The mass spectrometry core will provide proteomics and metabolomics resources to enable the three major P01 projects achieve success in uncovering the molecular mechanisms of Hamartoma syndromes and related cancers in the TSC1-TSC2 pathways for new drug targets and novel therapies. For proteomics, microcapillary tandem mass spectrometry (LC/MS/MS) services will include protein complex identification, post-translational modification (PTM) site mapping such as phosphorylation, ubiquitination, acetylation, etc. and the relative and absolute quantification of peptides/proteins including modified peptides using both stable isotope labeling (SILAC, ITRAQ, TMT) and label-free quantification [spectral counting, total ion current (TIC), multiple reaction monitoring (MRM)]. These studies will be performed from cell lines, xenografts in addition to in vivo tissue sources from mice and human tumors. For metabolomics, services will include polar metabolite profiling using selected reaction monitoring (SRM). We will profile cells, tumor tissues and biological fluids using both steady-state profiling and stable isotope labeled C glucose/glutamine and N flux experiments to determine which metabolic pathways are altered in cells harboring defects in the TSC pathways. For these studies, the core will use a Thermo Fisher Scientific hybrid linear Ion trap-Orbitrap XL-ETD mass spectrometer and an AB/SCIEX 5500 hybrid QTRAP triple quadrupole mass spectrometer. For phosphorylation studies, we will use a combination of CID and ETD fragmentation. The majority of these studies will take place from immunopurified (IP) protein complexes in the relevant TSC1-TSC2 pathways and from phosphopeptide enrichment with IMAC and TiOa. Using both hybrid ion trap-orbitrap mass spectrometry via spectral counting and average TIC in addition to triple quadrupole mass spectrometry via MRM, we will develop quantitative clinical assays that will aid in the mechanistic deduction of pathway activation. The core will further develop in-house software to improve our informatics infrastructure necessary to analyze the data from protein-protein interaction (PPI) and quantitative PTM studies. We will also utilize the drosophila PPI dataset from -300 bait-prey IP-LC/MS/MS experiments from 36 proteins in the insulin signaling pathway from the first granting period by overlapping the IP-MS data via reciprocal BLAST with mammalian PPI bait-prey datasets in the TSC1-TSC1 pathway to identify novel protein pathway members followed by biochemical validation for functional significance.

Public Health Relevance

The mass spectrometry core will provide proteomics and metabolomics services for the three major projects from cancer cells, tumor tissue and biological fluids using tandem mass spectrometry (LC/MS/MS). These services will support the discovery of novel pathways and drug targets for Hamartoma syndromes and related cancers stemming from defects in the tuberous sclerosis complex (TCS1-TSC2) genes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Program Projects (P01)
Project #
5P01CA120964-07
Application #
8567635
Study Section
Special Emphasis Panel (ZCA1-RPRB-O)
Project Start
Project End
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
7
Fiscal Year
2013
Total Cost
$172,953
Indirect Cost
$9,598
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Young, Nathan P; Kamireddy, Anwesh; Van Nostrand, Jeanine L et al. (2016) AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes. Genes Dev 30:535-52
Leonard, Paul G; Satani, Nikunj; Maxwell, David et al. (2016) SF2312 is a natural phosphonate inhibitor of enolase. Nat Chem Biol 12:1053-1058
Sumita, Kazutaka; Lo, Yu-Hua; Takeuchi, Koh et al. (2016) The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis. Mol Cell 61:187-98
Nathan, Neera; Tyburczy, Magdalena E; Hamieh, Lana et al. (2016) Nipple Angiofibromas with Loss of TSC2 Are Associated with Tuberous Sclerosis Complex. J Invest Dermatol 136:535-8
Koyama, Shohei; Akbay, Esra A; Li, Yvonne Y et al. (2016) Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun 7:10501
Mullarky, Edouard; Lucki, Natasha C; Beheshti Zavareh, Reza et al. (2016) Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers. Proc Natl Acad Sci U S A 113:1778-83
Cox, Andrew G; Tsomides, Allison; Kim, Andrew J et al. (2016) Selenoprotein H is an essential regulator of redox homeostasis that cooperates with p53 in development and tumorigenesis. Proc Natl Acad Sci U S A 113:E5562-71
Li, Ming; Tucker, Lynne D; Asara, John M et al. (2016) Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication. J Clin Invest 126:3117-29
Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien et al. (2016) Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress. Science 351:275-81
Ilagan, Erika; Manning, Brendan D (2016) Emerging role of mTOR in the response to cancer therapeutics. Trends Cancer 2:241-251

Showing the most recent 10 out of 229 publications