1. Rapid Detection System for Stress Responses - Use in Proteomic Analysis Nicola J. Holden (1), Maurice P. Gallagher (1), and Frank Fox (2). (1) Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK. (2) EA Biotech Ltd., Unit 4, Strathclyde Business Centre, 416 Hamilton Road, Flemington G72 7XR, Scotland, UK. The recent availability of the nucleic acid sequences for entire genomes of bacteria has provided an opportunity to link gene expression with complete cellular protein profiles. Such an approach which involves analysis of the cellular protein composition by two-dimensional gel electrophoresis, is referred to as proteomics. Potentially, this strategy has great importance for the pharmaceutical and food processing industries. It can be used to explore the global effects of toxic drugs, of sanitising agents and sanitising procedures or, of antibiotics on cells. Such applications may provide important information on drug design and specificity, cellular survival mechanisms, or strategies used by cells for host infection, for immune evasion or for combating therapeutic agents. The ability to analyse complex protein mixtures has benefited from improved procedures for performing two-dimensional electrophoresis (2-DE) under highly reproducible conditions and in particular, from the commercial availability of IEF strips with chemically fixed pH gradients. In addition, these improvements have been complemented by the development of highly sensitive methods for protein characterisation using tandem mass spectroscopy. Together this combination of approaches permit the identification of proteins by comparison of polypeptide fragments, revealed by tandem mass spectroscopy, with all the predicted coding regions from the genome of the organism examined. In essence then, the complement of proteins in the cell at any one time can be determined. Analysis of the dynamics of cellular responses, as opposed to the determination of the static protein composition of cells, requires evaluation of de novo protein synthesis and generally require pulse labelling the cells with radioactive amino acids, before and after exposure to the stimulus of interest. One of the limitations of this strategy is the time required for data acquisition, as the signals from 35 S-labelled proteins commonly takes several weeks to detect using X-ray film. In the present report, we describe the application a novel waxed-based system for enhanced autoradiography which is simple to apply and enhances both detection sensitivity and speed. Our studies show that application of EA-Wax TM enhances detection sensitivity to a level which equates with direct detection using a Molecular Dynamics phosphoimager. In contrast however, application of EA-Wax TM does not require costly phosphoimaging machinery. An outline of the basic procedure is described and a comparison of the results obtained by phosphoimaging and the EA- Wax TM system is illustrated. The cost and ease of use of this strategy makes it readily applicable as a standard approach for use in both academic and industrial environments. Introduction Materials and Methods Bacterial Strains and Growth Conditions S. typhimurium SL1344 was used for pulse labelling experiments. All cultures were grown aerobically, in Luria Broth (LB), at 37 o C. Pulse labelling and 2-D gel electrophoresis Strain SL1344 (Hosieth & Stocker, 1981) was grown at 37 o C to mid- exponential phase in Spitzizen minimal media, supplemented with histidine. Samples were then removed at specified times and labelled with 5mCi [ 35 S] methionine for 5 minutes at 37 o C. Cell were then lysed by sonication and the samples were processed using 2-DE, essentially as described by OFarrell (1975). For the first dimension samples were run on pre-cast 110mmResults Enhanced Autoradiography The EA-Wax system is a simple process which dramatically enhances the detection efficiencey of low energy radionuclides. In the current context it is used to enhance detection of samples which have been electroblotted onto PVDF membrane. However, it can be used with a variety of other flat matrices, such as paper, polyester-backed TLC plates, or cellulose nitrate filters. It is supplied in a simple to use kit format, containing sheets of EA-Wax and silicone release membrane (Figure 1). Figure 1. The EA-Wax Starter Kit The key components of the EA-Starter Kit are shown (panel A, left panel alongside the sheets of silicone release membrane and strips of EA-Wax (panel B, right). Application of the EA-Wax System EA-Wax contains a wax-embedded fluor which enhance autoradiography. Following 2-D electrophoresis, samples are electroblotted onto a suitable matrix such as PVDF membrane. After draining, the PVDF is then placed on top of a clean sheet of silicon release membrane. Strips of wax containing the fluor are then layered on top of the sheet of PVDF in a fairly even manner (Figure 2). Figure 2 Application of EA-Wax to PVDF. EA-Wax is shown adjacent to a sheet of PVDF membrane which has been placed on a sheet of silicon release membrane (panel A). Several strips of wax are removed and layered on the PVDF membrane (panel B). The number of strips required varies with the size of the membrane. polyacrylamide IPG strips (Amersham-Pharamacia), pH 3-10. IPG strips were pre-run at 300V, 1Vhr followed by 14,000V for 20Vhr. After separation, IPG strips were bonded onto a 1.5 x 150 x 180mm acrylamide slab gel for the second dimension and electrophoresed at 30mA for 16h at 4 o C. The gels were subsequently blotted onto PVDF membrane at 20V for 16h, 4 o C. Membranes were then imaged for 24h, using a Molecular Dynamic phosphoimager or were processed using the EA-Wax system described below. Following layering of the wax, the Silicon release membrane, PVDF and wax assembly are placed on a square glass plated which has been pre-heated to 60 o C and a second sheet of silicon release membrane is then layered on top of the assembly (Figure 3). Figure 3. Assembling the Sample with Wax A pre-heated glass plate is placed on a level stand (panel A, left). Shown also, is the Silicon Release Membrane/ PVDF/wax assembly which is layered on the glass plate. A second sheet of Silicon Release Membrane can then be applied to the top of the stack (panel B, right). Finally, a second pre-heated glass plate is placed on top of the stack and the sample is clamped with even compression, in order to spread the wax over the PVDF membrane (Figure 4). After a few minutes when the sample has cooled and the wax has solidified, the clamping system can be removed and the sample can be exposed to X-ray film in an autoradiography cassette which has been lined with aluminium foil to aid reflection. Figure 4. Compression of the wax. The completed assembly is shown, with the PVDF/wax assembly clamped between the sheets of Silicon Release Membrane and heated glass plates. A Comparison of EA-Wax with Detection by Phosphoimaging. The dynamics of cellular protein synthesis can be recorded by combining radioactive pulse labelling with 35 S-methionine together with 2-D gel electrophoresis. However, the time required for detection using autoradiography represent a rate-limiting step in the process. Whilst samples can generally be imaged in approximately 24h with a phosphoimager, image capture on X-ray film commonly takes 2-3 weeks. Nevertheless, this still represents a useful process as X-ray film images are generally sharper and so can be compared more readily using analysis software, such as the Phoretix package. In order to determine the value of the EA-Wax system, we compared the results of direct image analysis, using a Molecular Dynamics Phosphoimager, with data obtained after wax impregnation. In order to achieve this, cells of Salmonella were grown at 37 o C and radiolablelled with 35 S-methionine (as described in Materials and Methods). Samples were then sonicated and analysed by 2-D gel electrophoresis, prior to electroblotting onto PVDF membrane. The resulting membrane was then phosphimaged directly for 24 h. Subsequently the membrane was impregnated in EA-Wax as described above and was then exposed to autoradiography film for 24 h. The results of both treatments are shown (Figure 5). It can clearly be seen that wax treatment resulted in a major enhancement in the speed and sensitivity of detection by autoradiography and indeed, the images obtained using both systems were very similar. Analysing cellular response mechanisms using proteomics is a very powerful tool and has a wide range of application of relevance to the pharmaceutical and food industries. However, the time required for detection of 35 S labelled samples using autoradiography has been a limitation to the speed of analysis and a constraint for high throughout processing. Our studies have shown that use of EA-Wax can greatly improve this situation and can reduce exposure time from several weeks to less than 24h. EA-Wax therefore represents a relatively inexpensive and user friendly method for enhancing autoradiograph sensitivity, allowing detection to be performed with equivalent sensitivity to a phosphoimage detection system. S 35 labelled cells of Salmonella were fractionated by 2-D electrophoresis using IPG strips (pI 3-10; left to right) from Amersham-Pharmacia, prior to electroblotting onto a PVDF membrane. The membrane was imaged for 24h using a Molecular Dynamics Phosphoimager (panel A, left). Subsequently, the membrane was processed using the EA-Wax kit and exposed to X-ray film for 24 h (panel B, right). Figure 5. Comparison of 35 S detection using EA-Wax and Phosphoimaging. Conclusion References Hosieth, S. K., & B.A.D. Stoker. (1981) Nature. 291: 238-239. OFarrel, P.H. (1975). J. Biol. Chem. 4007-4021.