Technology Modules

We see the list of tasks of the technology development group as dynamically changing in response to the demands of the Biology Working Groups. Over the duration of the Center, new technology modules will be conceived and some of the initial ones will either be completed or phased out in response to ever-evolving requirements.

Technology Module A: Solid-phase stable isotope tag transfer chemistry - W. Andy Tao, P.I.

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 B: Kinase substrate identification via thiophosphate labeling and selective isolation of thiophosphorylated peptides - Yong Chi, P.I.

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 - Jeff Ranish, P.I.

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.

Technology Module E: Development of multiplexed high throughput peptide separation systems - Josh McBee & Kristian Swearingen, P.I.s

The objective of this module is to develop a comprehensive peptide separation platform for robust and reproducible peptide separations carried out at high throughput. During the first funding period, we will determine the most efficient way to increase sample throughput for proteomic analysis. The most efficient methods will be further developed in years 2 and 3. The research will proceed along two tracks: the first is based on ESI-MS/MS and the second is based on IM-MS/MS.

Technology Module F: Development of a mass spectrometric technology for quantitative high throughput protein analysis - Ulrike Kusebauch, Jeff Stevens, & Caroline Shu P.I.s

The objective of this module is to develop an analytical platform based on SRM mass spectrometry for the quantitative analysis of complex protein mixtures at high sample thoughput. In the first period of funding, we will implement a system for high throughput peptide analysis based on QTOF and QQQ technology. In years 2 and 3, the technology will be implemented in the HTPF and applied to projects of the BWG.

Technology Module G: Development of a suite of software tools for protein identification and quantification using mass spectra and tandem mass spectra - Jimmy Eng, P.I.

The objective of this module is to develop and disseminate a suite of software tools for the automated analysis of quantitative proteomics data. A main benefit of this suite will be the ability to standardize results obtained in different experiments, labs and centers, and therefore to make diverse data sets comparable and portable. The development of software tools for proteomics research is a top priority for the Center. In the initial phase, tools for peptide and protein identification and quantification will be concurrently developed as independent modules. After testing and validation, the individual modules will be merged into an integrated system in years 2 and 3 of the program. To train scientists in the use of the tools, the Center will periodically offer a training course.

Technology Module H: SBEAMS - A proteomics database - Eric Deutsch, P.I.

The objective of this module is to develop a relational database for the collection, storage and dissemination of proteomics data. In the initial phase of this project, the SBEAMS database will be implemented and tested at ISB as a research tool and as a tool to support the HTPF. In years 2 and 3, the database will be continually in process and disseminated to other centers.

Technology Module I: Expression and purification of peptides for the selection of AQUA Standards - Josh McBee, P.I.

The objective of this module is to develop reagents and technology required to carry out quantitative protein profiling experiments using SRM technology. In the early funding phase, the initial set of 5-10 proteins will be prepared for the purpose of selecting prototypic peptides specific for those proteins. In years 2 and 3, the number of proteins will be ramped up.

Technology Module: Enrichment and quantification of Post-translational modifications - Richard Rogers, P.I.

The objective of this module is to examine the global changes in post translational modification on a proteome-wide scale. We are currently using techniques to enrich for phosphopeptides and Ubiquitin-like modifications. During the first year of funding, we will examine changes in post-translational modification of proteins in bone marrow macrophages in response to Toll-like receptor agonists. In years 2 and 3, the analyses will be extended to quantifying post-translational modification changes that occur in mouse tissue during infection with bacterial pathogens and viruses.