263311 High Dynamic Range Proteomics

Tuesday, October 30, 2012: 8:30 AM
Fayette (Westin )
Phu Van and Jonathan Minden, Biological Sciences, Carnegie Mellon University, Pittsburgh, PA

Cells contain thousands of different types of proteins. These proteins exist in the cell at very different concentrations, from tens of copies per cell to millions of copies per cell, which represents a hundred-thousand-fold concentration range. The goal of comparative proteomics is to discover differences in protein expression patterns between cells, tissues and organisms grown under different conditions, with different genetic backgrounds, or at different stages of development or disease. Current proteome profiling methods are unable to detect and identify the full complement of proteins in a single experiment. This limitation is a serious impediment in many comparative proteomic analyses and is largely due to the fact that no detection system has a dynamic range that is well matched to the roughly 100,000-fold concentration range of cellular proteins. There are two general approaches to comparative proteomics experiments: peptide-centric and protein centric. Peptide-centric methods rely exclusively on mass spectrometers (MSs) for peptide identification and quantification. The dynamic range of typical MSs used for unbiased comparative proteomics is ~1,000. In protein-centric methods (which commonly involve fluorescently tagged proteins and difference-gel electrophoresis (DIGE)), protein quantification and identification are done separately by fluorescence imagers and MSs, respectively. Fluorescence imagers have a dynamic range of ~20,000. To quantify protein abundance over a 100,000-fold range, we developed a gel imaging system with a million-fold dynamic range, which is essential for detecting both low abundance proteins, such as transcription factors, and high abundance proteins, such as structural proteins, in the same experiment. To exploit the full capabilities of this new high dynamic range imaging system, we will discuss modifications to the protein labeling protocol and challenges to MS identification of low abundance proteins.

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