Global genome initiatives including the Human Genome Project have generated enormous amounts of information, spawned new technologies and catalyzed the emergence of a new discipline in biology. Systems biology attempts to build biological knowledge from the global analysis of biological systems and pathways from genes to proteins. To fuel this growing discipline, there is a need to develop means of global assessment that will ultimately clarify our understanding of protein function. Because both protein activity and turnover are closely tied to protein post-translational modification (e.g., phosphorylation and ubiquitination), new technologies are required for the large-scale identification, characterization, and quantification of protein post- translational modifications. Reversible protein phosphorylation is a general process affecting most cellular regulatory systems. Phosphorylation is critical to maintaining normal physiology;malfunctions in protein phosphorylation have been implicated in the etiology of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. Protein phosphorylation is an intense research area with great promise for improving human health. Technologies developed under the previous cycle of this grant allowed for the rapid, sensitive and accurate identification of thousands of phosphorylation sites from a single sample. This landmark achievement brings other important unsolved issues to light. Two of the most critical are addressed in this proposal - quantitative phosphorylation profiling and phosphorylation site occupancy.
For Aim 1, we will develop quantitative strategies to study dynamic phosphorylation on a near-global scale. This includes combining and integrating a robust stable isotope labeling strategy, new enrichment approaches, and new software tools with our existing platform.
For Aim 2, we will perform a highly focused set of experiments to globally profile phosphorylation differences after DNA damage in both yeast cells and human cell lines. These experiments will provide a biologically important proving ground for newly developed techniques and software. Finally, for Aim 3, we will develop and apply a strategy to determine the basal site occupancy levels for all kinases (87) in the tandem affinity purification (TAP) yeast library. In addition, site occupancy levels for a set of human kinases will be examined for DNA damage- and cell cycle-dependency.

Public Health Relevance

Protein phosphorylation is an intense research area with great promise for the improvement of human health. In addition to its critical role in normal physiology, malfunctions in protein phosphorylation have been implicated as a contributing factor in the causation of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. This proposal will provide new technologies to profile phosphorylation events between different cell states on a global scale.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG003456-07
Application #
8151104
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Ozenberger, Bradley
Project Start
2005-05-12
Project End
2012-11-30
Budget Start
2011-09-01
Budget End
2012-11-30
Support Year
7
Fiscal Year
2011
Total Cost
$426,977
Indirect Cost
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Chick, Joel M; Kolippakkam, Deepak; Nusinow, David P et al. (2015) A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides. Nat Biotechnol 33:743-9
Ræder, Helge; McAllister, Fiona E; Tjora, Erling et al. (2014) Carboxyl-ester lipase maturity-onset diabetes of the young is associated with development of pancreatic cysts and upregulated MAPK signaling in secretin-stimulated duodenal fluid. Diabetes 63:259-69
Tcherkezian, Joseph; Cargnello, Marie; Romeo, Yves et al. (2014) Proteomic analysis of cap-dependent translation identifies LARP1 as a key regulator of 5'TOP mRNA translation. Genes Dev 28:357-71
Chung, Jean-Ju; Shim, Sang-Hee; Everley, Robert A et al. (2014) Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility. Cell 157:808-22
Goranov, Alexi I; Gulati, Amneet; Dephoure, Noah et al. (2013) Changes in cell morphology are coordinated with cell growth through the TORC1 pathway. Curr Biol 23:1269-79
Wilson-Grady, Joshua T; Haas, Wilhelm; Gygi, Steven P (2013) Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods 61:277-86
McAllister, F E; Niepel, M; Haas, W et al. (2013) Mass spectrometry based method to increase throughput for kinome analyses using ATP probes. Anal Chem 85:4666-74
McAllister, Fiona E; Gygi, Steven P (2013) Correlation profiling for determining kinase-substrate relationships. Methods 61:227-35
Zhang, Xiaocui; Lavoie, Genevieve; Fort, Loic et al. (2013) Gab2 phosphorylation by RSK inhibits Shp2 recruitment and cell motility. Mol Cell Biol 33:1657-70
Pease, Brittany N; Huttlin, Edward L; Jedrychowski, Mark P et al. (2013) Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development. J Proteome Res 12:4028-45

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