Technology Modules

Technology Module A: Solid-phase stable isotope tag transfer chemistry

The objective of this module is to develop automatable and automated procedures for protein processing and stable isotope tagging. During the first period of funding, we will establish and validate experiments that will enable us to test different isotope tags and cleavable linkers, and start to systematically explore different chemistries. In years 2 and 3, the different chemistries will be optimized and commercialized.

Technology Module: Kinase substrate identification via thiophosphate labeling and selective isolation of thiophosphorylated peptides

The objective of this module is to develop a new technology for the systematic identification of protein kinase targets on a proteome-wide scale. We have developed a method to specifically label kinase substrates with thiophosphates in cell lysates, and then selectively isolate the thiophosphorylated peptides from complex mixtures. We are now advancing the technology by making the approach more physiological, thereby reducing false-positive identifications. Current efforts include combining the direct cell lysate-based method with the indirect cell-based approaches, as well as pursuing techniques that allows direct labeling of kinase targets in live cells.

Technology Module C: Selective isolation and isotope tagging of peptides with lipid modifications

The objective of this module is to develop a new technology for the analysis of protein lipid modifications on a proteome-wide scale. This project will build on the development of the solid-phase isotope tag strategy that is the subject of Technology Module A. No activities are planned for the first two years.

Technology Module D: Preparation of stable isotope tagged peptides for quantitative analysis of protein complexes

The objective of this module is to develop a new technology based on quantitative proteomics for the systematic and conclusive analysis of protein complexes, their components, stoichiometry, and dynamics of composition. During the first period of funding, we will establish basic protocols for the quantitative analysis of the components of protein complexes. In the first year, we will demonstrate that we can distinguish between specific and contaminating components of the complexes and that we can detect changes in abundance of complex components. The protocols will be transferred to the high throughput facility (HTPF) and applied to biology working group projects. In years 2 and 3, procedures to determine absolute quantification of complex components and, therefore, the stoichiometry of complexes will be developed and implemented.