New perspectives in the Escherichia coli proteome investigation.

Escherichia coli is a model organism for biochemical and biological studies as it is one of the best characterised prokaryote. Two-dimensional polyacrylamide gel electrophoresis, computer image analysis and different protein identification techniques gave rise, in 1995, to the Escherichia coli SWISS-2D PAGE database (http://www.expasy.ch/ch2d/). In the E. coli 3.5-10 SWISS-2D PAGE map, 40% of the E. coli proteome was displayed. The present study demonstrated that the use of narrow range pH gradients is able to potentially display up to a few copies of protein per E. coli cell. Moreover, the six new E. coli SWISS-2D PAGE maps (pH 4-5, 4.5-5.5, 5-6, 5.5-6.7, 6-9 and 6-11) presented here displayed altogether more than 70% of the entire E. coli proteome.

The 1999 SWISS-2DPAGE database update.

SWISS-2DPAGE (http://www.expasy.ch/ch2d/ ) is an annotated two-dimensional polyacrylamide gel electro-phoresis (2-DE) database established in 1993. The current release contains 24 reference maps from human and mouse biological samples, as well as from Saccharomyces cerevisiae, Escherichia coli and Dictyostelium discoideum origin. These reference maps have now 2824 identified spots, corresponding to 614 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence or any user-entered amino acids sequence. Last year improvements in the SWISS-2DPAGE database are as follows: three new maps have been created and several others have been updated; cross-references to newly built federated 2-DE databases have been added; new functions to access the data have been provided through the ExPASy proteomics server.

Two-dimensional electrophoresis resources available from ExPASy.

This paper describes the set of two-dimensional electrophoresis (2-DE) resources currently available from the ExPASy proteomics Web server. These resources include the SWISS-2DPAGE database, 2-DE software packages, 2-DE technical and educational services, as well as indexes and search engines for 2-DE related sites over the Internet.

A molecular scanner to automate proteomic research and to display proteome images.

Identification and characterization of all proteins expressed by a genome in biological samples represent major challenges in proteomics. Today's commonly used high-throughput approaches combine two-dimensional electrophoresis (2-DE) with peptide mass fingerprinting (PMF) analysis. Although automation is often possible, a number of limitations still adversely affect the rate of protein identification and annotation in 2-DE databases: the sequential excision process of pieces of gel containing protein; the enzymatic digestion step; the interpretation of mass spectra (reliability of identifications); and the manual updating of 2-DE databases. We present a highly automated method that generates a fully annoated 2-DE map. Using a parallel process, all proteins of a 2-DE are first simultaneously digested proteolytically and electro-transferred onto a poly(vinylidene difluoride) membrane. The membrane is then directly scanned by MALDI-TOF MS. After automated protein identification from the obtained peptide mass fingerprints using PeptIdent software (http://www.expasy.ch/tools/peptident.html + ++), a fully annotated 2-D map is created on-line. It is a multidimensional representation of a proteome that contains interpreted PMF data in addition to protein identification results. This "MS-imaging" method represents a major step toward the development of a clinical molecular scanner.

Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Studies of quantitative analysis of protein expression in Saccharomyces cerevisiae.

In the present study amino acid analysis is applied to quantitation of Saccharomyces cerevisiae proteome expression. The quantitation levels obtained are compared to data using densitometric analysis of silver or amido black staining and to the theoretical expression level (codon bias) of the identified proteins determined from their amino acid analysis (AAA). The results show that relative volume ratio (%vol) using Melanie II is a better parameter for spot quantitation than relative optical density ratio (%OD), and amino black staining provides good linearity within the range 1-100 pmol protein. However, AAA shows that theoretical expression levels are not well correlated with actual protein expression level, although there is better correlation when isoforms of the expressed protein are identified and included. It is concluded that amino acid analysis provides accurate protein quantitation and has a continuing role in proteome studies in terms of the rapid and inexpensive quantitation of proteins displayed on proteome maps. We do however recognize that in the context of future clinical applications and large-scale proteome discovery projects, quantitation and post-translational modification need to be analyzed by 'proteomatic' (i.e., proteome automatic bioinformatic analysis directly from the gel) techniques.

The SWISS-2DPAGE database: what has changed during the last year.

SWISS-2DPAGE (http://www.expasy.ch/ch2d/) is an annotated two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) database established in 1993. The current release contains 21 reference maps from human and mouse biological samples, as well as from Saccharomyces cerevisiae, Escherichia coli and Dictyostelium discoideum origin. These reference maps now have 2480 identified spots, corresponding to 528 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence. During the last year, the SWISS-2DPAGE has undergone major changes. Six new maps have been added, and new functions to access the data have been provided through the ExPASy server. Finally, an important change concerns the database funding source.

Towards an automated approach for protein identification in proteome projects.

The development of automated, high throughput technologies for the rapid identification of proteins is essential for large-scale proteome projects. While a degree of automation already exists in some stages of the protein identification process, such as automated acquisition of matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectra, efficient interfaces between different stages are still lacking. We report the development of a highly automated, integrated system for large scale identification of proteins separated by two-dimensional gel electrophoresis (2-DE), based on peptide mass fingerprinting. A prototype robotic system was used to image and excise 288 protein spots from an amido black stained polyvinylidene difluoride (PVDF) blot. Protein samples were enzymatically digested with a commercial automated liquid handling system. MALDI-TOF mass spectrometry was used to acquire mass spectra automatically, and the data analysed with novel automated peptide mass fingerprinting database interrogation software. Using this highly automated system, we were able to identify 95 proteins on the basis of peptide mass fingerprinting, isoelectric point and molecular weight, in a period of less than ten working days. Advantages, problems, and future developments in robotic excision systems, liquid handling, and automated database interrogation software are discussed.

'98 Escherichia coli SWISS-2DPAGE database update.

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.

Protein identification with N and C-terminal sequence tags in proteome projects.

Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein identification cannot be unambiguous unless an organism's complete sequence is available.

Current status of the SWISS-2DPAGE database.

The SWISS-2DPAGE database (http: //www.expasy.ch/ch2d/ch2d-top.html ) consists of two-dimensional polyacrylamide gel electrophoresis images, as well as textual descriptions of the proteins that have been identified on them. The current release contains 15 reference maps from human biological samples, as well as from Saccharomyces cerevisiae , Escherichia coli and Dictyostelium discoideum origin. These reference maps have 2088 identified spots, corresponding to 410 separate protein entries in the database, in addition to virtual entries for each SWISS-PROT sequence.

Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients.

A simple and inexpensive methacrylate rehydration chamber was built to accommodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 microL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.

Large-scale protein modelling and integration with the SWISS-PROT and SWISS-2DPAGE databases: the example of Escherichia coli.

Knowledge-based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software. To overcome these limitations, we developed the SWISS-MODEL server for automated knowledge-based protein modelling. The SWISS-MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three-dimensional structure. We have now used the software framework of the server to generate large collections of protein models, and established the SWISS-MODEL Repository, a new database for automatically generated and theoretical protein models. This repository is directly integrated with the SWISS-PROT and SWISS-2DPAGE databases through the ExPASy World Wide Web server (URL is http://expasy.hcuge.ch). Here we present an illustration of this process by an application to the Escherichia coli sequences.

The Dictyostelium discoideum proteome--the SWISS-2DPAGE database of the multicellular aggregate (slug).

The cellular slime mold Dictyostelium discoideum is a eukaryotic microorganism which has developmental life stages attractive to the cell and molecular biologist. By displaying the two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein map of different developmental stages, the key molecules can be identified and characterised, allowing a detailed understanding of the D. discoideum proteome. Here we describe the preparation of reference gel of the D. discoideum multicellular aggregate, the slug. Proteins were separated by 2-D PAGE with immobilised pH gradients (pH 3.5-10) in the first dimension and sodium dodecyl sulfate (SDS)-PAGE in the second dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride (PVDF) membranes and 150 spots were visualised by amido black staining. Protein spots were excised and 31 were putatively identified by matching their amino acid composition, estimated isoelectric point (pI) and molecular weight (M(r)) against the SWISS-PROT database with the ExPASy AAcompID tool (http:// expasy.hcuge.ch/ch2d/aacompi.html). A total of 25 proteins were identified by matching against database entries for D. discoideum, and another six by cross-species matching against database entries for Saccharomyces cerevisiae proteins. This map will be available in the SWISS-2DPAGE database.