Proteomics Core
Overview:
The purpose of the Proteomics Core is to provide state-of-the-art mass spectrometry-(MS)-based
technologies for obtaining accurate, comprehensive, and dynamic information about proteomes,
sub-proteomes, post-translational modifications and individual proteins. By obtaining this
detailed proteomics information, we enable researchers to gain novel insights into biological
complexity. Furthermore, this information is an essential component in our efforts to understand
how dynamic molecular networks respond to environmental cues. The capabilities of the Proteomics
Core will continue to be expanded in response to evolving research needs.
Capabilities:
Instruments
The Proteomics Core houses:
- Applied Biosystems ABI4000 Q-Trap;
- Thermo Finnigan LTQ linear ion trap (2005);
- Thermo Finnigan LCQ-DECA 3-D ion trap (2000);
- Applied Biosystems QSTAR Pulsar Q-TOF (2002);
- Applied Biosystems ABI 4700 MALDI TOF.
In addition, we have installed three new instruments in 2007, which were leveraged through other sources but significantly contribute to Center research capabilities:
- Waters Q-TOF Premier (2007);
- Waters Quattro Premier triple quadrupole (2007); and
- a Thermo Finnigan LTQ-Orbitrap (2007).
We have applied these technologies to provide insights into global protein expression, protein complex characterization, absolute quantification of proteins in complex mixtures, and identification of post-translationally modified proteins.
Development of New Methods
The Proteomics Core has worked to develop and apply new methods that allow us to obtain more detailed
information about dynamic proteomes. Much of this work has focused on the development of new methods to
characterize the dynamic composition of protein-DNA and low-affinity protein complexes. For example, we
have synthesized new amine reactive isotope tagging reagents (N-isotags) to provide comprehensive protein
tagging capability. We have used these isotags to characterize the composition of gene regulatory complexes
on a model promoter. Next, we will establish a more general and robust protocol to expand this capability
to characterize the composition of complexes that regulate gene expression in other systems such as the
peroxisome biogenesis system.
The Proteomics Core is working to develop an improved method for glycopeptide isolation. In the method,
glycopeptides derived from glycoproteins, are enriched by selective capture onto a solid support using
hydrazide chemistry, followed by enzymatic release of the peptides and subsequent analysis by tandem mass
spectrometry. Our approach provides optimized and robust selectivity for glycosylated peptides, improved
detection of glycoproteins especially hydrophobic membrane proteins, and enhanced MS detection of low-abundance
multiglycosylated proteins. This method is being used in the Center's
Disease Diagnostics research
and it will be adapted for use with microfluidics.
Centralization of Data and Software
We collaborate to support the integration of new datasets on protein interactions and gene expression with
existing datasets to facilitate the generation of dynamic interaction networks.
Plans:
The Proteomics Core plans to:
- Create a library of validated peptide fragments (MRM transitions) visible on the triple quadruple mass spectrometer.
- Analyze the yeast proteome following metabolic disturbance to study oleate responses at the levels of the chromatin, peroxisome and entire proteome using both shotgun and targeted quantitative MS approaches.
- Develop a set of transitions for yeast phospohopeptides that should allow sensitive analysis of the yeast phosphoproteome by targeted MRM MS.
- Develop the yeast expression system with heavy isotope labeling via heavy amino acids in culture (SILAC) to enable quantitative proteomics.
- Implement a powerful new integrated chemical crosslinking, mass spectrometric and computational database search strategy developed in the Aebersold laboratory for mapping the architecture of macromolecular assemblies.
- We will continue to collaborate with the Informatics Core to support the integration of new datasets on protein interactions and gene expression with existing datasets to facilitate the generation of dynamic interaction networks
- We will develop these tools and approaches in conjunction with projects to study organ specific blood biomarkers, yeast cell differentiation and organelle biogenesis and absolute quantification of Halobacterium proteins using spiked synthetic peptides.
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