The central hypothesis of this proposal is that continued production of aggregation prone proteins leads to age onset proteotoxicity and eventually disease. Two areas will be studied using proteomics technology. The first is to identify the proteins involved in disassembly/proteolysis of protein aggregates. By using newly developed assays in the Kelly laboratory, disassembly and proteolysis activities will be followed over the course of biochemical enrichment. Proteomics will be used during the enrichment process to follow and assess the course of enrichment and then finally to identify the proteins responsible for the activity. Detoxification activities are under the control of the transcription factors HSF-1 and DAF-16. We will identify the proteins whose expression levels are under the control of these transcription factors by using quantitative proteomics when the levels of HSF-1 and DAF-16 are reduced by RNAi. The methods, techniques, and experience are available in the proteomics core to conduct these analyses. The core will employ large-scale protein identification technology developed in our lab called MudPIT (Multi-dimensional Protein Identification Technology). To perform MudPIT experiments we have several different types of mass spectrometers that can be used including LTQ-Orbitrap, LTQ and LTQ-ETD (Electron Transfer Dissociation) systems. Each instrument has particular strengths that can be applied to these projects. The LTQ-Orbitrap produces high resolution and high mass accuracy data that is good for confident protein identifications, the discovery of post translational modifications, and accurate quantitation. The LTQ is a slightly more sensitive and faster scanning instrument than the hybrid and can be used when sensitivity and sequence coverage are important. The LTQ-ETD system uses a new dissociation technique that is good for fragmenting large polypeptides. This particular instrument may have an advantage when attempting to identify proteins from aggregates or proteins that do not digest well.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Program Projects (P01)
Project #
5P01AG031097-05
Application #
8429425
Study Section
Special Emphasis Panel (ZAG1-ZIJ-8)
Project Start
Project End
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
5
Fiscal Year
2013
Total Cost
$395,077
Indirect Cost
$183,130
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Roth, Daniela Martino; Hutt, Darren M; Tong, Jiansong et al. (2014) Modulation of the maladaptive stress response to manage diseases of protein folding. PLoS Biol 12:e1001998
Park, Sung Kyu Robin; Aslanian, Aaron; McClatchy, Daniel B et al. (2014) Census 2: isobaric labeling data analysis. Bioinformatics 30:2208-9
Greiner, Erin R; Kelly, Jeffery W; Palhano, Fernando L (2014) Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils. PLoS One 9:e105433
Bamberger, Casimir; Pankow, Sandra; Park, Sung Kyu Robin et al. (2014) Interference-free proteome quantification with MS/MS-based isobaric isotopologue detection. J Proteome Res 13:1494-501
Tsigelny, Igor F; Sharikov, Yuriy; Kouznetsova, Valentina L et al. (2014) Structural diversity of Alzheimer's disease amyloid-* dimers and their role in oligomerization and fibril formation. J Alzheimers Dis 39:583-600
Amschl, David; Neddens, Jorg; Havas, Daniel et al. (2013) Time course and progression of wild type ýý-synuclein accumulation in a transgenic mouse model. BMC Neurosci 14:6
Kim, Changyoun; Ho, Dong-Hwan; Suk, Ji-Eun et al. (2013) Neuron-released oligomeric *-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat Commun 4:1562
Yates 3rd, John R (2013) The revolution and evolution of shotgun proteomics for large-scale proteome analysis. J Am Chem Soc 135:1629-40
Toyama, Brandon H; Savas, Jeffrey N; Park, Sung Kyu et al. (2013) Identification of long-lived proteins reveals exceptional stability of essential cellular structures. Cell 154:971-82
Dusaban, Stephanie S; Purcell, Nicole H; Rockenstein, Edward et al. (2013) Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses. Proc Natl Acad Sci U S A 110:3609-14

Showing the most recent 10 out of 41 publications