Overview. The proteomics core provides cutting-edge proteomics capabilities to the Center, ISB and many collaborators locally and worldwide. Recent accomplishments include: development of analytical and computational tools enabling comprehensive and systematic analysis of proteomes, subproteomes, and post translational modifications;development of software suites for evaluation and validation of proteomic datasets;and the design and implementation of targeted quantitative mass spectrometry (MS) experiments to analyze proteomes and subcellular proteomes. The core equipped with state-of-the-art MS technologies (see Resources) and will continue to disseminate and promote tools for high quality quantitative data acquisition and rapid implementation of new technologies. The core provides training and assistance on MS operation and experimental design, and assists with the Proteomics Informatics course (See Education and Training). Because the core is so integral to Center research, we provide below a description of ongoing technology development. Quantitative Proteomics. The core is a world leader in the development and application of both label and label-free quantitative proteomics for expression profiling and analysis of macromolecular assemblages. For example, the core, in collaboration with the Aitchison group, developed a novel automated approach to quantify peptides in SILAC experiments (QTIPs) along with new approaches for isolation of macromolecular complexes. These approaches significantly improved the identification of in vivo relevant interactions and led to extensive definition of signaling networks involved in peroxisome induction"??. Analysis of post-translational modifications. The core continues to make significant contributions to the challenging analysis of post-translational modifications (PTMs) (e.g. phosphorylation and -glycosylation) through technologies developed for peptide isolation and identification of the modified sites. To address issues associated with the low abundance of phosphopeptides, the core has implemented fractionation methods for sample complexity reduction, and target enrichment strategies, (IMAC or Ti02), which have significantly increased the number of identified phosphopeptides from a few hundred to thousands. We have developed a cryolysis-based disruption, urea-solubilization methodology to minimize kinase or phosphatase activity and maintain the condition-specific phosphorylation status of the proteome. By combining metabolic labeling (SILAC) with biochemical, analytical, and computational approaches, we routinely identify key phosphoproteins that are significantly responsive to cellular perturbations at very low copy number. To further enhance the unequivocal identification of the phosphorylated site in phosphopeptides, the core implemented electron transfer dissociation (ETD) capabilities to produce fragmentation spectra that retain PTMs (e.g., labile phospho-serine/threonine bond). We have also developed software to interpret ETD spectra with statistical validation for incorporation into the Trans-Proteomic Pipeline (TPP).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Specialized Center (P50)
Project #
2P50GM076547-06A1
Application #
8517255
Study Section
Special Emphasis Panel (ZGM1-CBCB-3 (SB))
Project Start
Project End
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
6
Fiscal Year
2012
Total Cost
$496,409
Indirect Cost
$220,626
Name
Institute for Systems Biology
Department
Type
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98109
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