United Kingdom
Meeting Highlights
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.
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.
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.
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.
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.
Analysis of the Salmonella proteome by semi-automated two dimensional HPLC-mass spectrometry
Dr Nick Coldham, Food & Environmental Safety Dept, VLA
| Dymension - A new generation of 2D gel electrophoresis image analysis software | Dr. Paru Oatey - Syngene Ltd
| 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.
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