Transcript Nonlinear Dynamics and APB going further…
PerkinElmer Life Sciences Production Company Meeting - 1st February 2002
Progenesis
John Hoyland Product Manager - Bioinformatics
What is Proteomics?
• • • • “The total
PROTE
in complement of a gen
OME
.
” –
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.
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
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
2D Gel separation of Proteins
• To see the proteins we must stain them • ProXPRESS images both fluorescent and non fluorescent stains
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
Where is the bottleneck?
2D Gel Electrophoresis Protein Digestion MS Preparation Staining Image capture Feature Excision
842.5099
882.5312
MS Analysis Database
834.4492
762.4301
864.4393
770.4340
893.5501
787.4050
1051.6258
1008.6609
1301.6222
1226.5284
1479. 6781 1463.7367
1426.7256
1815.7591
1763.9192
1706.8923
1722.9282
1855.8650
Image Analysis Image Analysis
What was needed?
• A 2D gel analysis system – – – Automatic Reproducible High throughput
Progenesis - automatic
• Progenesis is a “hands-free”, walk-away automated 2D gel analysis package for medium to high throughput proteomics.
Smashing the bottleneck
Assume we have 50 protein samples to compare:
Process
Load gels in day and run in batches of 10 overnight Stain in Sypro Ruby overnight in batches of 10 Image gels using ProXPRESS 20 mins. per gel Analyse and compare gels with HT Analyser (2 hrs per gel) Analyse and compare gels with Progenesis (1 day total)
Total using HT Analyser or competitor Total using Progenesis (no competitors) Hands on time
4 hours 2 hours 5 hours 100 hours 7 hours
Elapsed time
} }6 days } 14 days 1 day
111 hrs/3 weeks 20 days 18 hrs/3 days 7 days 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
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
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
Objective
• Automatic objective spot splitting
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
Progenesis Analysis Summary
• Automated Analysis • Robust detection of every spot • Warping enhances matching
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
Export Picking List to ProPic
• Use Progenesis to find and select spots for picking – Built in ‘Export to ProPic’ option generates picking list
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 8 4 2 . 5 0 9 9 8 8 2 . 5 3 1 2 8 3 4 . 4 49 2 7 6 2. 4 3 01 7 7 0. 4 3 4 0 8 6 4. 4 3 93 8 9 3. 5 5 0 1 7 8 7 . 4 0 5 0 1 0 5 1 . 6 2 5 8 1 0 08 . 6 6 0 9 1 3 0 1 . 6 2 2 2 1 2 2 6. 5 2 8 4 1 4 7 9. 67 8 1 1 4 6 3 . 7 3 6 7 1 4 2 6 . 7 2 5 6 1 8 1 5 . 7 59 1 17 6 3. 9 1 92 1 7 0 6 . 89 23 17 22 . 9 2 8 2 1 85 5 . 8 6 5 0
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
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
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