Two-dimensional Gel Protein Database Research Articles

Identification of the oxidative stress proteome in the brain

The redox proteomics technique normally combines two-dimensional gel electrophoresis, mass spectrometry, and protein databases to analyze the cell proteome from various samples, thereby leading to the identification of specific targets of oxidative modification. Oxidative stress that occurs because of increased levels of reactive oxygen species and reactive nitrogen species can target most biomolecules, consequently leading to altered physiological function of the cells. Redox proteomics has identified oxidatively modified protein targets in various pathological conditions, consequently providing insight into the pathways involved in the pathogenesis of these conditions. This approach also can be used to identify possible protective mechanisms to prevent or delay these disorders.

TMIG-2DPAGE: a new concept of two-dimensional gel protein database for research on aging.

Cellular proteins of a normal human diploid fibroblast line (TIG-3) at various stages of replicative aging were resolved by horizontal isoelectric focusing on an immobilized pH gradient, followed by vertical sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Spot proteins were visualized by silver staining and quantitated by image processing. All corresponding spots were matched among two-dimensional gel images, and variation profiles in relative abundance of individual proteins during in vitro aging were classified into five categories, i.e., (i) increase, (ii) decrease, (iii) increase followed by decrease, (iv) decrease followed by increase, and (v) irregular or nonsignificant variation. The new concept of the Tokyo Metropolitan Institute of Gerontology two-dimensional gel protein database (TMIG-2DPAGE) was prepared from the above data to support research on cellular aging. The database was put on our World Wide Web home page at the URL of http://www.tmig.or.jp/2D/ to allow free access through the Internet. The individual protein data entries were linked to the standard spot protein map of the two-dimensional gel image in order to be accessible by clicking the mouse on it.

The maize two-dimensional gel protein database: towards an integrated genome analysis program

This paper describes the first maize database of proteins separated by two-dimensional electrophoresis. Fifty-six coleoptile proteins and 18 leaf proteins from two maize lines were partially microsequenced. Thirty-six proteins (49%) displayed high similarity with database proteins. Nine of these proteins, representing five different functions, had never been described in maize. No conclusive function could be found for 45 polypeptides (61% of the microsequenced proteins). In addition, an alternative identification method, based on amino acid analysis, allowed candidates to be proposed for 17 proteins out of 44 additional proteins analyzed in the coleoptiles. These results are stored in a database which also includes, when available, genetic information about the chromosomal location of structural genes and regulatory factors of proteins. This database is being used in the context of a project on the genetic mapping of the expressed genome in maize.

CDNA expression and human two-dimensional gel protein databases: towards integrating DNA and protein information.

The rapid progress in characterizing genes and mRNAs (expressed sequence tags, ESTs) as a result of the Human Genome Project makes it imperative to develop strategies to interface DNA mapping and sequencing data with protein information, as the latter orchestrate most cellular functions. Presently, the only technique able to resolve and record the thousands of proteins present in cells and tissues is two-dimensional (2-D) gel electrophoresis in combination with computer-aided technology to scan the gels, make synthetic images, assign numbers to individual spots as well as to enter qualitative and quantitative information. To date, comprehensive 2-D gel databases containing information about various properties of proteins (cellular localization, identification, regulatory properties, partial amino acid sequences, etc.) have been established (available on the internet: http:@biobase.dk/cgi-bin/celis). What remains is to provide a link between these data and the forthcoming information from the Human Genome Project. We are pursuing two approaches to achieve this goal: (i) microsequencing and mass spectrometry analysis of proteins resolved from 2-D gels and (ii) expression of cDNAs in the vaccinia virus expression system. Using the latter approach we have expressed about 60 cDNAs in human cells under conditions that faithfully reproduce post-translational trimmings and modifications of the proteins. The method, in combination with 2-D gel electrophoresis, allows precise matching of almost any cDNA to its protein product, irrespective of the protein abundance.

Molecular characterization of phagosomes.

The transformation of newly formed phagosomes into mature phagolysosomes is a process that involves a complex series of interactions between phagosomes and other vacuolar organelles. The machinery required by phagosomes to mediate these interactions is poorly understood. In this study, we allowed human and various rodent cells to take up latex beads whose density facilitates a simple purification of phagosomes using discontinuous sucrose gradients. With this system, we initiated a systematic study of phagosome proteins using two-dimensional gel electrophoresis and the currently available two-dimensional gel protein data bases. By this approach, we were able to recognize a group of polypeptides associated with mouse J774 phagosomes-phagolysosomes including annexin II, annexin VI, the beta-1 and beta-2 subunits of trimeric G proteins, and a group of actin-binding proteins. While the amount of annexin II associated to phagosomes was similar at all times of latex internalization, the levels of annexin VI were higher on late phagosomes. Phospholipid analysis of J774 phagosomes isolated at early and late time points during phagolysosome formation also revealed significant differences in their lipid composition. In the human phagosomes, we resolved over 200 polypeptides on the two-dimensional gels. These included the proteins described in the mouse, as well as 32 polypeptides that were found to be highly enriched in phagosomes, 15 of which are not present in the current data bases. The results demonstrate that the use of latex bead phagosomes is a powerful system to identify key molecules involved in phagolysosome biogenesis.

Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.

A comprehensive human keratinocyte two-dimensional (2-D) gel protein database has been established to study the expression levels and properties of the thousands of proteins that orchestrate various keratinocyte functions both in health and disease, cancer included. A major task in establishing such a database is to identify known proteins in the 2-D gel patterns as well as to reveal hitherto unknown proteins. To date, protein identification has been performed by one or a combination of the following methods: (i) comigration with known proteins, (ii) Western blotting using specific antibodies, (iii) microsequencing and (iv) vaccinia virus expression of full length cDNAs. Recently, the systematic identification of proteins has gained a new dimension with the advent of computer programs for searching peptide molecular mass databases with experimentally obtained peptide mass maps. Here we investigate this approach to identify proteins that are highly up- or down-regulated in simian virus SV40 transformed human keratinocytes (K14). Peptide mass maps of several proteins, including keratins 7, 8, 18 and 19 were obtained either by plasma desorption mass spectrometry (PDMS) analysis of high performance liquid chromatography (HPLC) purified peptides or by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of total digests. The results demonstrated that peptide mass maps can be used for a rapid and sensitive protein identification allowing fast screening of proteins recorded in 2-D gel databases. The mass spectrometric approach when combined with microsequencing strengthened identification, and added the possibility of full characterization of post-translational modifications and sequence variations.

Human cellular protein patterns and their link to genome DNA mapping and sequencing data: towards an integrated approach to the study of gene expression.

Analysis of cellular protein patterns by computer-aided two-dimensional gel electrophoresis together with recent advances in protein sequence analysis and expression systems have made possible the establishment of comprehensive two-dimensional gel protein databases that may link protein and DNA mapping and sequence information and that offer an integrated approach to the study of gene expression. With the integrated approach offered by two-dimensional gel protein databases it is now possible to reveal phenotype-specific protein(s), to microsequence them, to search for homology with previous identified proteins, to clone the cDNAs, to assign partial protein sequences to genes for which the full DNA sequence and the chromosome location are known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Comprehensive two-dimensional gel protein databases will provide an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways, and cytoskeletal systems, both under physiological and abnormal conditions, and are expected to lead to the identification of new regulatory networks. So far, about 20% (600 out of 2,980) of the total number of proteins recorded in the human keratinocyte protein database have been identified and we are actively gathering qualitative and quantitative biological data on all resolved proteins. Given the current improvements on microsequencing as well as the availability of specific antibodies, it seems feasible to expect that most known keratinocyte proteins will be identified in the very near future. This feast will reveal a wealth of new proteins that will become amenable to experimentation both at the biochemical and molecular biology level.

The human keratinocyte two-dimensional gel protein database: update 1993.

The master two-dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post-translational modifications. 763 proteins have been identified (protein name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP-binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one- and two-cell stages

A quantitative, high-resolution, two-dimensional gel protein database has been constructed for the mouse embryo. This database has been used to obtain a detailed accounting of the amount and relative timing of changes in protein synthesis that occur during the 1-, 2-, and 4-cell stages along with a description of the most prevalent developmentally regulated patterns of synthesis. We find that during these early stages of development the pattern of proteins synthesized changes to a much greater extent than previously appreciated. During the 1- and 2-cell stages, the majority (60% and 85%, respectively) of the analyzed proteins exhibit twofold or greater changes in their rates of synthesis. The periods of greatest change are the late 1-cell and mid 2-cell stages, during which an extensive remodelling of protein synthetic pattern occurs that is largely complete by 15 h following the first cleavage. Once this reprogramming is complete, very little change is observed during the late 2-cell and 4-cell stages. Cluster analysis of individual protein synthesis patterns reveals a limited number of coordinately regulated protein sets that are responsible for most of the changes observed during the 1- and 2-cell stages. During the 2-cell stage, one third of the proteins increase by an average of fivefold, another third decrease by an average of sevenfold, and 10% undergo transient changes in rates of synthesis. These patterns reflect the switch from zygotic to maternal mRNA utilization following transcriptional activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Microsequencing of proteins recorded in human two‐dimensional gel protein databases

Sixty-six human proteins recorded in the master transformed human epithelial amnion cells (AMA) (55) and keratinocyte (11) two-dimensional gel protein databases have been microsequenced since the last publication of the AMA database (Electrophoresis 1990, 12, 989-1071). Coomassie Brilliant Blue stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes and in situ digested with trypsin. The eluting peptides were separated by reversed-phase high performance liquid chromatography (HPLC), collected individually and sequenced. Computer searches using the FASTA and TFASTA programs from the Genetics Computer Group indicated that 29 of the analyzed polypeptides correspond to hitherto unknown proteins.

Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: Improved methods, applications and gene cloning strategies

The fields of protein chemistry and molecular biology are currently merging for study of biologically relevant events and conditions. To obtain partial sequences of microamounts of protein, efficient integration of high resolution separation and sequencing technologies is required. We report here on improved methods that allow extensive internal sequencing of 10 to 20 picomoles protein recovered from one- or two-dimensional gels. Each step of the standard protocol of Aebersold et al. (Proc. Natl. Acad. Sci. USA 1987, 84, 6970-6974) and the required instrumentation were examined and specifically adapted for use with submicrogram amounts of protein. Optimizations of in situ microdigests and liquid chromatography were needed for improved peptide recovery. Subsequent automated sequencing required subpicomole analysis. New methods for S-alkylation of gel-separated proteins and accurate identification of tryptophan-containing peptides were introduced to insure overall higher efficiencies. The acquired internal sequences facilitated cloning of the genes and several strategies are discussed. Applying our method, several proteins of unknown structure were sequenced and successfully identified or cloned. Internal sequences of submicrogram protein amounts, recovered from a single two-dimensional gel of Escherichia coli total protein (120 micrograms), allowed unambiguous identification of the spots but pre-gel enrichment will be required for analysis of most (90-95%) other spots. Integration of comprehensive two-dimensional gel protein databases with methods and strategies outlined here could potentially be an abundant source of DNA probes and markers useful for guidance of the human genome sequencing project and for analysis of the emerging vast amounts of data.