PerkinElmer Life Sciences PerkinElmer Life Sciences Production Company Meeting - 1st February 2002Production Company Meeting - 1st February 2002

ProgenesisProgenesis

John Hoyland

Product Manager - Bioinformatics

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.

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

FeatureExcision

2D Gel Electrophoresis

Staining Image capture

ProteinDigestion

Image Analysis

MS Preparation IdentificationMS Analysis

762.4301

770.4340

787.4050

834.4492

842.5099

864.4393

882.5312

893.5501

1008.6609

1051.6258

1226.5284

1301.6222

1426.7256

1463.7367

1479. 6781

1706.8923

1722.9282

1763.9192

1815.7591

1855.8650

IdentificationIdentification

Where is the bottleneck?

Image Analysis

Database

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

Process Hands on time Elapsed timeLoad gels in day and run in batches of 10 overnight 4 hours }Stain in Sypro Ruby overnight in batches of 10 2 hours }6 daysImage gels using ProXPRESS 20 mins. per gel 5 hours }Analyse and compare gels with HT Analyser (2 hrs per gel) 100 hours 14 daysAnalyse and compare gels with Progenesis (1 day total) 7 hours 1 dayTotal using HT Analyser or competitor 111 hrs/3 weeks 20 daysTotal using Progenesis (no competitors) 18 hrs/3 days 7 days

Assume 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

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

7 6 2. 4 3 01

7 7 0. 4 3 4 0

7 8 7 . 40 5 0

83 4 . 4 49 2

8 4 2 . 5 0 9 9

8 6 4. 4 3 93

8 8 2 . 5 3 1 2

8 9 3. 5 5 0 1

1 0 08 . 6 6 0 9

1 0 5 1 . 6 2 5 8

1 2 2 6. 5 2 8 4

1 3 0 1 . 6 2 2 2

1 4 2 6 . 7 2 5 6

1 4 6 3 . 7 3 6 7

1 4 7 9. 67 8 1

1 7 0 6. 89 23

17 22 . 9 2 8 2

17 6 3. 9 1 92

1 8 1 5 . 7 59 1

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

Delivering the future of Bioinformatics