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PubMed2005

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Antibodies↗
PubMed2003

Mapping the proteome of barrel medic (Medicago truncatula).

A survey of six organ-/tissue-specific proteomes of the model legume barrel medic (Medicago truncatula) was performed. Two-dimensional polyacrylamide gel electrophoresis reference maps of protein extracts from leaves, stems, roots, flowers, seed pods, and cell suspension cultures were obtained. Five hundred fifty-one proteins were excised and 304 proteins identified using peptide mass fingerprinting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Nanoscale high-performance liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry was used to validate marginal matrix-assisted laser desorption ionization time-of-flight mass spectrometry protein identifications. This dataset represents one of the most comprehensive plant proteome projects to date and provides a basis for future proteome comparison of genetic mutants, biotically and abiotically challenged plants, and/or environmentally challenged plants. Technical details concerning peptide mass fingerprinting, database queries, and protein identification success rates in the absence of a sequenced genome are reported and discussed. A summary of the identified proteins and their putative functions are presented. The tissue-specific expression of proteins and the levels of identified proteins are compared with their related transcript abundance as quantified through EST counting. It is estimated that approximately 50% of the proteins appear to be correlated with their corresponding mRNA levels.

Amino Acid Sequence↗
PubMed2005

The EH1 motif in metazoan transcription factors.

BACKGROUND: The Engrailed Homology 1 (EH1) motif is a small region, believed to have evolved convergently in homeobox and forkhead containing proteins, that interacts with the Drosophila protein groucho (C. elegans unc-37, Human Transducin-like Enhancers of Split). The small size of the motif makes its reliable identification by computational means difficult. I have systematically searched the predicted proteomes of Drosophila, C. elegans and human for further instances of the motif. RESULTS: Using motif identification methods and database searching techniques, I delimit which homeobox and forkhead domain containing proteins also have likely EH1 motifs. I show that despite low database search scores, there is a significant association of the motif with transcription factor function. I further show that likely EH1 motifs are found in combination with T-Box, Zinc Finger and Doublesex domains as well as discussing other plausible candidate associations. I identify strong candidate EH1 motifs in basal metazoan phyla. CONCLUSION: Candidate EH1 motifs exist in combination with a variety of transcription factor domains, suggesting that these proteins have repressor functions. The distribution of the EH1 motif is suggestive of convergent evolution, although in many cases, the motif has been conserved throughout bilaterian orthologs. Groucho mediated repression was established prior to the evolution of bilateria.

Amino Acid Motifs↗
PubMed2006

Bioinformatics for comprehensive finding and analysis of glycosyltransferases.

Bioinformatics is a very powerful tool in the field of glycoproteomics as well as genomics and proteomics. As a part of the Glycogene Project (GG project), we have developed a novel bioinformatics system for the comprehensive identification and in silico cloning of human glycogenes. Using our system, a total of 105 candidate human glycogenes were identified and then engineered for heterologous expression. Of these candidates, 38 recombinant proteins were successfully identified for their enzyme activity and substrate specificity. We also classified 47 out of 60 carbohydrate-active enzyme glycosyltransferase families into 4 superfamilies using the profile Hidden Markov Model method. On the basis of our classification and the relationship between glycosylation pathways and superfamilies, we propose the evolution of glycosyltransferases.

Algorithms↗
PubMed2005

PROTEIOS: an open source proteomics initiative.

SUMMARY: PROTEIOS is an initiative for the development of a comprehensive open source system for storage, organization, analysis and annotation of proteomics experiments. The PROTEIOS platform is based on commonly acknowledged principles for proteomics data publishing. AVAILABILITY: http://www.proteios.org

Algorithms↗
PubMed2003

The proteomics standards initiative.

The Proteomics Standards Initiative (PSI) aims to define community standards for data representation in proteomics and to facilitate data comparison, exchange and verification. Progress has been made in the development of common standards for data exchange in the fields of both mass spectrometry and protein-protein interaction. A proteomics-specific extension is being created for the emerging American Society for Tests and Measurements mass spectrometry standard, which will be supported by manufacturers of both hardware and software. A data model for proteomics experimentation is under development and discussions on a public repository for published proteomics data are underway. The Protein-Protein Interactions group expects to publish the Level 1 PSI data exchange format for protein-protein interactions soon and discussions as to the content of Level 2 have been initiated.

Biochemistry↗
PubMed2003

Public access for teaching genomics, proteomics, and bioinformatics.

When the human genome project was conceived, its leaders wanted all researchers to have equal access to the data and associated research tools. Their vision of equal access provides an unprecedented teaching opportunity. Teachers and students have free access to the same databases that researchers are using. Furthermore, the recent movement to deliver scientific publications freely has presented a second source of current information for teaching. I have developed a genomics course that incorporates many of the public-domain databases, research tools, and peer-reviewed journals. These online resources provide students with exciting entree into the new fields of genomics, proteomics, and bioinformatics. In this essay, I outline how these fields are especially well suited for inclusion in the undergraduate curriculum. Assessment data indicate that my students were able to utilize online information to achieve the educational goals of the course and that the experience positively influenced their perceptions of how they might contribute to biology.

Computational Biology↗
PubMed2006

Questions and answers concerning applicability of proteomics and transcriptomics in EMF research.

The applicability of high-throughput screening techniques of transcriptomics, proteomics and metabolomics in the search for biological and health effects of electromagnetic fields is a hotly debated issue. On the one hand, use of these modern screening technologies speeds up the discovery process and gives broader insight into biochemical events that follow the exposure to electromagnetic fields. On the other hand these modern screening technologies have the problem of reproducibility and variability between experiments and are prone to produce false positive results. These and other issues concerning the applicability of modern screening technologies were the topic of a workshop held at STUK in 2005 (30 October to 1 November) in Helsinki, Finland, and this Report summarizes the discussions at this workshop.

Databases, Genetic↗
PubMed2003

Integrating 'omic' information: a bridge between genomics and systems biology.

The availability of genome sequences for several organisms, including humans, and the resulting first-approximation lists of genes, have allowed a transition from molecular biology to 'modular biology'. In modular biology, biological processes of interest, or modules, are studied as complex systems of functionally interacting macromolecules. Functional genomic and proteomic ('omic') approaches can be helpful to accelerate the identification of the genes and gene products involved in particular modules, and to describe the functional relationships between them. However, the data emerging from individual omic approaches should be viewed with caution because of the occurrence of false-negative and false-positive results and because single annotations are not sufficient for an understanding of gene function. To increase the reliability of gene function annotation, multiple independent datasets need to be integrated. Here, we review the recent development of strategies for such integration and we argue that these will be important for a systems approach to modular biology.

Computational Biology↗
PubMed1998

European Functional Analysis Network (EUROFAN) and the functional analysis of the Saccharomyces cerevisiae genome.

Less than two yeras after the sequence of its genome was completed, the baker's yeast, Saccharomyces cerevisiae, is a leading organism in the rapidly growing field of functional genomics. Two thousands novel protein coding genes, nearly all of them "orphans", have already been disrupted by the coordinated efforts of a large consortium of European laboratories, EUROFAN, and other initiatives. The mutants are submitted to many specialized functional assays, and studies are performed in parallel at the transcriptome and the proteome levels. With a central repository of mutant yeast strains, and a centralized database, EUROFAN lays the foundations for the future of genomics with yeast serving both as a model and a tool.

DNA Transposable Elements↗
PubMed2004

Acid-labile surfactant improves in-sodium dodecyl sulfate polyacrylamide gel protein digestion for matrix-assisted laser desorption/ionization mass spectrometric peptide mapping.

Mass spectrometry (MS) together with genome database searches serves as a powerful tool for the identification of proteins. In proteome analysis, mixtures of cellular proteins are usually separated by sodium dodecyl sulfate (SDS) polyacrylamide gel-based two-dimensional gel electrophoresis (2-DE) or one-dimensional gel electrophoresis (1-DE), and in-gel digested by a specific protease. In-gel protein digestion is one of the critical steps for sensitive protein identification by these procedures. Efficient protein digestion is required for obtaining peptide peaks necessary for protein identification by MS. This paper reports a remarkable improvement of protein digestion in SDS polyacrylamide gels using an acid-labile surfactant, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (ALS). Pretreatment of gel pieces containing protein spots separated by 2-DE with a small amount of ALS prior to trypsin digestion led to increases in the digested peptides eluted from the gels. Consistently, treatment of gel pieces containing silver-stained standard proteins and those separated from tissue extracts resulted in the detection of increased numbers of peptide peaks in spectra obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS). Hence the present protocol with ALS provides a useful strategy for sensitive protein identification by MS.

Alkanesulfonates↗
PubMed2001

Molecular biology of nervous system tumors.

Many genetic alterations that contribute to CNS tumorigenesis and progression have been identified. One goal of such studies is to identify loci that would serve as diagnostic prognostic markers or both. A significant advance is the observation that chromosome 1p loss identified anaplastic oligodendroglioma and a subset of high-grade glioma patients who responded to chemotherapy and had longer survival times. Combined 1p and 19q loss was a predictor of prolonged survival of patients having pure oligodendrogliomas. Such markers eventually may be used to identify patients upfront who would benefit from treatment, while sparing patients who would not benefit. Although many molecular participants involved in the biologic pathways that promote proliferation, angiogenesis, and invasion have been elucidated, there are still many gaps in clinicians' knowledge. It is expected that the use of the human genome project information and databases such as SAGEmap, in combination with techniques such as cDNA arrays and proteomics, will facilitate greatly the identification of novel genes that contribute to CNS tumors. cDNA arrays and tissue arrays will permit the construction of CNS-specific screening tools that will permit the identification of tumor-specific mutations and alterations so that patient-specific therapies can be designed.

Cell Division↗
PubMed2002

Bioinformatics, functional genomics, and proteomics study of Bacillus sp.

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.

Bacillus↗
PubMed2004

Proteomic analysis of nuclear proteins from proliferative and differentiated human colonic intestinal epithelial cells.

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.

Caco-2 Cells↗
PubMed2004

Protein extraction from mature rice leaves for two-dimensional gel electrophoresis and its application in proteome analysis.

Sample preparation is crucial for extraction and higher resolution of proteins by two-dimensional gel electrophoresis (2-DE). In this study, we present an efficient protocol to extract proteins from mature rice leaves by minimizing the presence of nonprotein contaminants and by maximizing contact between the sample and extraction buffer. A combination of chemical and physical processes remarkably improved protein extraction for 2-DE. The efficiency of this protocol was demonstrated by comparison of the rice proteome at two developmental stages.

Databases as Topic↗
PubMed2007

Simple proteomic checks for detecting noncoding RNA.

Proper validation can accelerate sequence-based discovery of proteins and protein-coding genes. Databases currently contain a backlog of experimentally unverified gene models and tentative assignments of observed transcripts to coding or noncoding RNA. We present and apply a general principle, founded on base composition and the genetic code and validated here by bulk 2-D gels, that can improve the reliability of such classifications and of the algorithms or pipelines that lead to them.

Base Composition↗
PubMed2001

Predicting protein--protein interactions from primary structure.

MOTIVATION: An ambitious goal of proteomics is to elucidate the structure, interactions and functions of all proteins within cells and organisms. The expectation is that this will provide a fuller appreciation of cellular processes and networks at the protein level, ultimately leading to a better understanding of disease mechanisms and suggesting new means for intervention. This paper addresses the question: can protein-protein interactions be predicted directly from primary structure and associated data? Using a diverse database of known protein interactions, a Support Vector Machine (SVM) learning system was trained to recognize and predict interactions based solely on primary structure and associated physicochemical properties. RESULTS: Inductive accuracy of the trained system, defined here as the percentage of correct protein interaction predictions for previously unseen test sets, averaged 80% for the ensemble of statistical experiments. Future proteomics studies may benefit from this research by proceeding directly from the automated identification of a cell's gene products to prediction of protein interaction pairs.

Artificial Intelligence↗
PubMed2006

An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE.

Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues.

Databases, Protein↗