the collection of blood, the removal of abundant proteins using multicomponent immunodepletion system, and desalting procedure allow us to compile profiles of proteins occurring in low concentrations in the plasma and to improve the pattern generated during 2-DE.
Keywords: Human blood plasma; Two-dimensional gel electrophoresis (2-DE); Proteomics;

Agarose gel is the preferred electrophoretic medium currently used for separating high molecular mass (HMM) proteins (MW > 100 kDa). Agarose gels are widely used for both SDS-agarose gel electrophoresis and agarose isoelectric focusing (IEF). A two-dimensional gel electrophoresis method employing agarose gels in the first dimension (agarose 2-DE) that is sufficiently good at separating up to 1.5 mg of HMM proteins with molecular masses as large as 500 kDa has been used to separate proteins from various diseased tissues and cells. Although resolution of the agarose 2-DE pattern always depends on the tissue being analyzed, sample preparation procedures including (i) protein extraction with an SDS sample buffer; (ii) ultracentrifugation of a tissue homogenate; and (iii) 1% SDS in both stacking and separation gels of the second-dimension SDS-PAGE gel, are generally effective for HMM protein detection. In a comprehensive prostate cancer proteome study using agarose 2-DE, the HMM region of the gel was rich in proteins of particular gene/protein expression groups (39.1% of the HMM proteins but only 28.4% of the LMM ones were classified as transcription/translation-related proteins). Examples include transcription factors, DNA or RNA binding proteins, and ribosomal proteins. To understand oxidative stress-induced cellular damage at the protein level, a novel proteomic method, in which protein carbonyls were derivatized with biotin hydrazide followed by agarose 2-DE, was useful for detecting HMM protein carbonyls in tissues of both a diabetes model Ostuka Long-Evans Tokushima Fatty (OLETF) rat and a control Long-Evans Tokushima Otsuka (LETO) rat. In this paper, we review the use of agarose gels for separation of HMM proteins and disease proteomics of HMM proteins in general, with particular attention paid to our proteome analyzes based on the use of agarose 2-DE for protein separation followed by the use of mass spectrometry for protein identification.
Keywords: Reviews; Two-dimensional gel electrophoresis; Disease proteomics; High-molecular-mass proteins (HMM);

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.
Keywords: Protein digestion; Proteomics; Fourier transform ion cyclotron resonance mass spectrometry; LC/MS/MS; Monolithic column;

Cation exchangers for selective sorption of large proteins by O.V. Ochkur; A.A. Demin (231-235).
The sorption of bovine serum albumin, cytochrom c and fibrinogen on the series of carboxylic cation-exchangers with various concentrations of ionogenic groups has been investigated. The dependence of sorption selectivity on protein size and on concentration of ionogenic groups was demonstrated.
Keywords: Adsorption; Ion exchangers; Proteins; Competition;

Optimal operation conditions for protein separation in hydrophobic interaction chromatography by Andrea Mahn; M. Elena Lienqueo; Juan A. Asenjo (236-242).
Protein retention in hydrophobic interaction chromatography is determined by protein physicochemical properties and by system characteristics. In this paper we present an attempt to determine the optimal operation conditions that would allow the separation of binary protein mixtures. The statistically significant system variables were determined, and then empirical models were obtained which explained more than 92% of variability in dimensionless retention time based on salt properties, ionic strength of the initial eluent and substitution degree of the resin. These variables were optimized in order to achieve the maximum retention time difference between two proteins in a mixture. The optimum operation conditions as predicted by the models were tested experimentally, showing a good agreement with predicted separation. We concluded that it would be possible to determine the system conditions that allow the maximum separation of two proteins based on the main system properties. The methodology proposed here presents potential to be applied to partially characterized systems, however, it could be improved if protein's properties were included explicitly in the models.
Keywords: Hydrophobic interaction chromatography; Operation conditions; Optimization;

Solid phase proteomics: Dramatic reinforcement of very weak protein–protein interactions by Manuel Fuentes; Cesar Mateo; Benevides C.C. Pessela; Pilar Batalla; Roberto Fernandez-Lafuente; J.M. Guisán (243-250).
Very weak protein–protein interactions may play a critical role in cell physiology but they are not easily detectable in “in vitro” experiments. To detect these weak interactions, we have developed a strategy that included: (a) design of a rapid and very effective crosslinking of protein–protein complexes with poly-functional reagents; (b) selective adsorption of very large proteins on lowly activated ionic exchangers, based on the need of a multipoint physical adsorption to incorporate the proteins into the matrix; (c) purification by selective adsorption of protein–protein complexes formed by strong protein–protein interactions, via selective adsorption of the complexes on lowly activated ionic exchangers via multi-protein physical adsorption and leaving the non-associated proteins in the solution; (d) reinforcement of very weak protein–protein interactions by selective adsorption of the complex on lowly activated ionic exchange supports via a synergetic cooperation of the weak protein–protein interaction plus the interactions of both proteins with the support enabling the almost full shifting of the equilibrium towards the association position; (e) control of the aggregation state of proteins like BSA, formed by weak protein–protein interactions. In this last case, it seems that the interaction of the protein molecules placed on the borders of the aggregate with the groups on the support partially stabilizes the whole aggregate, although, some molecules of the aggregate cannot interact with the support. The size of the aggregates may be defined by controlling the concentration of ionised groups on the support: the less activated the supports are, the bigger the complexes. In this way, solid-phase proteomics could be a very interesting tool to detect weak protein–protein interactions.
Keywords: Multipoint adsorption; Stabilizaition of protein complex; Purification of protein complex;

Improvements in the search for potential biomarkers by proteomics: Application of principal component and discriminant analyses for two-dimensional maps evaluation by Ana María Rodríguez-Piñeiro; Francisco Javier Rodríguez-Berrocal; María Páez de la Cadena (251-260).
In this study, we evaluated if the application of multivariate analysis on the data obtained from two-dimensional protein maps could mean an improvement in the search for protein markers. First, we performed a classical proteomic study of the differential expression of serum N-glycoproteins in colorectal cancer patients. Then, applying principal component analysis (PCA) we assessed the utility of the 2-D protein pattern and certain subsets of spots as a tool to distinguish control and case samples, and tested the accuracy of the classification model by linear discriminant analysis (LDA). On the other hand we looked for altered spots by univariate statistics and then analysed them as a cluster by PCA and LDA. We found that those proteins combined presented a theoretical sensitivity and specificity of 100%. Finally, the spots with known protein identity were analysed by multivariate methods, finding a subgroup that behaved as the most obvious candidates for further validation trials.
Keywords: Two-dimensional gel electrophoresis; 2D-PAGE; Data evaluation; Multivariate statistics; Principal component analysis; Discriminant analysis;

Statistics for proteomics: Experimental design and 2-DE differential analysis by Jean-François Chich; Olivier David; Fanny Villers; Brigitte Schaeffer; Didier Lutomski; Sylvie Huet (261-272).
Proteomics relies on the separation of complex protein mixtures using bidimensional electrophoresis. This approach is largely used to detect the expression variations of proteins prepared from two or more samples. Recently, attention was drawn on the reliability of the results published in literature. Among the critical points identified were experimental design, differential analysis and the problem of missing data, all problems where statistics can be of help. Using examples and terms understandable by biologists, we describe how a collaboration between biologists and statisticians can improve reliability of results and confidence in conclusions.
Keywords: Experimental design; Differential analysis; Proteomics; Two-dimensional gel electrophoresis;

Rat plasma proteomics: Effects of abundant protein depletion on proteomic analysis by Thomas Linke; Sundari Doraiswamy; Earl H. Harrison (273-281).
The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and α1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2 D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2–3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7–8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.
Keywords: Plasma; Depletion; Immunoaffinity; SELDI-TOF MS; 2D SDS-PAGE; 2-Dimensional chromatography; Chromatofocusing; Reversed phase; Proteomics;

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis by Yasuhiro Kashino; Takeshi Harayama; Himadri B. Pakrasi; Kazuhiko Satoh (282-292).
In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-β-d-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.
Keywords: Membrane protein; Proteomics; IEF; Iso-electric focusing; Agarose; n-dodecyl-β-d-maltopiranoside;

Comparative proteomics of rat liver and Morris hepatoma 7777 plasma membranes by James G. Clifton; Xuesong Li; Werner Reutter; Douglas C. Hixson; Djuro Josic (293-301).
Plasma membranes from normal rat liver and hepatocellular carcinoma Morris hepatoma 7777 were selectively solubilized by use of different reagents. After selective solubilization, proteins were identified by nano-HPLC–electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). Using simple software, the patterns of proteins identified in membrane solubilizates from liver and hepatoma were compared. Proteins identified in Morris hepatoma 7777 and not in the corresponding membrane solubilizate from liver, mostly members of the annexin and heat shock protein families, are discussed as potential candidate markers for hepatocellular carcinomas.
Keywords: Liver; Morris hepatoma 7777; Plasma membrane; Mass spectrometry; Candidate biomarker;

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography–mass spectrometry by C. Cartoni; M.E. Schininà; B. Maras; R. Nonno; G. Vaccari; M. Di Bari; M. Conte; A. De Pascalis; S. Principe; F. Cardone; M. Pocchiari; U. Agrimi (302-306).
The conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPTSE) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC–MS method that allowed to demonstrate that in the brain of 109Met/Ile heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrPTSE with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.
Keywords: Prion protein; Quantitative method; Mass spectrometry;

Separation, characterization and identification of boar seminal plasma proteins by V. Jonáková; P. Maňásková; M. Tichá (307-314).
Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.
Keywords: Boar seminal plasma proteins; Separation methods; Identification and characterization of proteins; Binding properties of proteins; Reproduction; Spermadhesins;

Application of immunoproteomics in developing a Streptococcus iniae vaccine for olive flounder (Paralichthys olivaceus) by Gee-Wook Shin; K.J. Palaksha; Young-Rim Kim; Sung-Won Nho; Suk Kim; Gang-Joon Heo; Se-Chang Park; Tae-Sung Jung (315-322).
Streptococcus iniae is the major etiological agent of streptococcosis, which is responsible for hemorrhagic septicemia in fish, particularly olive flounder (Paralichthys olivaceus). In the present study, we sought to understand the pathogenicity and immunogenicity of S. iniae in order to develop a vaccine for streptococcosis. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting, was employed to investigate the pathogenicity and immunogenicity of two S. iniae isolates, Jeju-13 and Jeju-45, in olive flounder. The virulence of Jeju-13 was moderate whereas that of Jeju-45 was high. A vaccination trial with formalin-killed Jeju-45 demonstrated relatively low protection against the homologous isolate compared with the heterologous isolate. A significant difference in the secretion of extracellular products (ECPs) was noticed between the two S. iniae isolates. ECP antigens were highly immunogenic compared to those from whole cell lysates as determined by 2-DE immunoblot assay of Jeju-13 and Jeju-45 anti-sera collected from post-challenge survival fish. Furthermore, there were differences in the appearance of antigenic spots on 2-DE immunoblot profiles of ECPs of the respective sera. Interestingly, the mixture of killed-cells and concentrated ECPs from Jeju-45 led to significant protection against the homologous isolate of S. iniae in olive flounder. The present study demonstrates the usefulness of immunoproteomics in understanding the pathogenicity of S. iniae to aid the development of a vaccine for fish streptococcosis.
Keywords: Streptococcus iniae; Immunoproteome; Vaccine; Two-dimensional gel electrophoresis (2-DE); Immunoblotting; Extracellular products (ECPs);

Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1 by Dae-Hee Lee; Sung-Gun Kim; Yong-Cheol Park; Soo-Wan Nam; Kelvin H. Lee; Jin-Ho Seo (323-330).
The proteomic response of recombinant Escherichia coli producing human glucagon-like peptide-1 was analyzed by two-dimensional gel electrophoresis. Protein spots in two-dimensional gel could be identified by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their expression profiles were compared with those of nonproducing cells. Thirty-five intracellular proteins exhibited differential expression levels between the production and control strains. These changes reflected physiological responses to heterologous peptide production in recombinant E. coli. Specifically, physiological changes included the down-regulation of proteins involved in the central carbon metabolism, biosynthesis of cellular building blocks and peptides, and up-regulation of cell protection proteins and some sugar transport proteins. This comprehensive analysis would provide useful information for understanding physiological alterations to heterologous peptide production and for designing efficient metabolic engineering strategies for the production of recombinant peptides in E. coli.
Keywords: Escherichia coli; Proteomic response; Human glucagon-like peptide-1; Two-dimensional gel electrophoresis;

Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting by Tsai-Mu Cheng; Pei-Chen Huang; Ju-Pin Pan; Kuan-Yu Lin; Simon J.T. Mao (331-336).
Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3 mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.
Keywords: Polyphenol oxidase; Tyrosinase; Catechol; Immobilized-paper; Browning reaction; Natural PPO inhibitor;

The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; M r 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with M r 82 kDa) and a basic protein with M r 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM®) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2 M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5 M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with M r 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH3CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5 mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.
Keywords: Purification; Isolation; Cation-exchange chromatography; Short monolithic columns; HPLC; Pectin methylesterase; Polygalacturonase; Pectolytic enzymes; Tomato; Lycopersicon esculentum;

Extraction of wheat endosperm proteins for proteome analysis by William J. Hurkman; Charlene K. Tanaka (344-350).
Total protein extracts of wheat endosperm are widely used for the analysis of the highly abundant gliadins and glutenins. In this review, the most popular total endosperm extraction methods are compared for their effectiveness in proteome coverage. A drawback of total endosperm extracts is that the enormous dynamic range of protein abundance limits the detection, quantification, and identification of low abundance proteins. Protein fractionation is invaluable for improving proteome coverage, because it reduces sample complexity while enriching for specific classes of less abundant proteins. A wide array of techniques is available for isolating protein subpopulations. Sequential extraction is a method particularly suited for subfractionation of wheat endosperm proteins, because it takes advantage of the specific solubility properties of the different classes of endosperm proteins. This method effectively separates the highly abundant gliadins and glutenins from the much less abundant albumins and globulins. Subcellular fractionation of tissue homogenates is a classical technique for isolating membranes and organelles for functional analysis. This approach is suitable for defining the biochemical processes associated with amyloplasts, specialized organelles in the endosperm that function in the synthesis and storage of starch. Subproteome fractionation, when combined with 2-DE and protein identification, provides a powerful approach for defining endosperm protein composition and providing new insights into cellular functions.
Keywords: 2-DE; Albumins; Amyloplasts; Endosperm; Gliadins; Globulins; Glutenins; Proteome; Wheat;

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: A follow up by B.E. Maserti; C.M. Della Croce; F. Luro; R. Morillon; M. Cini; L. Caltavuturo (351-356).
With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3–8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.
Keywords: Citrus proteins; Narrow range immobilized pH gradient strips; Basic proteins; Low abundance proteins; C7BzO;

Purification and characterization of cassiicolin, the toxin produced by Corynespora cassiicola, causal agent of the leaf fall disease of rubber tree by Frédéric de Lamotte; Marie-Pierre Duviau; Christine Sanier; Robert Thai; Joël Poncet; Daniel Bieysse; Frédéric Breton; Valérie Pujade-Renaud (357-362).
Cassiicolin, a phytotoxin produced by the necrotrophic fungus Corynespora cassiicola, was purified to homogeneity from a rubber tree isolate. The optimized protocol involves reverse phase chromatography followed by size exclusion chromatography, with monitoring of the toxicity on detached rubber tree leaves. Cassiicolin appeared to be a peptide composed of 27 amino acids, glycosylated on the second residue, with a N-terminal pyroglutamic acid and 6 cysteines involved in disulfide bonds. Its molecular mass was estimated to be 2885 Da. No significant sequence homology with other proteins could be found. The availability of pure toxin in sufficient amount is a prerequisite for its structure determination, which is a key step in the understanding of the aggression mechanism.
Keywords: Cassiicolin; Corynespora cassiicola; Hevea brasiliensis; Toxin; Purification; Chromatography; Amino acid sequence; Mass spectrometry;