Alpha Project Phosphoproteomics
EMSL Project ID
17101
Abstract
The project goal is to use mass spectrometry to identify pathway proteins and the sites of their associated post-translational modifications including phosphorylation and ubiquitinylation in response to alpha factor. This goal will be achieved by the specific enrichment of pathway component proteins and their post-translationally modified forms (PTMs). To identify proteins and their PTMs, and to obtain relative quantification data, MSI/PNNL have assembled a diverse group of experimental approaches that includes affinity purification of individual proteins, high-throughput quantification strategies and novel labeling approaches.
Given the challenging nature of the project, we have developed multiple approaches to help accomplish our goals. Key players in the pheromone pathway are known to be low in abundance. Based on estimates from other systems, only 10-20% of each pathway protein is expected to be post-translationally modified in response to alpha factor. In addition, phosphorylation, a key post-translational modification in response to alpha factor, is difficult to analyze using mass spectrometric (MS) proteomic approaches because fragmentation data tends to be uninformative as a result of labile phosphoryl linkages (serine and threonine) during ionization (MS/MS).
The Alpha Project employs a multidisciplinary approach that focuses on the prediction of quantitative events in a biological system through the investigation of functional genomics in combination with computational research specifically applied to the G-protein receptor-coupled signal transduction pathway (STP) governing response to the alpha factor mating pheromone of haploid yeast cells. Experimental methods for the quantitation of the alpha factor-induced cellular response, including the formation of key intermediates in the STP, are being employed and monitored by the extremely sensitive and high-resolution method of reversed phase capillary liquid chromatography coupled with electrospray ionization Fourier transform ion cyclotron mass spectrometry (LC-FTCIR) at PNNL. The detection, identification, and quantitation, of specific protein phosphorylation events in the STP are key to unraveling the effects of dynamically, posttranslationally modified and unmodified proteins in the STP.
Project Details
Start Date
2006-02-02
End Date
2009-02-08
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members