(565e) Effective Separations: The Key for Proteomics

Proteomics seeks to study the total proteins expressed in any given system, whether by abundance, activity, structure, state of post translational or other modification or how these proteins interact with each other in networks or complexes. A key component to the proteomics workflow is separation. Crude proteomes are the consequence of the expression of thousands of genes, with complexities made even greater by the presence of PTMs and large variation in dynamic ranges (potentially exceeding 9 orders of magnitude). To address these dynamic range issues, proteomes need to be simplified in order to enable efficient analysis. The proteomics practitioner has a number of choices to make when seeking to examine a proteome and this then may dictate the ‘simplification’ process one chooses when performing a deeper analysis for systems biology endeavors. Traditionally, the choice was based on one-dimensional or two-dimensional gel electrophoresis, but this has now extended out to include various ‘flavors’ of chromatography and gas-phase fractionation in the mass spectrometer. This study seeks to demonstrate the advantages of selected (pre)separation strategies on identifying and quantifying proteomes from the three domains of life. The modern proteomics experiment relies heavily on the mass spectrometer (MS) and care must be taken to understand the relationship between the separation technology and the MS. Examples of post translation modification are given, as well as those that to seek to quantify the proteome using label-based methods such as iTRAQ. The impact of the separation strategy on the quality and quantity of data are provided.