Journal of Chromatography B (v.815, #1-2)

Preface by Z. Deyl (1).

Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.
Keywords: Comparative proteomics; Two-dimensional electrophoresis; Shotgun peptide sequencing;

Peptidomics, current status by Mikhail Soloviev; Paul Finch (11-24).
Characterisation of the complement of expressed proteins from a single genome is a central focus of the evolving field of proteomics. Traditional proteomics technologies were developed in the 20th century and are based on two-dimensional electrophoresis or multidimensional liquid chromatography. These facilitated functional genomics analysis, but they currently represent a significant bottleneck to progress in this area. We are now witnessing the development of novel alternative technologies for use in expression proteomics research. This review aims to familiarise the reader with the principles underlying the peptidomics approaches to proteomics research and provide examples of their applications.
Keywords: Peptidomics; Reviews;

Geometrical distortions in two-dimensional gels: applicable correction methods by T. Aittokallio; J. Salmi; T.A. Nyman; O.S. Nevalainen (25-37).
Two-dimensional electrophoresis (2-DE) provides a rapid means for separating thousands of proteins from cell and tissue samples in one run. Although this powerful research tool has been enthusiastically applied in many fields of biomedical research, accurate analysis and interpretation of the data have provided many challenges. Several analysis steps are needed to convert the large amount of noisy data obtained with 2-DE into reliable and interpretable biological information. The goals of such analysis steps include accurate protein detection and quantification, as well as the identification of differentially expressed proteins between samples run on different gels. To achieve these goals, systematic errors such as geometric distortions between the gels must be corrected by using computer-assisted methods. A wide range of computer software has been developed, but no general consensus exists as standard for 2-DE data analysis protocol. The choice of analysis approach is an important element depending both on the data and on the goals of the experiment. Therefore, basic understanding of the algorithms behind the software is required for optimal results. This review highlights some of the common themes in 2-DE data analysis, including protein spot detection and geometric image warping using both spot- and pixel-based approaches. Several computational strategies are overviewed and their relative merits and potential pitfalls discussed. Finally, we offer our own personal view of future trends and developments in large-scale proteome research.
Keywords: Geometrical distortions; Spot detection; Image warping;

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.
Keywords: Low abundance protein; MALDI-TOF; Gel electrophoresis;

Role of chromatographic techniques in proteomic analysis by Irina Neverova; Jennifer E. Van Eyk (51-63).
Proteomics, the characterization of the proteome, is conceptually simple but technically challenging. Development of such technologies as mass spectrometry, multidimensional protein separation, and DNA sequencing has allowed the new field of proteomics to flourish. Proteomic analysis relies on a set of techniques chosen on the basis of the biological question. In any proteomic analysis, the first and most important task is the separation of a complex protein mixture, i.e. the proteome. Chromatography, one of the most powerful methods of separation, employs one or more inherent characteristics of a protein—its mass, isoelectric point, hydrophobicity or biospecificity. This review emphasizes high-performance liquid chromatography as an integrated part of technologies used to study the proteome, discusses the capabilities and limitations of current instruments, and highlights the potential of multidimensional liquid chromatography in proteomic analysis.
Keywords: Multidimensional approach; Proteomics; Liquid chromatography–mass spectrometry;

Immunoproteomics by Jennifer L. Hess; Levi Blazer; Terence Romer; Lee Faber; R. Mark Buller; Michael D.P. Boyle (65-75).
A novel immunoproteomic assay, combining specificity of antibody with precision of mass spectral analysis is described, and a number of practical applications are presented. The assay is carried out in three steps. The first step of the assay involves antibody immobilization, using a bacterial Fc binding support. The second step is antigen capture and washing to remove non-specific binding. The third step involves analysis of the captured antigens by SELDI-TOF. The assay has many advantages in sensitivity, speed, and economy of reagents in detection of specific antigens or antibodies. In addition, under appropriate experimental conditions, semi-quantitative data may be obtained. By combining the increasing range of selective specific antibody reagents available, in part due to advances in antibody engineering technology, and the resolving power available, using mass spectrometry, immunoproteomics is a valuable technique in proteomic analysis. A number of examples of the application of this technique to analysis of biological systems are presented.
Keywords: Immunoproteomics;

Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein–DNA and protein–protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.
Keywords: Microarray technologies; Proteomics; Protein array; Protein interactions; High-throughput screening;

Survey of current protein family databases and their application in comparative, structural and functional genomics by Oliver Redfern; Alastair Grant; Michael Maibaum; Christine Orengo (97-107).
The last two decades have witnessed significant expansions in the databases storing information on the sequences and structures of proteins. This has led to the creation of many excellent protein family resources, which classify proteins according to their evolutionary relationship. These have allowed extensive insights into evolution and particularly how protein function mutates and evolves over time. Such analyses have greatly assisted the inheritance of functional annotations between experimentally characterised and uncharacterised genes. Moreover, the development of bioinformatics tools acts as a companion to the new technologies emerging in biology, such as transcriptomics and proteomics. The latter enable researchers to analyse gene expression profiles and interactions on a genome-wide scale, generating vast datasets of proteins, many of which include experimentally uncharacterised proteins. Protein family/function databases can be used to help interpret this data and allow us to benefit more fully from these technologies. This review aims to summarise the most popular sequence- and structure-based protein family databases. We also cover their application to comparative genomics and the functional annotation of the genomes.
Keywords: Databases; Proteomics; Genomics; CATH; Structural classifications;

System, trends and perspectives of proteomics in dicot plants by Ganesh Kumar Agrawal; Masami Yonekura; Yumiko Iwahashi; Hitoshi Iwahashi; Randeep Rakwal (109-123).
The first 3 years of the 21st century have seen the impact of plant proteomics on functional genomics that has enhanced our understanding, not only on the plant genome(s), but also more importantly, on the functional aspect of proteins. This is mainly due to availability of the complete genome sequence of the Arabidopsis thaliana—a dicotyledoneous (dicot) model plant—and technological advancements in proteomics. Proteomic analyses of a variety of dicot plants, including both Arabidopsis and the model legume species, barrel medic (Medicago truncatula), have greatly helped in an efficient separation, identification and cataloguing of a large number of proteins, and thereby defining their proteomes. Therefore, we have composed an inclusive review on dicot plant materials, as of February 2004, that provides system, trends and perspectives of proteomics in growth and development and the environment. The review is summarized and discussed as three individual, but interlinked, entities: Part I, technologies in proteome establishment (this review), Part II, proteomes of the complex developmental stages [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)], and Part III, unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)]. This review deals with the diverse proteomic technologies being used in proteome development of different dicot plants.
Keywords: Dicotyledoneous plants; Sample preparation; Techniques; 2-DGE; MS; Protein identification; Bioinformatics;

System, trends and perspectives of proteomics in dicot plants by Ganesh Kumar Agrawal; Masami Yonekura; Yumiko Iwahashi; Hitoshi Iwahashi; Randeep Rakwal (125-136).
This review is devoted to the proteomes of the complex developmental stages of dicotyledoneous (dicot) plant materials. The two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS), independently or in combination with each other, are propelling dicot plant proteomics to new discoveries and functions, with the establishment of tissue-specific and organelle proteomes, mostly in Arabidopsis thaliana and Medicago truncatula, revealing their complexity and specificity. These experimental proteomes have provided a good start towards the establishment of high-density 2-DGE reference maps and peptide mass fingerprint databases, for not only the model dicot plants, A. thaliana and M. truncatula, but also other important dicot plants, which will serve as a basis for proteomes of many other dicot plants and plant materials.
Keywords: Dicotyledoneous plants; Development; Proteomes; 2-DGE; MS;

System, trends and perspectives of proteomics in dicot plants by Ganesh Kumar Agrawal; Masami Yonekura; Yumiko Iwahashi; Hitoshi Iwahashi; Randeep Rakwal (137-145).
This review is devoted to the proteomics studies in dicotyledoneous (dicot) plants, such as Arabidopsis, Medicago, potato, soybean, and tomato, under the influence of the environment and at the functional and genetic relationship levels, where the two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS) have been instrumental in unraveling the proteomes affected therein. Abiotic and biotic stress responses, including the affect of allergens, the symbiotic interaction between the members of the Leguminoseae family and genera of nitrogen fixing bacteria, phosphoproteomics, and proteomics in revealing the genetic relationships between species and genera have been the subject of many proteomics studies, and these are discussed in this review. In all, these studies have complemented and extended the studies of developmental proteomics [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)].
Keywords: Dicotyledoneous plants; Proteomic studies; 2-DGE; MS; Protein identification;

Proteomic analysis of Korean ginseng (Panax ginseng C.A. Meyer) by Myung Hee Nam; Seung Il Kim; Jang Ryol Liu; Deok Chun Yang; Yong Pyo Lim; Kyung-Hoon Kwon; Jong Shin Yoo; Young Mok Park (147-155).
Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways operant in ginsenoside biosynthesis, or the genes involved therein. Proteomics analysis is an approach to elucidate the physiological characteristics and biosynthetic pathways of ginsenosides, main components of ginseng. In this review, we introduced the recent progress in proteomics studies of ginseng (Panax ginseng C.A. Meyer). We briefly reference the genomic analyses of P. ginseng, without which proteomics approaches would have been impossible. Functional genomics studies regarding secondary metabolism in P. ginseng are also introduced here, in order to introduce possible future prospects for further study.
Keywords: Panax ginseng; Proteomics; Araliaceae;

Comparative proteomic analysis of mammalian animal tissues and body fluids: bovine proteome database by Chiara D’Ambrosio; Simona Arena; Fabio Talamo; Luigi Ledda; Giovanni Renzone; Lino Ferrara; Andrea Scaloni (157-168).
Keywords: Reviews; Tissues; Fluids; Proteome; Proteomics; Bovine; Proteome database;

Proteomics of bronchoalveolar lavage fluid by R. Wattiez; P. Falmagne (169-178).
Lung diseases are essentially multi-factorial diseases that require a global analysis, and thus, cannot be understood through the sole analysis of individual or small numbers of genes. Proteome analysis has rapidly developed in the post-genome era and is now widely accepted as the obligated complementary technology for genetic profiling. It has been shown to be a powerful tool for the study of human diseases and for identifying novel prognostic, diagnostic and therapeutic markers. During last years, proteomic approaches applied to lung diseases are centred on the analysis of proteins in samples, such as cell cultures, biopsies and physiological fluids like serum and, especially, bronchoalveolar lavage fluid (BALF). BALF is presently the most common way of sampling the components of the epithelial lining fluid (ELF) and the most faithful reflect of the protein composition of the pulmonary airways. This review focuses on the state of the investigations of BALF proteome and its powerful contribution both to a better knowledge of the lung structure at the molecular level and to the study of lung disorders at the clinical level.
Keywords: Proteomics; Bronchoalveolar lavage fluid;

Proteomics analysis of human cerebrospinal fluid by Xianglin Yuan; Dominic M. Desiderio (179-189).
Cerebrospinal fluid (CSF) is secreted from several different central nervous system (CNS) structures, and any changes in the CSF composition will accurately reflect pathological processes. Proteomics offers a comprehensive bird’s eye view to analyze CSF proteins at a systems level. This paper reviews the variety of analytical methods that have been used for proteomics analysis of CSF, including sample preparation, two-dimensional liquid and gel electrophoresis, mass spectrometry, bioinformatics, and non-gel methods. The differentially expressed CSF proteins that have been identified by proteomics methods are discussed.
Keywords: Reviews; Proteomics; Cerebrospinal fluid;

Extraction and proteomic analysis of proteins from normal and multiple sclerosis postmortem brain by Jia Newcombe; Bodil Eriksson; Jan Ottervald; Yang Yang; Bo Franzén (191-202).
In this study, a reproducible fractionation procedure was developed to reduce levels of the abundant cytoskeletal proteins that are present in normal and pathological central nervous system (CNS) tissues. The fractionation and proteomic analysis techniques employed greatly facilitated comparison of the spectrum of proteins in normal postmortem brain with proteins in samples from patients with multiple sclerosis, an inflammatory demyelinating disease in which complex changes in protein expression occur as lesions develop. This approach may be of value for the proteomic identification and quantitation of proteins which undergo disease-related changes in CNS disorders, and also for protein expression studies on normal adult and developing CNS tissues.
Keywords: Central nervous system; Human brain; Postmortem; Proteomics; Protein extraction; Multiple sclerosis; MS;

Revealing urologic diseases by proteomic techniques by Seung-won Lee; Kum-Il Lee; Jin Young Kim (203-213).
Proteomics, as the study of the proteomes of tissues and body fluids, has recently been introduced as a tool for revealing urologic diseases. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and surface-enhanced laser desorption/ionzation (SELDI) are two techniques used in proteomic studies. Among the many urologic diseases, the malignancies including prostate cancer, bladder cancer, and renal cancer are the subjects most often selected for proteomic analysis. Poor reproducibility is one of the difficulties that must be overcome in order for proteomic technology to be a robust tool.
Keywords: Review; Proteomics; Urologic diseases;

Proteomics of breast carcinoma by Richard I. Somiari; Stella Somiari; Stephen Russell; Craig D. Shriver (215-225).
Beast cancer is the most diagnosed cancer in women, accounting for approximately 40,000 deaths annually in the USA. Significant advances have been made in the areas of detection and treatment, but a significant number of breast cancers are detected late. The advent of proteomics provides the hope of discovering novel biological markers that can be used for early detection, disease diagnosis, prognostication and prediction of response to therapy. Several proteomics technologies including 2D-PAGE, 2D-DIGE, ICAT, SELDI-TOF, MudPIT and protein arrays have been used to uncover molecular mechanisms associated with breast carcinoma at the global level, and a number of these technologies, particularly the SELDI-TOF hold promise as a proteomic approach that can be applied at the bedside for discovering protein patterns that distinguish disease and disease-free states with high sensitivity and specificity. Laser microdissection, a method for selection of homogenous cell populations, coupled to 2D-DIGE or MudPIT constitute a new proteomics-based paradigm for detecting disease in pathology specimens and monitoring disease response to therapy. This review describes proteomics technologies, and their application in the proteomic analysis of breast carcinoma.
Keywords: Reviews; Proteomics;

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS–PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed. These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins. We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome. The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four. Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as 35S-methionine or the stable isotope, 15N.
Keywords: 15N-Whole cell labeling; Membrane protein proteomics; Bacillus subtilis; SecA;

Proteomics of Halophilic archaea by Won-A Joo; Chan-Wha Kim (237-250).
Halophilic archaea is a member of the Halobacteriacea family, the only family in the Halobacteriales order. Most Halophilic archaea require 1.5 M NaCl both to grow and retain the structural integrity of the cells. The proteins of these organisms have thus been adapted to be active and stable in the hypersaline condition. Consequently, the unique properties of these biocatalysts have resulted in several novel applications in industrial processes. Halophilic archaea are also to be useful for bioremediation of hypersaline environment. Proteome data have expended enormously with the significant advance recently achieved in two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The whole genome sequencing of Halobacterium species NRC-1 was completed and this would also provide tremendous help to analyze the protein mass data from the similar strain Halobacterium salinarum. Proteomics coupled with genomic databases now has become a basic tool to understand or identify the function of genes and proteins. In addition, the bioinformatics approach will facilitate to predict the function of novel proteins of Halophilic archaea. This review will discuss current proteome study of Halophilic archaea and introduce the efficient procedures for screening, predicting, and confirming the function of novel halophilic enzymes.
Keywords: Halophilic archaea; Proteomics; Bioinformatics; Prediction of function; Enzymes;

Proteomics and physiology of erythritol-producing strains by Yong-Cheol Park; Do-Yup Lee; Dae-Hee Lee; Hyo-Jin Kim; Yeon-Woo Ryu; Jin-Ho Seo (251-260).
In-depth knowledge bases on physiological properties of microbes are required to design a better microbial system at a gene level and to develop an industrially viable process in an optimized scheme. Proteomic analyses of industrially useful microorganisms are particularly important for achieving such objectives. In this review, industrial application of erythritol in food and pharmaceutical areas and proteomic techniques for erythritol-producing microbes were presented. Proteomic technologies for erythritol-producing strains such as Candida magnoliae contained protein or peptide sample preparation for two-dimensional electrophoresis and mass spectrometry, analysis of proteome with matrix assisted laser desorption-ionization/time-of-flight mass spectrometry, liquid chromatography/electrospray ionization/tandem mass spectrometry and similarity searching algorithms. The proteomic information was applied to predict the carbon metabolism of erythritol-synthesizing microorganisms.
Keywords: Review; Proteomics; Candida magnoliae; Erythritol;

Several strategies carried out for the application of proteomic methodologies to species and populations are summarized. Species of the genus Mytilus are used as a model organism to explain these strategies. The proteomics needed to differentiate populations, species, following some different approaches are provided. Moreover, there is an explanation of when it is most critical to carry out a rigorous analysis of this type. Sample preparation, selection of the most appropriate tissue of this organism for a comparative analysis, two-dimensional gels, computer analysis, mass spectrometry (MS) combined with two-dimensional electrophoresis (2-DE) are described. Then, the inconveniences of working with species like those of the genus Mytilus that are poorly represented in databases will be presented along with a description of how to approach this problem. Likewise, the review will include the strategy to follow when dealing with species, like mussels, that present a high degree of genetic polymorphism. The different protein-expression-based strategies used to approach the problem of differentiating Mytilus species will also be shown. Examples are presented to illustrate the use of 2-DE and MS to differenciate populations, species in taxonomic analysis.
Keywords: Mytilus; Proteomics; Taxonomy; 2-DE;

Methods of comparative proteomic profiling for disease diagnostics by Laura F. Steel; Brian B. Haab; Samir M. Hanash (275-284).
The recent development of numerous technologies for proteome analysis holds the promise of new and more precise methods for disease diagnosis. In this review, we provide an overview of some of these technologies including two-dimensional gel electrophoresis (2DE), historically the workhorse of proteomic analysis, as well as some newer approaches such as liquid phase separations combined with mass spectrometry, and protein microarrays. It is evident that each method has its own strengths and weaknesses and no single method will be optimal in all applications. However, the continuing development of innovative strategies for protein separation and analysis is providing a wealth of new tools for multi-dimensional protein profiling that will advance our capabilities in disease diagnostics and our understanding of disease pathology.
Keywords: Comparative proteomic profiling; Disease diagnostics; Proteins biomarkers;

Asthma, the ugly duckling of lung disease proteomics? by René Houtman; Edwin van den Worm (285-294).
The human respiratory system represents a vital but vulnerable system. It is a major target for many diseases such as cancer and asthma. The incidence of these diseases has increased dramatically in the last 40–50 years. In the search for possible new therapies, many experimental tools and methods have been developed to study these diseases, ranging from animal models to in vitro studies. In the last decades, genomic and proteomic approaches have gained a lot of attention. After the major scientific breakthroughs in the field of genomics, it is now widely accepted that to understand biological processes, large-scale protein studies through proteomics techniques are required. In the battle against lung cancer, the proteomics approach has already been successfully implemented. Surprisingly, only a few proteomics studies on the ever-increasing global asthma problem have been published so far. And although proteomics also has its limitations and experimental difficulties, in our opinion, proteomics can definitely contribute to the understanding of a complex disease such as asthma. Therefore, the additional values and possibilities of proteomics in asthma research should be thoroughly investigated. A close collaboration between the different scientific disciplines may eventually lead to the development of new therapeutic strategies against asthma.
Keywords: Asthma; Lung disease proteomics; Respiratory system;

An antibody-secreting B cell is derived from a lymphoid stem cell through a series of developmental stages: progenitor B cell (pro-B cell), precursor B cell (pre-B) cell, immature B cell and mature B cell stage. The gene rearrangement of antigen receptor genes and their expression on the cell surface are crucial regulation steps cells to develop to the next stage. This control mechanism occurs at the sequential manner during the B cell development. Proteomic approach using two-dimensional (2-D) gel electrophoresis and mass spectrometry makes possible to analyze the expression pattern of total proteins at each developmental stage and isolate proteins differentially expressed in B cells. Some transcriptional factors such E2A and Pax5 are expressed at the earlier stages of B cell development and repressed at later developmental stages. RAD52-related protein and chromatin assembly factor 1 are dominantly expressed at early B cells undergoing DNA rearrangement. These comparative analyses of total proteins in each B cell can provide some crucial information to understand the molecular basis of B cell ontogeny.
Keywords: Proteomics; B cell development; Gene rearrangement;

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites by Eung-goo Lee; Jae-hoon Kim; Yong-seung Shin; Gee-wook Shin; Young-rim Kim; K.J. Palaksha; Dae-yong Kim; Itsuro Yamane; Yong-hwan Kim; Gon-sup Kim; Myung-deuk Suh; Tae-sung Jung (305-314).
Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M r between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin α- and β-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.
Keywords: Neospora caninum; Toxoplasma gondii; Proteome analysis; Two-dimensional gel electrophoresis; Mass spectrometry; Antigen spots;

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by oxo-compounds, particularly glucose, ribose, glyoxal and glutardialdehyde, have been investigated using a set of modern chromatographic and electrophoretic separation methods. High-performance liquid chromatography (HPLC) alternatively with UV spectrophotometric (diode array) or mass spectrometric (MS) detection, polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue staining detection, and capillary zone electrophoresis (CZE) with UV spectrophotometric detection have been employed for the investigation of the chemical and structural changes of BSA caused by its reaction with the above oxo-compounds exhibiting different degree of reactivity. The extent of modifications was found to be dependent on the nature of the oxo-compound used and progressed in the glucose < ribose < glyoxal < glutaraldehyde order. With the aid of HPLC/UV/MS and CZE/UV tryptic peptide mapping and amino acid analysis of both unmodified and modified BSA it was revealed that the mildest modification resulted from the reaction of BSA with glucose, in this case presumably only monofunctional derivatives have arisen, whereas the most intensive modifications were found after BSA reaction with glutardialdehyde, which resulted in high degree of both inter- and intra-molecular cross-linking, due to which no unmodified peptides were detected after tryptic cleavage of BSA modified by this agent.
Keywords: Bovine serum albumin; Oxo-compounds; Non-enzymatic posttranslational modifications;

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate by Johan Malmström; Jason Marchese; Peter Juhasz; Laurel Pukac; Morris Karnovsky; György Marko-Varga; Gunilla Westergren-Thorsson (333-342).
Certain structures of heparan sulfate (HS) inhibit cell proliferation of fibroblasts. Whether this inhibition is dependent on inhibition of mitogenic signaling pathways or nuclear translocation of HS is unknown. In this study we investigated possible mechanism(s) and structural requirements by which antiproliferative glycosaminoglycans exert their effects on mitogen-activated protein kinase (MAP kinase) phosphorylation, a key intermediate in cell signaling, followed by quantitative proteomic analysis of nuclear proteins by stable isotope coded affinity tags, multidimensional chromatography and tandem mass spectrometry. Serum starved human lung fibroblasts were stimulated with serum, platelet derived growth factor (PDGF-BB) or epidermal growth factor (EGF) in the presence of structurally different glycosaminoglycans. Antiproliferative heparan sulfate with a high content of 2-O-sulfated iduronic acid (IdoA-2SO4) and heavily sulfated glucosamine, and the structurally related glycosaminoglycan heparin inhibited significantly serum stimulated MAP kinase phosphorylation, by at least 80% when stimulated by serum and HS6. We hypothesized that the inhibition of the MAP kinase pathway will have effect in the nuclear proteome. Therefore an isotope coded affinity tag (ICAT™) reagent labeling of nuclear proteins and tandem mass spectrometry was applied, resulting in the identification and quantification of 206 proteins. Several nuclear proteins were found to be induced or repressed due to HS stimulation, where the repression EBNA-2 co-activator and the induction of PML protein were of special interest. These results show that heparan sulfate with high content of (IdoA-2SO4) and heavily sulfated glucosamine specifically inhibits MAP kinase activation with a subsequent change in the nuclear proteome of the fibroblast.
Keywords: Cell growth; Fibroblast; Heparan sulfate; Mitogen-activated protein kinase (MAP kinase); Signal transduction;

Author Index (343-344).

Keyword Index (345-348).