1. PerkinElmer Life Sciences Production Company Meeting - 1st February 2002 Progenesis John Hoyland Product Manager - Bioinformatics

2. What is Proteomics? “The total PROTEin complement of a genOME. ” –M. Wilkins et al. Electrophoresis 1995, 16, 1090-1094 Proteome describes the complete set of proteins that is expressed, and modified following expression, by the entire Genome in the lifetime of a cell. –In essence, it describes the complement of proteins expressed by a cell at any one time. Proteomes are highly dynamic Proteomics is the study of protein properties on a large scale to obtain a global, integrated view of disease processes, cellular processes and networks at the protein level.

3. Why are Proteins important? Proteins are actively involved in a large number of disease and genetic medical conditions Proteins are a primary target for new drugs to combat these conditions If we can identify the proteins which are either missing, or are present when they should not be, we have a good chance of developing a drug to correct it. In order to identify individual proteins, we must first separate them to analyse them independently

4. How do we separate Proteins? 2D Gel Electrophoresis –Different Proteins have different sizes or molecular weights –Different Proteins have different charges or pH However: –Some Proteins have the same size or molecular weight –Some Proteins have the same charge or pH We therefore need to separate them for both size and charge –Separate in the X dimension for charge –Separate in the Y dimension for size This gives us a 2 dimension (2D) map of the protein spots which can be imaged and analysed

5. 2D Gel separation of Proteins To see the proteins we must stain them ProXPRESS images both fluorescent and non- fluorescent stains

6. Comparing images of gels Now we have separated them, we may need to compare the images of large numbers of gels to see which proteins are present and which are missing Looking for the differences in 50 or 100 gels with between two and four thousand protein spots on each is not unusual Making these comparisons is also NOT EASY and very time consuming. It is the major bottleneck in Proteomics

7. Feature Excision 2D Gel Electrophoresis StainingImage capture Protein Digestion Image Analysis MS Preparation Identification MS Analysis 7 6 2.4 3 01 770.4340 78 7.4 05 0 834.4492 842.5099 864.4393 882.5312 893.5501 1008.660 9 1051.6258 1 226.5284 1301.6222 1426.7256 1463.736 7 1479.6781 170 6. 8 923 1722.9282 1763.9192 1815.7591 1855.8 6 50 Identification Where is the bottleneck? Image Analysis Database

8. What was needed? A 2D gel analysis system –Automatic –Reproducible –High throughput

9. Progenesis - automatic Progenesis is a “hands-free”, walk-away automated 2D gel analysis package for medium to high throughput proteomics.

10. Smashing the bottleneck èAssume we have 50 protein samples to compare: Using HT Analyser, elapsed time and hands on time are similar so this is a full time job for a skilled user - maximum throughput 50 gels per MONTH Using Progenesis, elapsed time is only 1 week with only 3 days skilled user time - - maximum throughput 100gels per WEEK

11. The Analysis Wizard Select Gels to Analyse Group them Define Reference Gel Add Filters Define Background Subtraction Add Warping and Matching Define Normalisation Save this Protocol

12. Automated spot detection Automation – the key to high throughput Every spot is detected accurately and objectively Walk away with confidence that each image is treated using the same processes

13. Objective Automatic objective spot splitting

14. More targets than ever before Progenesis delivers: –More true spots, more targets –Objective and operator independent Increase in detected expression from 2D gels Traditional - auto 465 spots …with edit 432 spots Progenesis 686 spots

15. Progenesis Analysis Summary Automated Analysis Robust detection of every spot Warping enhances matching

16. Identification of interesting proteins Proteins that look like they have been up regulated or down regulated can be selected for further analysis Spots are cut out of the gel by ProPic, our automated spot cutting robot

17. Export Picking List to ProPic Use Progenesis to find and select spots for picking –Built in ‘Export to ProPic’ option generates picking list

18. Final Identification After processing to separate the proteins from the gel, they are analysed in a Mass Spectrometer The Mass Spectrometer separates and quantifies all elements in the protein Each protein has a unique ‘fingerprint’ of constituents which may be checked against massive databases to identify it 7 6 2.4 3 01 770.4340 78 7.4 05 0 834.4492 842.5099 864.4393 882.5312 893.5501 1008.660 9 1051.6258 1 226.5284 1301.6222 1426.7256 1463.7367 1479.6781 1706.8923 1722.9282 1763.9192 1815.7591 1855.8650

19. Target Accounts Target accounts Pharmaceutical Companies with Proteomic research facilities University and academic core proteomics facilities Any Proteomic group analysing more than 10-15 gels per week We have four specialists supporting Progenesis sales one in Europe, two in USA/Can and one Asia/Pacific

20. Is it value for money? Many researchers think so –In the first 6 months we have over 70 leads worldwide –Sold 3 copies in first 6 months Pharmaceutical Companies –It now costs around $800M to get a major new drug on the market –If Progenesis reduces this by a fraction of 1% it saves its cost Compare Progenesis automation with old manual methods –If a Proteomic facility is running 100 gels per week –Progenesis pays for itself in just 10 weeks

21. Finally:- At the start I mentioned 2 bottlenecks The second revolves around managing the huge amounts of data generated by Proteomics The next project will be Progenera, a databasing and data mining package from Nonlinear Dynamics Progenera will be on the market by September 2002

22. Delivering the future of Bioinformatics