Recent advances in proteomics have provided tools for protein characterization that allow sampling of proteins in the important biological context of protein complexes. We propose to apply high sensitivity and high dynamic range protein characterization by mass spectrometry to identify proteins, detect posttranslational modifications and quantify relative and absolute protein levels. A hypothesis of this proposal is that quantitative measurements of protein abundances in tissue or cell culture samples will reveal roles of the target proteins. Further, the complexes are at the cell-cell or organelle-organelle interface and thus are part of a membrane micro domain. The lipids in these domains organize proteins, serve as signal reservoirs, donate or trap reactive oxygen species products and are intimately involved with the function of the proteins. A second hypothesis is that the difference in lipid profile of the micro domains with age, or manipulation, will reveal details of the protein lipid relationship.
Aim 1) to identify proteins isolated in protein complexes obtained with immuno- or affinity extractions, a) Use MALDI TOP and TOF/TOF to rapidly confirm the ID of proteins and guide sample preparations, b) Use LC/MS/MS on the LTQ-FT to improve protein identification coverage with capillary HPLC and nanoUPLC. c) Improve information content from LC/MS/MS experiments 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 post translational modifications in protein complexes, a) Use LC/MS/MS on LTQ-FT or QTOF to extend protein coverage and find modified peptides. b) Use LCMS on LTQ-FT or QTOF for quantitation by SILAC, 16O/18O labeling, or spiked internal standards c) Improve data handling from LC/MS.
Aim 3) Profile lipids in raft or membrane preparations from membranes at different ages, a) Profile sphingo- and phospholipids using head group specific MS/MS scans, b) Improve the quantitation and data handling in sphingolipid profiling. We hope to identify proteins/lipids that are most relevant to the aging process and oxidative stress using feature selection algorithms. In addition, our lipid and protein data will be used in a systems biology context to identify biological networks and pathways affected by aging and oxidative stress via mapping the proteins/lipids to known pathways.
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