2D Electrophoresis; The Way Forward

Hatfield, Hertfordshire
Friday, 18 November 2005

2D Electrophoresis; The Way Forward
Friday, 18 November 2005 09:30 - 16:30 (GMT)

Fielder Conference Centre
Hatfield Business Park
Hatfield
Hertfordshire
AL10 9FL
United Kingdom

Map and Directions

"This meeting is to examine the current 2-D gel electrophoresis technologies that are currently on the market, and how they are being utlizied within the scientific community. How has 2DE developed over the years and where is it going?" - Dr Ayesha De Souza (meetings chair)


9:00 – 9:40        Registration – Tea, coffee and biscuits

 

9:40 – 9:55        Introduction by the Chair: Dr Ayesha De Souza

9.55 - 10.25      
2D gels and drug safety evaluation: applications, limitations and alternatives
Dr Neil
Kitteringham - Liverpool University
Adverse drug reactions are a major cause of patient morbidity and drug attrition - they cost the NHS about £500 million a year.  New insights into their mechanisms should lead to development of better tests for safety evaluation during drug development.  Using examples from our work in Liverpool, I will highlight the benefits of a global perspective when investigating ADRs using 2D electrophoresis. In addition, I will flag up the shortcomings of the technique for certain applications, and indicate where alternative MS-based methodologies may have advantages over the conventional approach.


10:25 – 11:00
   Morning tea/coffee

 

11:00 – 11:30    Current sample preparation and fractionation methods in Proteomics

Dr Timothy Cross - Bio-Rad Laboratories Ltd

The information content of 2DE is heavily influenced by both a proper sample preparation and fractionation strategy. Sample preparation dramatically affects both reproducibility and resolution of 2-D gels. This presentation is meant to provide a broad overview of the principles and recent developments of sample preparation tools prior to the first step of 2DE. Examples from three strategies for sample preparation, based on solution chemistry, chromatography and electrophoresis, will be discussed in detail as well as used to illustrate how these key areas can be applied to general-purpose sample cleanup and sample fractionation for enrichment of low abundance proteins.

11:30 – 12:00    Proteomic analysis of the heart using 2D electrophoresis
Professor Michael Dunn - Proteome Research Centre - Conway Institute of Biomolecular and Biomedical Research - University College Dublin
Heart disease resulting in chronic heart failure is one of the major causes of morbidity and mortality.  For many patients the only long term therapy is cardiac transplantation.  We have used proteomics based on the use of 2D electrophoresis to investigate alterations in protein expression associated with the diseased heart and in response to cardiac transplantation.  These studies have given new insights into the molecular basis of heart disease and rejection following transplantation.  In addition we have used a 2DE based strategy to develop minimally invasive biomarkers of acute and chronic rejection.

12:00– 13:15     Lunch

 

13:15 – 13:45    Improving the resolution of target proteins with automated gradient gel casting for two-dimensional electrophoresis

Dr Robert Mount – NextgenSciences, UK

2D electrophoresis has been the work horse of proteomics for many years. Advances in the first dimension separation especially the introduction of IPG strips has resulted in increased reproducibility.

 

The availability of narrow range IPG strips has made significant improvements to the resolution.

However, the second dimension separation has been largely been neglected. Everyone is aware that gradient gels offer increased resolution but casting gradients is difficult to do accurately and reproducibly. I will demonstrate the advantage of gradient gels and introduce the a2DEoptimzer - a system that enables gradient gels to be cast easily and reproducibly.

 
13:45 – 14:15   
Spicing Up Proteomics with Splicing
Professor J. Godovac-Zimmermann - The Rayne Institute, London
In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.


14:15 –14:45     Sample preparation and 2D electrophoresis at the CMMI core facility
Dr. Judit Nagy - Imperial College London

Within the CMMI, proteomics is currently employed to investigate bacterial pathogens. In addition to the CMMI projects the Core Facility is supporting several large initiatives where high-through-put equipment is essential. The facility primarily uses 2D gel-electrophoresis for high-through-put protein separation (IPGphor manifold, multiphor systems for 1st dimension, the Dalt II for second dimension separation). 2D images are obtained either by the Fuji FLA 5000 laser scanner or by a UMAX scanner. Gel images are analysed by the Nonlinear 2D evolution software.Several examples of sample preparation methods will be discussed and their effect on protein separation and 2D electrophoresis.


14:45 – 15.15    Afternoon Tea/Coffee

 

15:15 – 15:45   Analysis of the Salmonella proteome by semi-automated two dimensional HPLC-mass spectrometry
Dr Nick Coldham, Food & Environmental Safety Dept, VLA
The proteome of Salmonella Typhimurium has been characterised by 2-dimensional HPLC mass spectrometry to determine the effectors of the multiple antibiotic resistance (MAR) phenotype.  A short overview of MAR will be provided followed by technical details for proteome and protein expression analysis by 2D-HPLC-MS.   Changes in the expression of protein effectors which affect the molecular flux of antibiotics and may form the basis of a test for MAR mutants will be presented.   The application of proteomics to detect protein expression of enzymes involved in fatty acid biosynthesis and mutation in FabI for resistance to the disinfectant triclosan will also be discussed.


15:45 – 16:15  Dymension - A new generation of 2D gel electrophoresis image analysis software
Dr. Paru Oatey - Syngene Ltd

16:15 Close


 


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