Recent advances in proteomics have provided tools for protein characterization that allow sampling ofproteins in the important biological context of protein complexes. We propose to apply high sensitivity andhigh dynamic range protein characterization by mass spectrometry to identify proteins, detectposttranslational modifications and quantify relative and absolute protein levels. A hypothesis of thisproposal is that quantitative measurements of protein abundances in tissue or cell culture samples will revealroles of the target proteins. Further, the complexes are at the cell-cell or organelle-organelle interface andthus are part of a membrane micro domain. The lipids in these domains organize proteins, serve as signalreservoirs, donate or trap reactive oxygen species products and are intimately involved with the function ofthe proteins. A second hypothesis is that the difference in lipid profile of the micro domains with age, ormanipulation, will reveal details of the protein lipid relationship.
Aim 1) to identify proteins isolated in proteincomplexes obtained with immuno- or affinity extractions, a) Use MALDI TOP and TOF/TOF to rapidly confirmthe ID of proteins and guide sample preparations, b) Use LC/MS/MS on the LTQ-FT to improve proteinidentification coverage with capillary HPLC and nanoUPLC. c) Improve information content from LC/MS/MSexperiments on the LTQ-FT or QTOF instruments using MALDI search engines on LC/MS1 'survey' data, d)Develop an alternative protein ID validation strategy.
Aim 2) To quantify stoichiometry and detect posttranslational modifications in protein complexes, a) Use LC/MS/MS on LTQ-FT or QTOF to extend proteincoverage and find modified peptides. b) Use LCMS on LTQ-FT or QTOF for quantitation by SILAC, 16O/18Olabeling, or spiked internal standards c) Improve data handling from LC/MS.
Aim 3) Profile lipids in raft ormembrane preparations from membranes at different ages, a) Profile sphingo- and phospholipids using headgroup specific MS/MS scans, b) Improve the quantitation and data handling in sphingolipid profiling. Wehope to identify proteins/lipids that are most relevant to the aging process and oxidative stress using featureselection algorithms. In addition, our lipid and protein data will be used in a systems biology context toidentify biological networks and pathways affected by aging and oxidative stress via mapping theproteins/lipids to known pathways.
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