Gene-protein database of Escherichia coli K-12: edition 3

The first two editions of the E. coli Gene-Protein Index were published to provide identifications of protein spots resolved by two-dimensional gel electrophoresis as the products of known genes. This third edition has been expanded to include information about genes and proteins gained directly from two-dimensional gel analysis--including information about protein spots not yet characterized genetically or biochemically--and is therefore more properly called a cellular protein database. An alpha-numeric designation has been uniquely assigned to each of the 616 polypeptide spots in the current database. To this, information is linked about the polypeptide's identification (protein name, gene name, Enzyme Commission--EC number), location on reference gels (x-y coordinates), genetics (Genbank code, DNA sequence reference), biochemistry (molecular weight, isoelectric point), and physiology (steady state level of the protein as a function of media and temperature, membership in various regulons and stimulons).     

The gene-protein database of Escherichia coli: edition 5.

The gene-protein database of Escherichia coli is both an index relating a gene to its protein product on two-dimensional gels, and a catalog of information about the function, regulation, and genetics of individual proteins obtained from two-dimensional gel analysis or collated from the literature. Edition 5 has 102 new entries--a 15% increase in the number of annotated two-dimensional gel spots. The large increase in this edition was accomplished in part by the use of a new method for expression analysis of ordered segments of the E. coli genome, which has resulted in linking 50 gel spots to their genes (or open reading frames) and another 45 to specific regions of the chromosome awaiting the availability of DNA sequence information. Communication of information from the scientific community resulted in additional identifications and regulatory information. To increase accessibility of the database it has been placed in the repository at the National Center for Biotechnology Information (NCBI) at the National Library of Medicine under the name ECO2DBASE. It will be updated twice yearly. This edition of the gene-protein database is estimated to contain entries for one-sixth of the protein-encoding genes of E. coli.     

Two-dimensional electrophoresis analysis of proteomics based on image feature and mathematical morphology

Protein analysis techniques, including 2-D electro- phoresis, image analysis, biological mass spectrometry, database search, etc., are fundamental technologies of proteomics, a front area in biochemistry and life sci- ences[1―3]. Among them image analysi…     

A review of the CLIP system for the quantitative analysis of two-dimensional electrophoresis gels

This paper reviews the CLIP image processing system for the complete analysis of two-dimensional electrophoresis images. The analysis problem for two-dimensional gel images can be broken down into three issues: segmentation of individual gel images, alignment and comparison of pairs of gel images, and information storage and retrieval. This paper describes these problems and reviews how the CLIP system handles each of them. Segmentation is the location and isolation of each protein spot on an individual gel image and also the extraction of individual spot data such as position, area and volume. There are three basic stages: background field correction, noise filtering, spot detection and information extraction. Alignment and comparison of gel images involves matching protein spots between two gels. This can be quite difficult because there is not a simple relationship which can transform one gel image onto another. The database issues concern storing all the information which has been obtained from the above operations such that retrieval of this information can be readily performed. The advantage of the CLIP system over others is speed of processing. CLIP series computers use one processor for every pixel of the camera image such that image processing algorithms run in parallel. The main disadvantage is in the cost of these machines. With the declining trend in the cost of parallel processors, these machines will become more and more viable alternatives. This papers reviews the algorithms for the analysis of two-dimensional gels. It is shown that CLIP is flexible enough to perform more than one type of algorithm for a particular operation.     

Towards a two-dimensional database of common human proteins.

A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.     

Human liver protein map: a reference database established by microsequencing and gel comparison.

This publication establishes a reference human liver protein map obtained with immobilized pH gradients. By microsequencing, 57 spots or 42 polypeptide chains were identified. By protein map comparison and matching (liver, red blood cell and plasma sample maps), 8 additional proteins were identified. The new polypeptides and previously known proteins are listed in a table and/or labeled on the protein map, thus providing a human liver two-dimensional gel database. This reference map can be used to identify protein spots on other samples such as rectal cancer biopsies.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

Exploratory data analysis groupware for qualitative and quantitative electrophoretic gel analysis over the Internet-WebGel

Many scientists use quantitative measurements to compare the presence and amount, of various proteins and nucleotides among series of one- and two-dimensional (1-D and 2-D) electrophoretic gels. These gels are often scanned into digital image files. Gel spots are then quantified using stand-alone analysis software. However, as more research collaborations take place over the Internet, it has become useful to share intermediate quantitative data between researchers. This allows research group members to investigate their data and share their work in progress. We developed a World Wide Web group-accessible software system, WebGel, for interactively exploring qualitative and quantitative differences between electrophoretic gels. Such Internet databases are useful for publishing quantitative data and allow other researchers to explore the data with respect to their own research. Because intermediate results of one user may be shared with their collaborators using WebGel, this form of active data-sharing constitutes a groupware method for enhancing collaborative research. Quantitative and image gel data from a stand-alone gel image processing system are copied to a database accessible on the WebGel Web server. These data are then available for analysis by the WebGel database program residing on that server. Visualization is critical for better understanding of the data. WebGel helps organize labeled gel images into montages of corresponding spots as seen in these different gels. Various views of multiple gel images, including sets of spots, normalization spots, labeled spots, segmented gels, etc. may also be displayed. These displays are active and may be used for performing database operations directly on individual protein spots by simply clicking on them. Corresponding regions between sets of gels may be visually analyzed using Flicker-comparison (Electrophoresis 1997, 18, 122-140) as one of the WebGel methods for qualitative analysis. Quantitative exploratory data analysis can be performed by comparing protein concentration values between corresponding spots for multiple samples run in separate gels. These data are then used to generate reports on statistical differences between sets of gels (e.g., between different disease states such as benign or metastatic cancers, etc.). Using combined visual and quantitative methods, WebGel can help bridge the analysis of dissimilar gels which are difficult to analyze with stand-alone systems and can serve as a collaborative Internet tool in a groupware setting.     

Determination of protein spots separated by two-dimensional polyacrylamide gel electrophoresis

A simple method for quantitating proteins in the spots on two-dimensional polyacrylamide gel electropherograms is described. The system consists in three steps: (1) O'Farrell's two-dimensional gel electrophoresis of the proteins to be analysed; (2) staining of the gels with Coomassie brilliant blue; and (3) determination of the area and integrated density of the stained spots by the Joyce Loebl Magiscan-1 image analysis system. The method can be used for the determination of proteins in the range 0.5-100 micrograms/cm2; the amount of protein involved in most spots detected by the staining method actually falls within this range. As the minimum spot diameter that can easily be handled by the method is about 2 mm, as much as 30 ng of protein in such a spot can be determined. The method can also be applied to autoradiograms.     

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.     

Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis

We report a method using a fully automated chip-based nanoelectrospray system for two-dimensional (2-D) gel sample analyses with mass spectrometric detection. The automated nanoelectrospray system, consisting of the NanoMate and electrospray ionization (ESI) chip, serves as both an autosampler and nanoESI source. This infusion system aspirates samples from a 96-well plate using disposable pipette tips and then delivers these samples sequentially to an ESI chip. This chip is a fully integrated monolithic device consisting of a 10x10 array of nozzles. The automated nanoelectrospray system is easily controlled through software, permitting the user to select the number of samples to be analyzed, the volume of sample to aspirate, the spray voltage, and analysis time. The system offers all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without analyte carryover. The system was used for a protein identification study of 2-D gel spots of both Escherichia coli and yeast crude cell extracts. The identification of 50 spots from E. coli crude cell extract and 27 spots from yeast extract is presented, demonstrating the powerful combination of the automated nanoESI system, the Thermo Finnigan LCQ Deca ion-trap mass spectrometer, and SEQUEST search software. In addition, the effects of silver staining and colloidal Coomassie blue staining of 2-D gel spots on the detection sensitivity and protein sequence coverage are compared and discussed. Furthermore, the comparison results using the multiwell microscale preparation kit versus manual extraction for in-gel samples are presented.     

Superimposing two-dimensional gels to study genetic variation in malaria parasites.

Two-dimensional polyacrylamide gel electrophoresis is a valuable tool for studying genetic variation in the human malaria parasite, Plasmodium falciparum. It involves examining the position of protein spots in gel produced from different isolates. Some spots have been seen to vary, while others have had a constant position in all isolates so far examined. These invariant spots provide a reference frame to compare variations in other spots. This paper discusses the usefulness of digital image handling, warping and superimposition in a personal computer environment. Rather than produce a fully automatic interpretation system, we show how the computer may be used as a tool for manipulating gel images, although interpretation of the gels' features remains with the human expert. Autoradiographs are scanned on a desktop scanner, and the images in digital form can be displayed on a monitor attached to a personal computer. The coordinates of the invariant spots on each of several gels are identified by the user. Each of the gels is then warped so that the invariant spots of all the gels coincide as closely as possible. The variable spots are then examined. We have used both affine warping transformations, which match the invariant spots as closely as possible, and thin plate spline transformations, which match them exactly. Colour superimposition proved a useful way of examining the gels.     

Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999).

By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).     

Plasma protein map: an update by microsequencing.

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.     

Two-dimensional protein map of human vastus lateralis muscle

Protein samples of human vastus lateralis muscle were analyzed by two-dimensional gel electrophoresis, using immobilized pH gradients encompassing several pH regions in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. More than 500 protein spots on each gel were detected by silver staining, of which 150 were excised, digested in-gel with trypsin and characterized by matrix assisted laser desorptioin/ionization-mass spectrometry and tandem electrospray mass spectrometry. Using these techniques, 124 spots including contractile proteins and metabolic enzymes were identified. This database should provide a valuable resource for the definition of the functional properties of muscle fibres and for assessment of the influence of e.g., hypoxia, ischaemia and ageing on protein level.     

Identification of proteins from Escherichia coli using two-dimensional semi-preparative electrophoresis and mass spectrometry

Escherichia coli is a gram-negative bacterium that causes sepsis and infections of the nervous system, and the digestive and urinary tracts. The availability of the complete nucleotide sequence encoding the E. coli K-12 genome has made this organism an excellent model for proteomic studies. Semi-preparative two-dimensional electrophoresis, including liquid phase isoelectric focusing (IEF), one-dimensional sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and gel elution, have for the first time been used in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), electrospray tandem mass spectrometry and database searching for rapid separation of proteins from a uropathogenic strain of E. coli. The identity of 30 proteins, including the membrane protein nmpC, was obtained using this approach.     

Two-dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases

A rapid and reproducible method of two-dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer-personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.