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
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
Technology Module E: Development of multiplexed high throughput peptide
- 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
- 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.
Technology Modules Staff