A key objective of phosphoproteomic analyses is to obtain an in-depth understanding of protein phosphorylation and signaling networks in cells. However, despite the great progress that has been made over the past few years, our ability to analyze such signaling pathways is still greatly limited by a number of serious technical challenges. The long term goal is to develop novel proteomics strategies valuable for understanding on the molecular level the role of phosphorylation in regulating cell proliferation. The objective in this application is to devise and optimize effective and efficient methodologies and strategies for the analyses of tyrosine kinase phosphoproteomes, using a dual functional tyrosine kinase Syk as the model system. This approach will utilize functional, soluble polymers for the efficient and inclusive isolation of phosphopeptides coupled with an integrated chemical, mass spectrometric, and computational strategy for the identification of specific tyrosine kinase substrates and for quantitative phosphoproteomics. We will focus on Syk-dependent signaling and establish protocols that will ultimately provide a powerful method to dissect any signaling pathway regulated by protein-tyrosine phosphorylation. Guided by strong preliminary data, this objective will be achieved by pursuing three specific aims: 1) Develop a proteomic platform for the analysis of protein phosphorylation. We will primarily develop, characterize and optimize functionalized soluble polymers designed for the affinity and chemical isolation of phosphopeptides;2) Identification of specific tyrosine-kinase substrates and their phosphorylation sites;and 3) In-depth analyses of tyrosine kinase phosphoproteomes in several cancer types. We will develop and apply our quantitative proteomic technologies for the identification and quantification of tyrosine-phosphorylation sites in several cancer types. This project is innovative because it will develop a series of novel analytical techniques to directly identify novel tyrosine kinase substrates and phosphorylation sites that will provide investigators with a significantly expanded capability to dissect signaling pathways. The developed techniques will provide detailed knowledge at the molecular level on precisely how tyrosine kinases are involved in cell signaling under different physiological conditions through dynamic changes in protein phosphorylation. It is also expected that, by identifying a kinase's direct substrates, the proposed research will provide a powerful tool to conveniently map kinase networks.

Public Health Relevance

The proposed studies to develop a series of analytical techniques have potential applicability for investigators in the molecular signaling field. The tools will allow them to examine important roles of tyrosine kinase in multiple signaling pathways. The proposed research will provide unique strategies and materials that will strengthen the important contribution that proteomics can make to our understanding of cancer biology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088317-04
Application #
8537209
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2010-09-01
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2013
Total Cost
$286,263
Indirect Cost
$94,715
Name
Purdue University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Xue, Liang; Arrington, Justine V; Tao, W Andy (2016) Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics. Methods Mol Biol 1355:263-73
Iliuk, Anton; Li, Li; Melesse, Michael et al. (2016) Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers. Chembiochem 17:900-3
Wang, Linna; Yang, Li; Pan, Li et al. (2015) Time-Resolved Proteomic Visualization of Dendrimer Cellular Entry and Trafficking. J Am Chem Soc 137:12772-5
Iliuk, Anton B; Tao, W Andy (2015) Universal non-antibody detection of protein phosphorylation using pIMAGO. Curr Protoc Chem Biol 7:17-25
Gendrin, Claire; Lembo, Annalisa; Whidbey, Christopher et al. (2015) The sensor histidine kinase RgfC affects group B streptococcal virulence factor expression independent of its response regulator RgfA. Infect Immun 83:1078-88
Pan, Li; Wang, Linna; Hsu, Chuan-Chih et al. (2015) Sensitive measurement of total protein phosphorylation level in complex protein samples. Analyst 140:3390-6
Iliuk, Anton; Jayasundera, Keerthi; Wang, Wen-Horng et al. (2015) In-Depth Analyses of B Cell Signaling Through Tandem Mass Spectrometry of Phosphopeptides Enriched by PolyMAC. Int J Mass Spectrom 377:744-753
Searleman, Adam C; Iliuk, Anton B; Collier, Timothy S et al. (2014) Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer. Electrophoresis 35:3463-9
Xue, Liang; Wang, Pengcheng; Cao, Pianpian et al. (2014) Identification of extracellular signal-regulated kinase 1 (ERK1) direct substrates using stable isotope labeled kinase assay-linked phosphoproteomics. Mol Cell Proteomics 13:3199-210
Jayasundera, Keerthi B; Iliuk, Anton B; Nguyen, Andrew et al. (2014) Global phosphoproteomics of activated B cells using complementary metal ion functionalized soluble nanopolymers. Anal Chem 86:6363-71

Showing the most recent 10 out of 38 publications