Most recent update
27 July 1998
Contents
Database structure.
Clickable spot maps
Enlarged gel sections
Spot tables
Haemophilus isolates examined
- Isolate origins
- HI64443
- H. influenzae - Eagan
- H. influenzae - Rd
- H. haemolyticus
- H. parahaemolyticus
- H. parainfluenzae (1391)
- H. parainfluenzae (7857)
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Haemophilus 2D Protein Database
Introduction
The following describes the methods used to compare the 2D protein profiles obtained for different members of the Haemophilus genus.
Contents
Objectives
The primary objective for developing the Haemophilus 2D protein database is to provide a comprehensive view of gene expression for this group of human pathogens. It is intended to link the proteins resolved by 2D-PAGE to the genome nucleotide sequence determined for the Rd strain of H. influenzae. The data derived will be applied to the analysis of global gene expression of H. influenzae under defined conditions. The data currently available have been used to examine the extent of protein variation among H. influenzae isolates as well as other members of the Haemophilus genus.
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Database
Assembly
The strategy used to assemble the current 2D database and its application to compare bacterial isolates has been described elsewhere [Cash et al. (1995) Electrophoresis, 16, 135-148] and is illustrated in Figure 1 for the analysis of two bacterial isolates, A and B. The following approach has been used. A single isolate of H. influenzae is used as the reference strain for the database - this is a nontypable clinical H. influenzae isolate, designated HI-64443. This bacterial isolate is analysed in parallel with all Haemophilus spp. isolates that are to be incorporated into the 2D protein database. The proteins from each new isolate to be included into the database are co-electrophoresed with proteins extracted from HI-64443 in order to optimise the matching of the protein profiles. Under these conditions it is possible to accurately link proteins identified for each new bacterial isolates into the database based on their ability to co-migrate with proteins identified for HI-64443.
At present, this approach is limited in that, although proteins in new bacterial isolates which co-migrate with those in HI-64443 are accurately recorded in the full database, proteins which do not co-migrate with proteins in HI-64443 can only be characterised at the level of individual experiments. Thus, in the example described above proteins unique for isolate A cannot be matched to unique proteins identified for isolate B unless these two isolates are directly compared by co-electrophoresis. As the number of bacterial isolates included in the database increases the pairwise matching of all isolates becomes too large to be practicable.
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Analysis
of 2D protein profiles
Preparation of the reference 2D protein profile
The reference 2D protein profile is derived from three independent analyses of HI-64443 proteins processed through the standard preparative and analytical protocols. One of the three protein profiles is taken as the Master gel to which the remaining two protein profiles are matched. The gel image shown elsewhere on this WWW site for the reference protein profile is that of the Master gel. Proteins not resolved in the Master profile but resolved on the other gels are added to the reference profile using routines from within the analytical computer software. Thus, a reference protein map is built up containing data on all proteins detected on the three individual gels.
Matching new Haemophilus spp. isolates to the database
Each new bacterial isolate to be included into the database is compared to HI-64443 within a single batch of 2D gels. The proteins for HI-64443 and the new bacterial isolate are analysed independently and by co-electrophoresis with each other. These three conditions are analysed in duplicate within the same batch of gels. All of the gels are then processed for computer-assisted spot detection and matching - the use of co-electrophoresis improves the fidelity of the profile matching and provides absolute confirmation on whether or not proteins from different bacteria co-migrate. Once these data have been combined, one of the protein profiles of HI-64443 is selected and matched to the reference 2D protein profile. In this manner, it possible to link the proteins resolved for a specific bacterial isolate to the reference 2D protein profile first by co-electrophoresis with HI-64443 and then through a sample of HI-64443 analysed within the same batch of gels. It is the latter profile that is matched to the reference 2D protein profile.
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Accessing
the data
The data which can be accessed by outside users includes information on the reference gel 2D protein profile as well as data on protein matches between the Haemophilus isolates analysed to date.
The reference gel data can be viewed either as a spot list or gel images with associated spot maps. The spot list is presented as a table of all proteins recorded on the reference 2D gel profile. The spot number indicates the number assigned to each spot as it is added to the gel using the computer software. The occurrence of the protein on each of the three gels comprising the reference 2D protein profile is indicated by a "+" in the appropriate column of the table. The spot volume is determined by the computer software and is the average volume for the protein on those gels in which it is detected. The spot volumes are determined from images captured using a video camera and have not, at present, been normalised in any manner. The final two columns present the isoelectric points and molecular weights for the bacterial proteins as determined by comparison with the mobilities of marker proteins - see protocols. These values are determined by routines from within the computer software and are only scored for those proteins migrating within the range of the marker proteins. Hypertext links are available from this table to link with further data availbale on protein co-migration with those members of the genus we have characterised by 2D-PAGE.
Spot maps for the reference gel are accessed using a full image of the Master gel. Selection of one of the quadrants on this gel presents an enlargement of that region of the gel together with two corresponding spot maps. These are the "Gel Map" and the "Reference Gel Map". The Gel Map shows the proteins detected for the Master gel and indicates the protein number stated in the spot list. Those proteins indicated by the filled red symbols - without a number - are protein spots added from one of the other gels making up the reference profile. The Reference Gel Map shows a diagram of the 2D protein profile with all of the proteins indicated by a number. Since this can produce a crowded diagram, specific regions (indicated by the blue rectangles) can be selected to provide a further enlargement of the protein map.
Accessing data on matching proteins among the Haemophilus isolates examined to date is made by selecting spots from the HI64443 reference image. This is currently limited to the selection of the most abundant proteins resolved for HI64443. The data provided by this route includes the experimentally determined isoelectric point and molecular weight as well as the abundance of the selected protein in the HI64443 reference protein profile. Data on matching proteins only provides information on whether or not a co-migrating protein was present. If it is not possible to establish (for experimental reasons) the co-migration of a protein between HI64443 and the other Haemophilus isolates then this is indicated by "n.d.". In the near future it is intended to include a clickable map based on the Rd strain of H. influenzae to specify proteins. These data may be also be accessed via hypertext links in the spot list table.
The spot files accessed via the clickable spot maps will also contain data on protein identities and synthesis under defined conditions as they become available.
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Last Modified: Monday September 27th, 1999