Covalent modification of proteins by ubiquitin-like-proteins (Ubls) such as SUMO has been recognized as an important regulatory mechanism for most biological pathways, many of which are of great medical relevance. A comprehensive knowledge of Ubl-modification of proteins is therefore critical to understand how fundamental biological processes are regulated and to find new strategies for the treatment of a variety of diseases. To reach this goal, we propose to develop and apply tools to isolate and identify sumoylated proteins jn a proteomic scale. This will allow us to detect changes in sumoylation patterns in a variety of biological and medically relevant settings. The strategy we propose is to express SUMO fused to a tandem affinity-tag and generate a purified fraction of sumoylated proteins by a two-step affinity purification. The purified fractions will be analyzed by a combination of multidimensional liquid chromatography and tandem mass spectrometry [to generate sumoylation profiles. Such profiles will be valuable in identification of proteins that are modified with SUMO but more importantly, comparison of profiles generated from cells in different physiological or developmental states will help to identify important regulatory factors.
The first aims will use yeast as a model system to detect quantitative changes in sumoylation patterns during |the different cell cycle stages and a cell cycle checkpoint arrest.
Aim 2 attempts to adapt the approach from /east to mammalian cells and to quantitatively analyze changes in SUMO-1 modifications in response to a checkpoint-induced cell cycle arrest. This proposal is focused on identification of sumoylation targets, but we believe that the proposed strategy can applied to other modifications with ubiquitin-like proteins without any significant changes and will form the foundation for the generation of a variety of Ubl modification profiles. ? ?
| Flick, Karin; Kaiser, Peter (2009) Proteomic revelation: SUMO changes partners when the heat is on. Sci Signal 2:pe45 |
| Booher, Keith R; Kaiser, Peter (2008) A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteins. Biotechnol J 3:524-9 |
| Kaiser, Peter; Meierhofer, David; Wang, Xiaorong et al. (2008) Tandem affinity purification combined with mass spectrometry to identify components of protein complexes. Methods Mol Biol 439:309-26 |
