Journal of Chromatography B (v.771, #1-2)

Preface (1).

Detection technologies in proteome analysis by Wayne F. Patton (3-31).
Common strategies employed for general protein detection include organic dye, silver stain, radiolabeling, reverse stain, fluorescent stain, chemiluminescent stain and mass spectrometry-based approaches. Fluorescence-based protein detection methods have recently surpassed conventional technologies such as colloidal Coomassie blue and silver staining in terms of quantitative accuracy, detection sensitivity, and compatibility with modern downstream protein identification and characterization procedures, such as mass spectrometry. Additionally, specific detection methods suitable for revealing protein post-translational modifications have been devised over the years. These include methods for the detection of glycoproteins, phosphoproteins, proteolytic modifications, S-nitrosylation, arginine methylation and ADP-ribosylation. Methods for the detection of a range of reporter enzymes and epitope tags are now available as well, including those for visualizing β-glucuronidase, β-galactosidase, oligohistidine tags and green fluorescent protein. Fluorescence-based and mass spectrometry-based methodologies are just beginning to offer unparalleled new capabilities in the field of proteomics through the performance of multiplexed quantitative analysis. The primary objective of differential display proteomics is to increase the information content and throughput of proteomics studies through multiplexed analysis. Currently, three principal approaches to differential display proteomics are being actively pursued, difference gel electrophoresis (DIGE), multiplexed proteomics (MP) and isotope-coded affinity tagging (ICAT). New multiplexing capabilities should greatly enhance the applicability of the two-dimensional gel electrophoresis technique with respect to addressing fundamental questions related to proteome-wide changes in protein expression and post-translational modification.

Proteomic tools for biomedicine by Valerie C Wasinger; Garry L Corthals (33-48).
Proteomic tools measure gene expression, protein activity and interactions of biological events at the protein level. Proteins are the major catalysts of biological functions and contain several dimensions of information that collectively indicate the actual rather than the potential functional state as indicated by mRNA analysis. Measurements can be made in terms of protein quantity, location, and time-point. For the future we see a further integration of existing and new technologies for proteomics from a wide range of areas of biochemistry, chemistry, physics, computing science and molecular biology. This will further advance our knowledge of how biological systems are built up and what mechanisms control these systems. However, the potential of proteomics to comprehensively answer all biological questions is limited as only protein activity is measured. A unification of genomics, proteomics, and other technologies is needed if we are to start to understand the complexity of biological function in the context of disease and health.

Separation techniques for high-molecular-mass proteins by Masamichi Oh-Ishi; Tadakazu Maeda (49-66).
Many high-molecular-mass (HMM) proteins (MW>100 kDa) are known to be involved in cytoskeleton, defence and immunity, transcription and translation in higher eukaryotic organisms. Even in the post-genomic era, purification of HMM protein is the first important step to analyze protein composition in a tissue or a cell (proteomics), to determine protein tertiary structure (structural biology), and to investigate protein function (functional genomics). To separate a HMM protein from a protein mixture, ions, chaotropes (urea and thiourea), detergents and protease inhibitors in extraction media and buffer solutions either for liquid chromatography or for gel electrophoresis should be carefully chosen, since HMM proteins tend to be aggregates under denatured condition and their long polypeptide chains are easily attacked by intrinsic proteases during separation procedure. Among many liquid chromatography techniques, affinity chromatography either with sequence-specific DNA for transcription factor, or with monoclonal antibody specific for myosin heavy chain has been used for preparative isolation of the respective HMM proteins. Though SDS–PAGE could analyze the size and the quantity of megadalton proteins, the resolution of HMM proteins is relatively poor. A newly developed pulse SDS–PAGE would be able to raise the resolution of HMM proteins compared with the conventional SDS–PAGE. The 2-DE method is not particularly suitable in analyzing HMM proteins larger than 200 kDa. However, a 2-DE method that uses an agarose IEF gel in the first dimension (agarose 2-DE) has been shown to produce significant improvements in 2-DE separation of HMM proteins larger than 150 kDa and up to 500 kDa.
Keywords: High-molecular-mass proteins;

Separation technologies for glycomics by Jun Hirabayashi; Ken-ichi Kasai (67-87).
Progress in genome projects has provided us with fundamentals on genetic information; however, the functions of a large number of genes remain to be elucidated. To understand the in vivo functions of eukaryotic genes, it is essential to grasp the features of their post-translational modifications. Among them, protein glycosylation is a central issue to be discussed, considering the predominant roles of glycoproteins in cell–cell and cell–substratum recognition events in multicellular organisms. In this context, it is necessary to establish a core strategy for analyzing glycosylated proteins under the concept of the “glycome” [Trends Glycosci. Glycotechnol. 12 (2000) 1]. Though the term glycome should be defined, in analogy to the genome and proteome, as “a whole set of glycans produced in a single organism”, here we propose a glycome project specifically focusing on glycoproteins. Principal objectives in the project are to identify: (1) which genes encode glycoproteins (i.e. genome information); (2) which sites among potential glycosylation sites are actually glycosylated (i.e. glycosylation site information); (3) what are the structures of glycans (i.e. structural information); and (4) what are the effects (functions) of glycosylation (functional information). For these purposes, two affinity technologies have been introduced. One is named the “glyco-catch method” to identify genes encoding glycoproteins [Proteomics 1 (2001) 295], and the other is the recently reinforced “frontal affinity chromatography” [J. Chromatogr. A 890 (2000) 261]. By the former method, genes that encode glycoproteins as well as glycosylation sites are systematically identified by the efficient combination of conventional lectin-affinity chromatography and contemporary in silico database searching. The following three actions have been devised for rapid and systematic characterization of glycans: (1) mass spectrometry to acquire exact mass information; (2) 2-D/3-D mapping to obtain refined chemical information; and (3) reinforced frontal affinity chromatography to determine affinity constants (K a-values) for a set of lectins. Pyridylaminated glycans are used throughout the characterization processes. In this review, the concept and strategy of glycomic approaches are described referring to the on-going glycome project focused on the nematode Caenorhabditis elegans.

Functional proteomics; current achievements by Mitsuaki Yanagida (89-106).
This review presents the current improvements in functional proteomic strategies and their research applications. Proteomics has emerged as an indispensable methodology for large-scale and high-throughput protein analyses in the post-genome era. Functional proteomics, the comprehensive analysis of proteins with special attention to their functions, is a powerful and useful approach for investigations in the life and medical sciences. Various methods have been developed for this purpose, expanding the field further. This important technology will not only provide a wealth of information on proteins, but also contribute synergistically to the understanding of life with other systematic technologies such as gene chips.

Separation and identification of human heart proteins by D Jäger; P.R Jungblut; U Müller-Werdan (131-153).
Heart failure is not a uniform disease entity, but a syndrome with various causes, including hypertension, ischemia and congenital heart disease, cardiomyopathy, myocarditis and intoxication. During the recent years a number of molecular and cellular alterations have been identified in the diseased heart, but a direct causative link between these changes and functional impairment, medical responsiveness, progression of the disease and the patients’ outcome remains to be established. After an accumulation of large amounts of DNA sequence data in genomic projects, scientists have now turned their attention to the central executors of all programs of life, the proteins. In complementation of the genomic initiatives, proteomics based approaches have lined up not only for large-scale identification of proteins and their post-translational modifications, but also to study the function of protein complexes, protein–protein interactions and regulatory and signalling cascades in the cellular network. In concert with genomic data functional proteomics will hold the key for a better understanding and therapeutical management of cardiovascular diseases in the future.
Keywords: Human heart proteins; Eucaryotic elongation factor-2;

Proteomic analysis of striated muscle by Robert J. Isfort (155-165).
The techniques collectively known as proteomics are useful for characterizing the protein phenotype of a particular tissue or cell as well as quantitatively identifying differences in the levels of individual proteins following modulation of a tissue or cell. In the area of striated muscle research, proteomics has been a useful tool for identifying qualitative and quantitative changes in the striated muscle protein phenotype resulting from either disease or physiological modulation. Proteomics is useful for these investigations because many of the changes in the striated muscle phenotype resulting from either disease or changes in physiological state are qualitative and not quantitative changes. For example, modification of striated muscle proteins by phosphorylation and proteolytic cleavage are readily observed using proteomic technologies while these changes would not be identified using genomic technology. In this review, I will discuss the application of proteomic technology to striated muscle research, research designed to identify key protein changes that are either causal for or markers of a striated muscle disease or physiological condition.

Towards a high resolution separation of human cerebrospinal fluid by Albert Sickmann; Wilma Dormeyer; Stefanie Wortelkamp; Dirk Woitalla; Wilfried Kuhn; Helmut E Meyer (167-196).
Human cerebrospinal fluid is an ultrafiltrate of plasma that is largely produced by the choroid plexus. It consists of a mixture of anorganic salts, various sugars, lipids and proteins from the surrounding brain tissues. The predominant proteins in cerebrospinal fluid are isoforms of serum albumin, transferrin and immunoglobulins, representing more than 70% of the total protein amount. A rough overview of the protein compounds of human cerebrospinal fluid including their respective concentrations is given by Blennow et al. [Eur. Neurol. 33 (1993) 129]. In contrast, the aim of this work is to display the detailed protein composition of CSF by two-dimensional gel electrophoresis and to identify both high and low concentrated proteins using different mass spectrometry techniques. This extensive overview of proteins in human cerebrospinal fluid will be highly relevant for clinical research. Furthermore, the comparison of 2D gels will help to analyze the standard protein variability in CSF of healthy persons and detect specific protein variations of patients with various neurological diseases (e.g., Alzheimer’s disease, Huntington’s chorea). Sample preparation for two-dimensional gel electrophoresis must include concentration and desalting steps such as precipitation and ultrafiltration due to the high amount of salts, sugars and lipids and the low total amount of protein of 0.3–0.7 μg/μl present in human CSF. Up to now we were able to identify more than 480 spots from suchlike generated 2D gels using MALDI- and ESI-mass spectrometry.
Keywords: Proteins;

Proteomic map and database of lymphoblastoid proteins by Michel Caron; Naima Imam-Sghiouar; Florence Poirier; Jean-Pierre Le Caër; Valerie Labas; Raymonde Joubert-Caron (197-209).
Advances in genomics have led to the accumulation of an unprecedented amount of data, giving rise to a new field in biochemistry, proteomics. We used a combination of two dimensional gel electrophoresis, analysis and annotation using third-generation software, and mass spectrometry to establish the proteome maps of lymphoblastoid B-cells, a prerequisite for analysis of drug effects and lymphocyte cell diseases. About 1200 protein spots were detected and characterised in terms of their isoelectric point, molecular mass and expression. The present status of proteomic technologies, as well as a description of the usefulness of human hematopoietic cells proteomic database are discussed.
Keywords: Lymphoblastoid proteins;

Proteomic analysis of dental tissues by Michael J Hubbard; Jew C Kon (211-220).
Teeth are highly refined structures formed by several types of specialised cell. Tooth formation embraces many areas of biomedical interest, including cellular mechanisms for calcium handling, protein secretion and mineralised tissue production. Proteomics offers great potential to elucidate these cellular roles, and to establish their relevance to general cell types. Here we review our proteomic investigations of dental enamel formation, covering both the approaches taken and some findings of general biomedical relevance.

Database of bronchoalveolar lavage fluid proteins by Isabelle Noël-Georis; Alfred Bernard; Paul Falmagne; Ruddy Wattiez (221-236).
Bronchoalveolar lavage during fiberoptic bronchoscopy is extensively used for investigating cellular and biochemical alterations of the epithelial lining fluid in various lung disorders. Two-dimensional electrophoresis (2-DE) offers the possibility to simultaneously display and analyze proteins contained in bronchoalveolar lavage fluid (BALF). We present the current status of 2-DE of BALF samples with an updated listing of the proteins already identified and of their level and/or posttranslational alterations in lung disorders. Alternatives to 2-DE of BALF samples and future prospects of proteomics to unravel lung functions and pathologies are discussed.
Keywords: Proteins;

Alteration of ribosomal protein maps in herpes simplex virus type 1 infection by Jean-Jacques Diaz; Stéphane Giraud; Anna Greco (237-249).
At present, the effect of herpes simplex virus infection on the entire proteomes of infected cells is very poorly documented. Following several studies performed over the past few years, the modifications of a sub-cellular fraction induced by herpes simplex virus type 1 can be documented. These studies were performed in order to characterize the virally-induced modifications of a major component of the translational apparatus, the ribosomes. The very basic nature of most of the ribosomal proteins renders them very difficult to separate using isoelectric focusing (IEF). Therefore these studies were achieved using several different but related two-dimensional electrophoretic systems which allowed several two-dimensional ribosomal protein maps to be built. Comparison of the ribosomal protein maps built from non-infected cells with those built from infected cells demonstrated that infection by herpes simplex virus type 1 (HSV-1) induces important modifications of ribosomes: (i) non-reversible phosphorylation of ribosomal protein S6; (ii) unusual phosphorylation of several proteins of the small and the large subunits; and (iii) association of viral and cellular proteins to the ribosomal fraction. An overview of these published studies is presented in this review.
Keywords: Poly(A)-binding proteins; Viral proteins;

The analysis of Helicobacter pylori proteins is a demanding task for the elucidation of virulence factors, antigens and vaccines, all important for diagnosis, therapy and protection. In the “pre-genomic era” the purification of proteins was mostly performed by using various techniques such as detergent treatment of the bacterial cells, ultra-centrifugation, various chromatographic methods, antibody detection, N-terminal sequence determination and finally cloning and identification of the corresponding gene. In this review, the most representative methods used for purification, separation and identification of H. pylori proteins will be presented as well as some important developments in the “post-genomic era” that have improved the performance of these characterisation techniques.
Keywords: Proteins;

Bioinformatics, functional genomics, and proteomics study of Bacillus sp. by Supachok Sinchaikul; Boonyaras Sookkheo; Supachai Topanuruk; Hsueh-Fen Juan; Suree Phutrakul; Shui-Tein Chen (261-287).
The ability of bioinformatics to characterize genomic and proteomic sequences from bacteria Bacillus sp. for prediction of genes and proteins has been evaluated. Genomics coupling with proteomics, which is relied on integration of the significant advances recently achieved in two-dimensional (2-D) electrophoretic separation of proteins and mass spectrometry (MS), are now important and high throughput techniques for qualifying and analyzing gene and protein expression, discovering new gene or protein products, and understanding of gene and protein functions including post-genomic study. In addition, the bioinformatics of Bacillus sp. is embraced into many databases that will facilitate to rapidly search the information of Bacillus sp. in both genomics and proteomics. It is also possible to highlight sites for post-translational modifications based on the specific protein sequence motifs that play important roles in the structure, activity and compartmentalization of proteins. Moreover, the secreted proteins from Bacillus sp. are interesting and widely used in many applications especially biomedical applications that are the highly advantages for their potential therapeutic values.

Protein mapping in rat basophilic leukaemia cells by K.M. Carroll; E.M. Carey; B.A. Helm (289-301).
This review discusses some of our recent studies on rat basophilic leukaemia (RBL) cells, a model cell line for mast cell function. Our interest in these cells is a consequence of the role played by mast cells in the allergic response. Thus far we have described the identification of several spots, and their pI/M r co-ordinates. Here we describe how we can further decipher the mast cell proteome using a variety of staining/immuno-blotting procedures. We demonstrate the sensitivity of staining procedures and immuno-blotting using an anti-phosphotyrosine antibody. Our aim is to contribute to the ever-expanding two-dimensional gel and phosphoprotein databases currently available.

Proteome database of hepatocellular carcinoma by Rosa C.M.Y Liang; Jason C.H Neo; Siaw Ling Lo; Gek San Tan; Teck Keong Seow; Maxey C.M Chung (303-328).

Lactic acid bacteria and proteomics: current knowledge and perspectives by Marie-Christine Champomier-Vergès; Emmanuelle Maguin; Michel-Yves Mistou; Patricia Anglade; Jean-François Chich (329-342).
Lactic acid bacteria (LAB) are widely used in the agro-food industry. Some of the LAB also participate in the natural flora in humans and animals. We review here proteomic studies concerning LAB. Two methods of research can be distinguished. In the first one, a systematic mapping of proteins is attempted, which will be useful for taxonomy and to function assignment of proteins. The second one focuses particularly on proteins whose synthesis is induced by various environmental situations or stresses. However, both approaches are complementary and will give new insights for the use of bacteria in industry, in human health and in the struggle against bacterial pathogens. Interest in LAB is growing, showing thus an increasing concern of their rational use and one can foresee in the near future an increasing use of proteomics as well as genomics.
Keywords: Lactic acid bacteria;

Proteomics of allergens by Marie Tichá; Věra Pacáková; Karel Štulı́k (343-353).
The present state of proteomics research is generally outlined and the character of allergenic compounds briefly elucidated. The principles of experimental approaches to isolation, purification, identification and characterization of allergens and to monitoring of their biological activity are described, with emphasis on the most modern methods. Selected examples are given for illustration and important results are summarized in tables.
Keywords: Allergens;

Author Index (369-370).