Progress 01/03/09 to 09/30/14
Outputs
Target Audience: With specific tasks achieved at the end of the project, we have developed important techniques and strategies significant for multiple research areas. Besides several obvious approaches such as publications and conference presentations, we have disseminated information quickly through networking with relevant research groups who critically need the technology. Purdue University has the state-of-the-art facilities in the interdisciplinary Purdue Discovery Park that can develop, pilot test and disseminate new techniques. The PI was a fellow of Bindley Bioscience Center (BBC) at Purdue Discovery Park and engage faculties on and off campus to utilize the techniques developed by the funding support. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This interdisciplinary project has led to the advancement of modern bioanalytical capabilities and providing unique opportunities to train graduate and undergraduate students in interdisciplinary approaches to essential biochemical questions. Our research group has continuously strived for integration of research and education through multiple programs, projects, activities at Purdue University. How have the results been disseminated to communities of interest? We have generated high quality data and more tangible products, such as databases and reagents, centered around the state-of-the-art mass spectrometry, for this important field. Besides several obvious approaches such as publications and conference presentations, we will disseminate information quickly through networking with relevant research groups who critically need the technology. Purdue University has a strong research team that involves the measurement of basic signaling pathways in human, animals, and plants in response to physiological stimuli, developmental state, and genetic modifications. Proteomics data will greatly complement information obtained in molecular biology, thus provide key insights for biochemical mechanisms. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During this funding period, we have focused on the further development of PolyMAC with different metal ions and also the development of a new visualization technique for phosphoproteins in gels in high specificity: (I) Global Phosphoproteomics of Activated B cells Using Complementary Metal Ion Functionalized Soluble Nanopolymers. Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Since spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13,000 unique phosphopeptides with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration. The manuscript based on this study has recently been published on Anal. Chem. (II) Specific visualization and identification of phosphoproteome in gels. The applicability of gel-based proteomic strategies in phosphoproteomics has been largely limited by the lack of technologies for specific detection of phosphoproteins in gels. Here for the first time we report a strategy for simultaneous Visualization and Identification of Phosphoproteome in gels (VIPing) through coupling specific detection of phosphoproteins with protein identification and phosphorylation site mapping by tandem mass spectrometry. The core of the strategy is a novel compound multifunctionalized with a titanium ion (IV) for outstanding selectivity towards phosphorylated residues, a fluorophore for visualization, and a biotin group for phosphopeptide enrichment. The sensitivity and specificity of the VIPing strategy was demonstrated using standard protein mixtures and complex cell extracts, and the method was applied to study the phosphorylation changes of an essential tyrosine kinase Syk and interacting proteins upon B-cell stimulation. The novel technique provides a powerful platform for gel-based phosphoproteomic studies. This study was recently published in Anal.Chem.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jayasundera, K.B., A.B. Iliuk, A. Nguyen, R. Higgins, R.L. Geahlen and W.A. Tao. 2014. Global phosphoproteomics of activated B cells using complementary metal ion functionalized soluble nanopolymers. Anal. Chem. 86: 6363-6371.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Wang, L., L. Pan and W.A. Tao. 2014. Specific visualization and identification of phosphoproteome in gels. Anal. Chem. 86: 6741-6747.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Biochemists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has primarily led to the advancement of modern analytical capabilities and providing unique opportunities to train graduate and undergraduate students in interdisciplinary approaches to essential biochemical questions. Our research group has continuously strived for integration of research and education through multiple programs, projects, activities at Purdue University. How have the results been disseminated to communities of interest? With more specific and sensitive strategies to isolate proteins of biological significance, we have generated high quality data and more tangible products, such as databases and reagents, for this important field. Besides several obvious approaches such as publications and conference presentations, we will disseminate information quickly through networking with relevant research groups who critically need the technology. Purdue University has a strong research team that involves the measurement of basic signaling pathways in human, animals, and plants in response to physiological stimuli, developmental state, and genetic modifications. Proteomics data will greatly complement information obtained in molecular biology, thus provide key insights for biochemical mechanisms. What do you plan to do during the next reporting period to accomplish the goals? We plan to further develop the technology and strategy to identify direct substrates of serine/threonine kinases. Furthermore, we are applying the approach to different biological systems in which specific kinase-substrate relationship can be mapped.
Impacts
What was accomplished under these goals?
During this funding period, we have focused on the development of novel proteomic strategies, termed Kinase Assay Linked Phosphoproteomics (KALIP), to identify direct kinase substrates of Syk and other model kinases. The initial study of the project which was published in PNAS last year (PNAS 109:5615, 2012) has outlined the novel strategy to understand signaling mechanisms. There have been many attempts on high throughput approaches to identify kinase substrates but it has remained a challenge. By linking a sensitive in vitro kinase assay with in vivo phosphoproteomics, the new proteomic strategy is remarkably efficient in identifying multiple direct substrates of a spleen tyrosine kinase, SYK. This study has essentially opened a new research direction in our group and greatly assisted multiple researchers, such as co-inveistigator Dr. Robert Geahlen, for their discoveries in the lab. After the initial report, we greatly expanded the strategy by improving the technique, introducing a chemistry-based method for phosphoprotein detection, and broadening its applications in signaling transduction: Identified Syk’s direct substrates at the protein level. Here we describe a new proteomic strategy called protein Kinase Assay Linked Phosphoproteomics (proKALIP) suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the re-phosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein kinase assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine kinases. The manuscript based on this study has recently been accepted to publish on Mol. Cell. Proteomics. Developed a universal label-free quantitation method for phosphoproteomics. We have introduced a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. This study was recently published in Mol Cell Proteomics.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Xue L, Geahlen RL, and Tao WA (2013). Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics. Mol Cell Proteomics June 22. Epub.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wang P, Xue L, Batelli G, Lee S, Hou YJ, Van Oosten MJ, Zhang H, Tao WA, Zhu JK (2013). Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action. Proc Natl Acad Sci U S A. 110:11205-10.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Xue L, Wang P, Wang L, Renzi E, Radivojac P, Tang H, Arnold R, Zhu JK, and Tao WA* (2013). Quantitative measurement of phosphoproteome response to osmotic stress in Arabidopsis based on Library-Assisted eXtracted Ion Chromatogram (LAXIC). Mol Cell Proteomics 12:2354-69.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Hu L, Yang L, Lipchik AM, Geahlen RL, Parker LL, Tao WA (2013). A Quantitative Proteomics-based Competition Binding Assay to Characterize pITAM-Protein Interactions. Anal Chem. 85(10):5071-7.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: During the funding period, we introduced a novel multiplexed assay for the simultaneous quantitation of protein phosphorylation and total protein concentration on 96-well microplates. The use of a multi-functionalized, water-soluble nanopolymer termed "pIMAGO" (phospho imaging) and a protein antibody has allowed us to accurately and quantitatively measure levels of protein phosphorylation normalized by protein amount. In recent years, Titanium ions (IV) have been demonstrated to exhibit outstanding selectivity toward binding phosphorylated residues. In our lab, we successfully implemented a novel homogenous platform based on func-tionalized water-soluble nanopolymers in the form of den-drimers conjugated with multiple Titanium ions (IV) for either phosphopeptide enrichment prior to mass spectrometric analyses or phosphoprotein detection on microplates. Here, we further modify the dendrimer with infrared (IR) fluorescent dyes to allow direct fluorescence detection with high sensitivity and coupled with general antibody detection for unique applications in multiplexed phosphorylation analyses. Because of the much smaller size (~15 kDa) of the pIMAGO reagent as compared to that of a typical antibody (~150 kDa), competition for epitopes is reduced facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of a 96-well microplate. Furthermore, the nanopolymer's multiple functional groups per molecules effectively amplify fluorescent signals and thus greatly improve the detection sensitivity for low abundant phosphoproteins. we applied the unique pIMAGO assay to measure differential phosphorylation of a protein in a complex biological system. We chose three breast cancer lines in which the amount of phosphorylated Syk could be modulated: MCF-7 cells lacking any Syk expression (Syk -/-), MCF-7 cells expressing exogenous Syk tagged by enhanced green fluorescent protein (Syk-EGFP) and then treated with or without trace hydrogen peroxide, and MCF-7 cells expressing exogenous Syk-EGFP with an added nuclear localization signal (NLS) at the C-terminus (Syk-EGFP-NLS). To test whether the pIMAGO assay was capable of quantifying the extent of tyrosine-phosphorylation of Syk-EGFP under these different cellular conditions, we immunoprecipitated Syk-EGFP protein from lysates of MCF7 cells either lacking Syk, expressing Syk-EGP and treated without or with hydrogen peroxide, or expressing Syk-EGFP-NLS. The beads/protein complexes were thoroughly washed to elimi-nate non-specific binding and Syk-interacting proteins. After elution, Syk-EGFP was immobilized onto the microplate for the pIMAGO assay as described above. Samples derived from MCF-7 cells lacking Syk (Syk -/-) served as a negative control. The level of Syk phosphorylation in the different cell states was calculated along with protein normalization. Treatment of Syk-EGFP-expressing cells with hydrogen peroxide significantly increased the amount of the Syk phosphorylation by ~10 fold, consistent with previous observations that Syk can be activated through hydrogen peroxide induced oxidative stress in cells. PARTICIPANTS: Colaborators: Robert Geahlen Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Lakshmi Rajagopal, Children's Hospital at Seattle and University of Washington Jin-rong Xu, Department of botany and plant pathology, Purdue University TARGET AUDIENCES: Biochemists interested in tyrosine kinase signaling pathways. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Protein phosphorylation, one of the most ubiquitous post-translational modifications, has been implicated in the regu-lation of almost all aspects of a cell's life. Aberrant phosphor-ylation dynamics within the cell contribute to the onset and development of many malignances. Therefore, considerable effort has been devoted to profiling protein phosphorylation under different cellular conditions. Currently, a majority of studies report phosphorylation events that fail to distinguish changes in phosphorylation from protein expression. Recent studies indicated that nearly 25% of what appears to be dif-ferential protein phosphorylation is actually due to the changes in protein expression. Thus, more accurate meas-urements of actual phosphorylation changes normalized by protein expression changes are needed for the correct inter-pretation of comprehensive phosphorylation dynamics. Here we have presented dendrimer-based water soluble nanopolymers functionalized with Ti (IV) ions and infrared fluorescent tags. Coupled with general antibody detection, the design led to unique applications in the simultaneous measurements of protein expression levels and phosphorylation. The technique, in theory, can measure ab-solute phosphorylation level on a protein provided that a calibration curve using known amount of the protein and phosphorylation can be constructed. Because it is difficult to construct such a calibration curve, the utilization of the method will be most likely in measuring changes in protein phosphorylation under different states. The technique may not be applicable to hyper-phosphorylated proteins in which only a specific single site changes its phosphorylation status, thus not significantly affecting overall phosphorylation level of the protein. The novel technique, however, is highly at-tractive to applications in which a good quality phospho-specific antibody is not available. The ability to facilitate the finding of new kinase and phosphatase substrates, screen kinase inhibitors, or profile changes in endogenous levels of phosphorylation without site microenvironment prejudice or safety concerns will be tremendously valuable for many research groups.
Publications
- 1: Pan L, Iliuk A, Yu S, Geahlen RL, Tao WA. Multiplexed Quantitation of Protein Expression and Phosphorylation Based on Functionalized Soluble Nanopolymers. J Am Chem Soc. 2012 Oct 24. [Epub ahead of print] PubMed PMID: 23088311.
- 2: Chu H, Puchulu-Campanella E, Galan JA, Tao WA, Low PS, Hoffman JF. Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps. Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12794-9.
- 3: Iliuk A, Liu XS, Xue L, Liu X, Tao WA. Chemical visualization of phosphoproteomes on membrane. Mol Cell Proteomics. 2012 Sep;11(9):629-39.
- 4: Xue L, Wang WH, Iliuk A, Hu L, Galan JA, Yu S, Hans M, Geahlen RL, Tao WA. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates. Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5615-20.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: During the reporting period, we have focused on the development of a novel proteomic tool, Polymer-based Metal-ion Affinity Capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach is based on a metal-ion functionalized soluble nanopolymer to chelate phosphopeptides in a homogeneous aqueous environment. I) We demonstrate in this study that the PolyMAC method offers a rapid process for the recovery of phosphopeptides. The entire isolation procedure including chelation of phosphopeptides, recovery of PolyMAC-phosphopeptide complexes on agarose beads, washing, and elution requires less than 30 min while still retaining exceptionally high selectivity, reproducibility and recovery yields that are superior to the commonly used TiO2 method. PolyMAC is generally applicable for the isolation of phosphopeptides modified on serine, threonine or tyrosine. II) To test this PolyMAC strategy, we probed the phosphotyrosine proteome of invasive breast cancer cells expressing the Syk protein-tyrosine kinase and identified 794 sites of tyrosine phosphorylation, 514 of which were largely dependent on the expression of the kinase. GO annotation of the phosphoproteome identified prominent subclasses of substrates involved in important processes that regulate cell growth, motility and survival. The large number of tyrosine phosphorylated cytoskeletal (17%) and plasma membrane (10%) proteins is consistent with the described roles for Syk in modulating cancer cell motility, invasion and cytoskeletal rearrangements. The identification of substrates important in the regulation of apoptosis also is consistent with the reported role for Syk in the enhancement of cell survival. In addition, 27% of the phosphotyrosine-containing proteins were annotated to the nucleus, consistent with previous studies suggesting important roles for the kinase in the nucleus. The most abundant category for the molecular function of substrates was protein binding (44%), which is consistent with the known role of Syk and tyrosine-phosphorylation in regulating protein-protein interactions. In addition, we have initiated an integrated proteomic strategy that combines a sensitive kinase reaction with phosphoproteomics of endogenous phosphorylation for identifying direct substrates of tyrosine kinases. A novel in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides as substrates that have been derived directly from cellular kinase substrates and then dephosphorylated in vitro. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. We identified 63 and 23 direct substrates of the spleen tyrosine kinase (Syk) specific to B cells and breast cancer cells, respectively. PARTICIPANTS: Cooperator: Robert Geahlen Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Lakshmi Rajagopal, Children's Hospital at Seattle and University of Washington Jin-rong Xu, Department of botany and plant pathology, Purdue University TARGET AUDIENCES: Biochemists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes. Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells. Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry is still a considerable challenge due to the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. A drawback of most phosphoproteomic approaches is a less than satisfactory reproducibility and selectivity during the phosphopeptide enrichment step. We reasoned that inconsistencies are due largely to the heterogeneity of solid phase extraction-based isolation methods as represented by approaches that use IMAC or metal oxides. Heterogeneous isolation conditions can suffer from nonlinear kinetic behavior, unequal distribution of and/or access to functional groups, and solvation problems. The PolyMAC approach instead allows the isolation of low abundant phosphopeptides through chelation of Ti(IV) in a homogeneous, aqueous solution. A high concentration of metal ions in solution is achieved through the immobilization of the ions on soluble polymers for fast chelation. The preparation of PolyMAC reagents and their use for phosphopeptide isolation could be easily automated for high reproducibility and high throughput. The PolyMAC reagents are much more amenable to analysis using NMR, MS and other spectroscopic methods than TiO2 or IMAC reagents. The identification of sites of tyrosine phosphorylation on important components of signaling networks offers exciting new experimental targets in cancer research. Protein-tyrosine kinases play vital roles in human cancer, most often as the products of dominant, transforming genes. In contrast, Syk appears to play an unusual role in a subset of cancers by restricting their metastatic behavior, but the full repertoire of substrates for Syk, especially in epithelial cells, is not known. Interestingly, the majority of the phosphorylation sites identified in this study were characterized by a high abundance of acidic amino acid residues surrounding the phosphorylated tyrosine. While it is not yet known which of the substrates are most important for mediating Syk's effects on epithelial cell function, it is intriguing that known and verified substrates such as MAPRE1 and TRIP4 are involved in activities that are known to be modified as a function of Syk's expression.
Publications
- Iliuk A, Jayasundera K, Schluttenhofer R, Tao WA (2011). Functionalized soluble nanopolymers for phosphoproteome analysis. Methods Mol Biol. 790(5): 277-85.
- Iliuk AB, Hu L, Tao WA (2011). Aptamer in bioanalytical applications. Anal Chem. 83(12): 4440-52.
- Hu L, Iliuk A, Galan J, Hans M, Tao WA (2011). Identification of drug targets in vitro and in living cells by soluble nanopolymer-based proteomics. Angew. Chem. Int. Ed. 50(18): 4133-36.
- Iliuk A, Martinez J, Hall MC, Tao WA (2011). Phosphorylation assay based on functionalized soluble nanopolymer. Anal. Chem. 83(7): 2767-74.
- Liu W, Iliuk A, Tao WA, Ding S (2011). Identifying protein complexes by affinity purification and mass spectrometry analysis in the rice blast fungus. Fungal Genomics. Chapter 11. Methods in Molecular Biology. Vol 722. Humana Press, New York.
- Galan J, Paris L, Zhang H, Min J, Adler J, Geahlen RL and Tao WA (2011). Identification of Syk-interacting proteins using a novel amines-specific isotopic tag and GFP nanotrap. J. Am. Soc Mass Spec. 22(2): 319-28.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Signal transduction pathways modulated by protein-tyrosine kinases are often difficult to dissect because of complexities of interacting networks, a large number of substrates and low levels of phosphorylation. MS-based phosphoproteomics is presently the most powerful tool for profiling dynamic phosphorylation events on a global scale. A drawback of most phosphoproteomic approaches is a less than satisfactory reproducibility and selectivity during the phosphopeptide enrichment step. We reasoned that inconsistencies are due largely to the heterogeneity of solid phase extraction-based isolation methods as represented by approaches that use IMAC or metal oxides. Heterogeneous isolation conditions can suffer from nonlinear kinetic behavior, unequal distribution of and/or access to functional groups, and solvation problems. This presents a serious problem for the recovery of low-abundant phosphopeptides from complex samples. The PolyMAC approach instead allows the isolation of low abundant phosphopeptides through chelation of Ti(IV) in a homogeneous, aqueous solution. A high concentration of metal ions in solution is achieved through the immobilization of the ions on soluble polymers for fast chelation. Then, in a second step, the PolyMAC-phosphopeptide complexes are gathered from the solution by covalent coupling to a solid support. Besides the hydrazine-aldehyde coupling pair described here, a variety of other highly efficient pairs can be envisioned including azide-alkyne (click chemistry), thiol-idoacetyl, thiol-maleimide, thioester-cysteine, etc. The preparation of PolyMAC reagents and their use for phosphopeptide isolation could be easily automated for high reproducibility and high throughput. The density of functional groups (Ti(IV) and aldehyde) on the surface of the dendrimer can be adjusted during the synthetic steps. The PolyMAC reagents are much more amenable to analysis using NMR, MS and other spectroscopic methods than TiO2 or IMAC reagents. In addition, the dendrimer can be functionalized with different metal ions such as Fe(III) or Ga(III). Our data sets suggest that PolyMAC-Ti may exhibit selectivity for singly phosphorylated peptides, an observation documented by others using TiO2 or ZrO2 for the isolation of phosphopeptides. As a solution, a strategy that uses IMAC and TiO2 separation strategies in sequence has been proposed for the separation of multi-phosphorylated and then monophosphorylated peptides. An analogous strategy could be adopted easily by the sequential use of PolyMAC-Fe and PolyMAC-Ti. PolyMAC is generally applicable for the isolation of phosphopeptides modified on serine, threonine or tyrosine. By combining PolyMAC with an anti-phosphotyrosine enrichment step, we have developed a strategy for the in-depth analysis of tyrosine phosphoproteomes. PARTICIPANTS: Cooperator: Robert Geahlen Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Biochemists working with proteomics and breast cancer research
Impacts
Based on the PolyMAC strategy, we probed the phosphotyrosine proteome of invasive breast cancer cells expressing the Syk protein-tyrosine kinase and identified 794 sites of tyrosine phosphorylation, 514 of which were largely dependent on the expression of the kinase. GO annotation of the phosphoproteome identified prominent subclasses of substrates involved in important processes that regulate cell growth, motility and survival. The large number of tyrosine phosphorylated cytoskeletal (17%) and plasma membrane (10%) proteins is consistent with the described roles for Syk in modulating cancer cell motility, invasion and cytoskeletal rearrangements. The identification of substrates important in the regulation of apoptosis also is consistent with the reported role for Syk in the enhancement of cell survival. In addition, 27% of the phosphotyrosine-containing proteins were annotated to the nucleus, consistent with previous studies suggesting important roles for the kinase in the nucleus. The most abundant category for the molecular function of substrates was protein binding (44%), which is consistent with the known role of Syk and tyrosine-phosphorylation in regulating protein-protein interactions. The identification of sites of tyrosine phosphorylation on important components of signaling networks offers exciting new experimental targets in cancer research. Protein-tyrosine kinases play vital roles in human cancer, most often as the products of dominant, transforming genes. In contrast, Syk appears to play an unusual role in a subset of cancers by restricting their metastatic behavior, but the full repertoire of substrates for Syk, especially in epithelial cells, is not known. A previous study identified a number of proteins potentially phosphorylated on tyrosine in Syk-expressing breast cancer cells, but sites of phosphorylation were not determined. Interestingly, the majority of the phosphorylation sites identified in this study were characterized by a high abundance of acidic amino acid residues surrounding the phosphorylated tyrosine, which is consistent with the known substrate specificity of Syk. While it is not yet known which of the substrates are most important for mediating Syk's effects on epithelial cell function, it is intriguing that known and verified substrates such as MAPRE1 and TRIP4 are involved in activities (i.e., regulation of microtubule structure and activation of NF-kB) that are known to be modified as a function of Syk's expression. Further work will be needed to characterize fully the role of these and other substrates in the Syk-dependent regulation of epithelial cell metabolism.
Publications
- Burnside K, Lembo A, de Los Reyes M, Iliuk A, Binhtran NT, Connelly JE, Lin WJ, Schmidt BZ, Richardson AR, Fang FC, Tao WA and Rajagopal L (2010). Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase. PLoS ONE 5: e11071.
- Iliuk AB, Martin VA, Alicie BM, Geahlen RL and Tao WA (2010). In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion functionalized soluble nanopolymers. Mol Cell Proteomics. 9: 2162-2172.
- Ding SL, Liu W, Iliuk A, Ribot C, Vallet J, Tao A, Wang Y, Lebrun MH, Xu JR (2010). The Tig1 Histone Deacetylase Complex Regulates Infectious Growth in the Rice Blast Fungus Magnaporthe oryzae. Plant Cell 22(7): 2495-2508.
- Sanchez EE, Lucena SE, Reyes S, Soto JG, Cantu E, Lopez-Johnston JC, Guerrero B, Salazar AM, Rodriguez-Acosta A, Galan JA, Tao WA and Perez JC (2010). Cloning, expression, and hemostatic activities of a disintegrin, r-mojastin 1, from the mohave rattlesnake (Crotalus scutulatus scutulatus). Thromb Res. 126(3): e211-19.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: We have developed a new reagent and a novel strategy termed Polymer-based Metal-ion Affinity Capture (PolyMAC) for the isolation of phosphopeptides with exceptionally high reproducibility, selectivity and sensitivity. This approach is based on a metal-ion functionalized soluble nanopolymer to chelate phosphopeptides in a homogeneous aqueous environment. We have prepared and characterized the PolyMAC reagents and compare these with existing separation techniques that employ IMAC or TiO2. We applied this approach for the analysis of complex systems and demonstrate that the PolyMAC technology greatly facilitates the characterization of the spleen tyrosine kinase (Syk)-dependent phosphoproteome of malignant breast cancer cells. A line of highly invasive MDA-MB-231 cells, which normally lack Syk, was generated in which the expression of an enhanced green fluorescence protein (EGFP)-tagged form of the kinase could be induced by treatment with tetracycline (Tet). Because sites of tyrosine phosphorylation are much less abundant than sites of serine or threonine phosphorylation, we coupled an immuno-affinity step to the PolyMAC method to enrich for phosphotyrosine containing peptides. Syk-negative and Syk-EGFP-expressing MDA-MB-231 cells (- and + Tet) were grown to 50% confluency. Western blotting analyses indicated a marked elevation of tyrosine phosphorylation in cells expressing Syk-EGFP. Cell lysates were digested with trypsin. Phosphotyrosine-containing peptides were immunoprecipitated with anti-phosphotyrosine antibodies linked to agarose beads, eluted under mildly acidic conditions, subjected to PolyMAC-Ti enrichment, and analyzed by nano-flow LC-MS/MS. To evaluate the efficiency of isolation of phosphotyrosine-containing peptides, we labeled phospho-angiotensin II with one of two amine-specific isotope tagging reagents of different molecular mass. The "light" isotope tagged phospho-angiotensin II (10 fmol) was added to the sample prior to enrichment. After enrichment, 10 fmol of phospho-angiotensin II labeled with the "heavy" isotope tag was added prior to the LC-MS/MS analysis. As shown in Figure 3D, greater than 90% of the light isotope tagged phospho-angiotensin was recovered as measured by the ratio of the light (m/z,MH2+= 620.299) to heavy (m/z, MH2+ = 623.316) phosphopeptides. In contrast, TiO2 was only able to recover ~10% of the light isotope-labeled phosphopeptide. This measurement of phosphopeptide enrichment and recovery indicates that the PolyMAC method provides an extremely efficient approach for the analysis of sites of tyrosine phosphorylation. PARTICIPANTS: Anton Iliuk, Graduate student in Biochemistry Victoria A. Martin, Graduate student in the Department of Medicinal chemistry & Molecular Pharmacology Bethany M. Alicie, Graduate student in the Department of Medicinal chemistry & Molecular Pharmacology Robert Geahlen, Collaborator, Department of Medicinal chemistry & Molecular Pharmacology TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A much larger number of phosphotyrosine-containing peptides was identified in samples from cells induced to express Syk-EGFP as compared to Syk-deficient cells (820 versus 377 unique phosphopeptides), consistent with the Western blotting data. Selectivity for the recovery of phosphopeptides using the PolyMAC-Ti reagent was excellent. Overall, we identified 514 unique sites of tyrosine phosphorylation on 458 different proteins that were present only in samples from Syk-EGFP expressing cells. To complement identification, we also examined the exacted ion chromatograms (XICs) to quantify the relative abundance of phosphopeptides recovered from the two samples. After signal normalization using spiked standard phosphopeptides, we measured the relative abundance of tyrosine phosphopeptides from cells expressing or lacking Syk. The majority of phosphopeptides that were not identified as being present in one sample also were not detectable in the corresponding raw MS data. Only a small fraction of these phosphopeptides were detected with extremely low intensity and poor MS/MS spectra. These data indicated that, although lack of identification of a phosphopeptide is not evidence for its absence, it is sufficient to reflect a change of phosphorylation within a signaling pathway. The further analysis of these tyrosine phosphoproteomes will provide a unique opportunity for researchers to gain insights into important signaling pathways in breast cancer cells. Gene ontology (GO) analyses of proteins with enhanced tyrosine phosphorylation in Syk-induced cells grouped by molecular function, biological process and subcellular localization indicate roles for the kinase in a variety of cellular locations, functions and processes (Figure 4). Possible cellular signaling pathways influenced by the expression of Syk were further assessed using the induced phosphorylated proteins as input. Our data revealed elevated levels of tyrosine phosphorylation in a number of cancer-related networks, notably cancer cell movement and cell death. To confirm the identification and novel functions of tyrosine-phosphorylated proteins, we selected several phosphoproteins to explore further, including a microtubule-associated protein MAPRE1, a nuclear transcriptional coactivator TRIP4/ASC-1, a cell polarity protein SCRIB, and a microtubule-based motor protein Dynactin. Tyrosine phosphorylated proteins were immunoprecipitated from lysates of MDA-MB-231 cells lacking or expressing Syk-EGFP using anti-phosphotyrosine antibodies. The resulting immune complexes were examined by Western blotting with antibodies against MAPRE1, TRIP4, SCRIB, or Dynactin. The phosphoproteins were enriched in anti-phosphotyrosine immune complexes prepared from Syk-expressing cells. Similarly, the phosphorylation of each protein on tyrosine was confirmed by the Western blotting of anti-protein immune complexes with anti-phosphotyrosine antibodies.
Publications
- Iliuk A, Galan J, Tao WA* (2009). Playing tag with quantitative proteomics. Anal Bioanal Chem. 393(2):503-13.
- Silvestroni A, Jewell KA, Lin W-J, Connelly JE, Ivancic MM, Tao WA and Rajagopal L (2009). Enrichment and identification of serine/threonine phosphopeptides from the human pathogen Streptococcus agalactiae. J. Proteome Res. 8(5): 2563-74.
- Salazar AM, Guerrero B, Cantu B, Cantu E, Rodriguez-Acosta A, Perez JC, Galan JA, Tao WA, Sanchez EE (2009).Venom variation in hemostasis of the southern Pacific rattlesnake (Crotalus oreganus helleri): Isolation of hellerase. Comp Biochem Physiol C Toxicol Pharmacol. 149(3): 307-16.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Classical methods for identifying protein kinase substrates are based on making an educated guess and then incubating the purified candidate substrate with a purified protein kinase, usually in the presence of [γ-32P]ATP. Although this technique has been extremely useful in the past, it cannot be used where there is little or no knowledge of the likely substrate. We have devised a novel proteomic approach to identify specific tyrosine kinase substrates. The strategy is based on the generation of a pool of highly enriched, tyrosine-phosphorylated (pY) proteins as candidate substrates. A tyrosine phosphatase will be added to dephosphorylate the substrates. Then 5'-p-fluorosulfonylbenzoyl adenosine, a protein kinase inhibitor, will be added to inhibit irreversibly kinase activity in the mixture. After removing the phosphatase and excess inhibitor, the mixture will be incubated with the recombinant, active tyrosine kinase of interest, ATP (or ATP analog for engineered kinases), and MgCl2. Finally the mixture will be digested and phosphopeptides will be enriched followed by LC-MS/MS to identify phosphopeptides and sites of phosphorylation. Identified substrates in vitro will be confirmed with other biochemical methods in vitro and in cell culture. We will use Syk as the model kinase to develop this strategy. The identification of several known Syk substrates will confirm the success of this technology. At the same time, the identification of novel Syk substrates will be vital to our understanding of different signaling pathways involving Syk in distinctive cell types. PARTICIPANTS: Anton Iliuk, Graduate student in Biochemistry. He works on the development of dendrimer-based reagents for phosphopromics. He is currently supported as a graduate teaching assistant. Hua-Jie Zhang, postdoctoral associate. He works on the synthesis of new chemical reagents for quantitative proteomics and phosphoproteomics. He is partially supports by the agency. Rachel schluttenhofer, undergraduate student in Biochemistry, starts her research since her first year and continues to carry out research in this subject. She earns credit hours and the Purdue Discovery Park internship. NO salary from the agency. TARGET AUDIENCES: Biochemists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The crucial step to identify kinase substrate is to have an efficient method to isolate phosphopeptides. To provide a more effective and reproducible method for phosphopeptide isolation, we designed a novel reagent, termed soluble polymer-based metal ion affinity capture (PolyMAC) with different immobilized metal ions (i.e., PolyMAC-Zr and PolyMAC-Fe), to provide a homogeneous environment for more efficient isolation of phosphopeptides. PolyMAC-Zr is a multi-functionalized dendrimer whose surface is derivatized with phosphonate groups to chelate Zr(IV) ions. The dendrimer surface is also functionalized with hydroxylamine to provide a "handle" with which to isolate the dendrimer-phosphopeptide complex. PolyMAC-Zr was characterized using NMR and, MS and ICP-MS. The resulting reagent was compared to two existing reagents, Fe(III)-NTA resin and TiO2, for its ability to separate a standard peptide mixture consisting of angiotensin II (m/z 1046) and phosphorylated angiotensin II (m/z 1126). The phosphopeptide was captured by PolyMAC-Zr immediately after its addition to the peptide mixture. In contrast, it required over 15 min for TiO2 and over 30 min for Fe(III)-NTA resin (IMAC) to capture the majority of the phosphopeptide. Some phosphopeptides were lost during the washing step with NTA-Fe resin. The PolyMAC-Zr-phosphopeptide conjugate was then captured on aldehyde-functionalized beads through highly efficient conjugation to the hydroxylamine handle. The phosphopeptide was eluted efficiently with a yield over 90% while recovery was much lower for the Fe-NTA resin and TiO2. These data demonstrate that the capture and release of phosphopeptides using PolyMAC-Zr is specific and more efficient than with either Fe-NTA resin or TiO2. The results are most likely due to the homogenous nature of the PolyMAC-Zr agent and the phosphonate-Zr(IV) complex that provides specific binding to phosphate groups on peptides.
Publications
- Tao WA (2007). Soluble polymers-based isotopic labeling (SoPIL): a new strategy to discover protein biomarkers Expert Review of Proteomics, 4(5), 603-7.
- Timmer JC, Enoksson M, Wildfang E, Zhu W, Igarashi Y, Denault J, Ma Y, Chang Y-H, Mast AE, Eroshkin A, Osterman A, Smith J, Tao WA, Salvesen GS (2007). Profiling constitutive proteolytic events in vivo. Biochem. J. 407(1):41-8.
- Galan JA, Guo M, Sanchez EE, Cantu E, Rodriguez -Acosta A, Perez JC, and Tao WA (2008) Quantitative analysis of snake venom by soluble polymer-based isotope labeling, Mol. Cell. Proteomics, 7(4), 785-799.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: We devised a novel proteomics strategy named Solution Polymer-based Isotopic Labeling (SoPIL). We demonstrated the superior reactivity and efficiency of SoPIL reagents for the identification and quantification of Cys-containing peptides in standard proteins and complex mixtures. The novel strategy based on soluble polymers allows the derivatization reaction to be carried out in a homogeneous environment for maximum yield. In the second step, polymers and immobilized samples will be isolated on a solid phase through a highly efficient reaction between functional groups on the soluble polymer and on the solid phase. The large concentration ratios of reactive/binding groups to 'handles' facilitates the solution reaction to capture samples on the soluble polymer, while eliminating the step to remove excess reagents. For the 'handle' group, we seek to engineer a pair of biologically inert coupling
partners on dendrimers and on the solid phase, respectively, that would react at practical rates at submillimolar concentrations and at high yield. Examples include the Staudinger ligation of azides with triaryl phosphines, the formation of a Schiff's base between ketone/aldehyde-hydrazine groups, and copper(I) catalyzed azide-alkyne cycloaddition.
PARTICIPANTS: Jacob Adler: Currently senior undergraduate student. He works part-time in my lab through credit hours and fellowship. He works on the synthesis of novel proteomics reagents for the project. Jacob Galan: Now a third year graduate student. He works on the method development for the project. Minjie Guo: formally postdoctor in my group. She worked on the synthesis of new reagents for the project. Kam To: formally senior undergraduate student. He worked part-time in my lab through credit hours and fellowship. He worked on the method development for the project.
Impacts
We have examined several types of bio-orthogonal coupling reactions. The click chemistry was found to be generally biocompatible and highly efficient (the reaction can be completed in minutes). Dendrimer is selected as the soluble nanopolymer of choice because of its high structural, chemical homogeneity, high density in functional groups, and its capability to permeate cell walls for potential applications to proteomics in living cells. The SoPIL strategy is being extended to functionalize the surface of soluble nanopolymers with nucleophiles (e.g., SH groups). The reagent has adequate 'SH' concentration to efficiently achieve Michael addition in a homogeneous environment, resulting in the immobilization of serine and threonine phosphopeptides on the polymer surface. In the next step, dendrimers are captured on the solid phase through click chemistry and non-phosphopeptides are removed by
extensive washing. In the final step, peptides that are formerly serine and threonine phosphopeptides are recovered into the solution with isotope tags for identification and quantification by LC-MS/MS. We devised a novel proteomics strategy named Solution Polymer-based Isotopic Labeling (SoPIL). We demonstrated the superior reactivity and efficiency of SoPIL reagents for the identification and quantification of Cys-containing peptides in standard proteins and complex mixtures. The novel strategy based on soluble polymers allows the derivatization reaction to be carried out in a homogeneous environment for maximum yield. In the second step, polymers and immobilized samples will be isolated on a solid phase through a highly efficient reaction between functional groups on the soluble polymer and on the solid phase. The large concentration ratios of reactive/binding groups to 'handles'
facilitates the solution reaction to capture samples on the soluble polymer, while eliminating the step to remove excess reagents. For the 'handle' group, we seek to engineer a pair of biologically inert coupling partners on dendrimers and on the solid phase, respectively, that would react at practical rates at submillimolar concentrations and at high yield. Examples include the Staudinger ligation of azides with triaryl phosphines, the formation of a Schiff's base between ketone/aldehyde-hydrazine groups, and copper(I) catalyzed azide-alkyne cycloaddition.
Publications
- Enoksson, M., J. Li, M.M. Ivancic, J.C. Timmer, E. Wildfang, A. Eroshkin, G.S. Salvesen and W.A. Tao. 2007. Identification of proteolytic cleavage sites by quantitative proteomics. J. Proteome Res. 6(7):2850-2858.
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Progress 10/01/05 to 09/30/06
Outputs
Metabolic syndrome is a clinical entity characterized by obesity, hypertension, hypertriglyceridemia, low serum HDL cholesterol, and either diabetes mellitus type 2 or glucose intolerance. Metabolic Syndrome greatly increases the likelihood of developing coronary artery disease (CAD) that is the most common type of heart disease and the leading cause of death in the United States in both men and women. The goal of this study is to develop novel proteomics technologies and conduct comprehensive examination of signaling, in particular serine, threonine, and tyrosine phosphorylation in CASMC of a swine model, that links metabolic syndrome and CAD. We devised a novel proteomics strategy named Solution Polymer-based Isotopic Labeling (SoPIL). We demonstrated the superior reactivity and efficiency of SoPIL reagents for the identification and quantification of Cys-containing peptides in standard
proteins and complex mixtures. The novel strategy based on soluble polymers allows the derivatization reaction to be carried out in a homogeneous environment for maximum yield. In the second step, polymers and immobilized samples will be isolated on a solid phase through a highly efficient reaction between functional groups on the soluble polymer and on the solid phase. The large concentration ratios of reactive/binding groups to 'handles' facilitates the solution reaction to capture samples on the soluble polymer, while eliminating the step to remove excess reagents. For the 'handle' group, we have examined several types of bio-orthogonal coupling reactions. The click chemistry was found to be generally biocompatible and highly efficient (the reaction can be completed in minutes). Dendrimer is selected as the soluble nanopolymer of choice because of its high structural, chemical homogeneity, high
density in functional groups, and its capability to permeate cell walls for potential applications to proteomics in living cells. The SoPIL strategy is being extended to functionalize the surface of soluble nanopolymers with nucleophiles (e.g., SH groups). The reagent has adequate 'SH' concentration to efficiently achieve Michael addition in a homogeneous environment, resulting in the immobilization of serine and threonine phosphopeptides on the polymer surface. In the next step, dendrimers are captured on the solid phase through click chemistry and non-phosphopeptides are removed by extensive washing. In the final step, peptides that are formerly serine and threonine phosphopeptides are recovered into the solution with isotope tags for identification and quantification by uLC-MS/MS.
Impacts
In the United States, an estimated 47 million people have metabolic syndrome that is characterized by obesity, hypertension, hypertriglyceridemia, low serum HDL cholesterol, and either diabetes mellitus type 2 or glucose intolerance. Metabolic Syndrome greatly increases the likelihood of developing coronary artery disease (CAD) that is the most common type of heart disease and the leading cause of death in the United States in both men and women. The goal of this proposal is to develop corresponding novel proteomics technologies and conduct comprehensive examination of signaling that links metabolic syndrome and CAD using a novel swine model.
Publications
- Cooks, R. G., Chen, H., Eberlin, M. N., Zheng, X., Tao, W. A., Polar acetalization and transacetalization in the gas phase: the Eberlin Reaction, Chem. Rev., 2006, 106, 188-211.
- Tao, W. A., Bodenmiller, B., Aebersold, R., Characterization of post-translational modifications: undertaking the phosphoproteome, in Method Express-Proteomics, O'Connor, D. ed., Scion Publishing, London, 2006.
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Progress 10/01/04 to 09/30/05
Outputs
Active ABL kinase was purchased from commercial source (Upstate) which was expressed from bacterial E. Coli culture. Its substrate, abltide, is a 9-residue peptide. Treating abltide with Abl kinase resulted in phosphorylated peptide or thiophosphopeptide, respectively, when ATP or ATP-gamma-S was added into the in vitro kinase essay solution. The kinase uses ATP-gamma-S less efficiently than ATP. However, with the presence of transitional metal ions (such as Mn2+), efficient thiophosphorylation of Abltide by Abl kinase was achieved. We are working on designing a solid-phase based reagent used to capture thiophosphorylated peptides. The solid phase reagent contains an acid-labile linker, a stable isotope tag, and a thiol-reactive group. Such solid-phase reagent is highly desirable to efficiently capture low-abundant peptides. Sequence analyses, including the site of phosphorylation, will be achieved by micro-capillary liquid chromatography-tandem mass spectrometry
(microLC-MS/MS) using the new Agilent nanoflow HPLC system coupled with a linear quadrupole ion trap mass spectrometer (LTQ). Some inconsistency has occurred to us on the capture of thiophosphopeptides. We reason that due to low abundance of these thiophosphopeptides, the capture efficiency cannot be guaranteed in every occasion. In the light of the difficulty, we synthesized a soluable polymer support-based chemical reagent for the capture of thiophosphopeptides. It is based on a synthetic polymer-dendrimer. Dendrimer has been seen wide applicatios in drug delivery and combinatorial synthesis. we believe low amounts of sample presents solid phase extraction/approaches a non-trivial problem due to their heterogeneous nature. Dendrimers as soluable support, on the other hand, can provide homogenous reactions betwen dendrimer and thiophosphpeptide. The identification of phosphoproteins and sites of phosphorylation will be achieved by searching MS/MS spectra against the human protein
database using the SEQUEST algorithm. A modification of thiophosphate group (+96) on tyrosine residues allows us to detect sites of phosphorylation.
Impacts
Protein kinases play pivotal roles in many cellular processes such as cell proliferation, metabolism, survival and apoptosis. Several protein tyrosine kinases are known to be activated in cancer cells and to drive tumour growth and progression. Understanding the role of any particular kinase (normal or oncogenic) has remained one of the major challenges. We reason that the identification of the substrates of each kinase in a cell should provide a basis for understanding the complex cellular functions and consequently also provide a blueprint for novel targets for drug discovery. Here, we propose to develop quantitative proteomic approaches to identify substrates of kinases. Using an ATP analog (ATP-gamma-S), we introduce a thiophosphate group on the phosphorylation sites, followed by the solid-phase capture of thiophosphopeptides. LC-MS and database searching will be used to identify thiophosphorylated peptides and their corresponding phosphorylated proteins. Coupled
with stable isotope labeling, it allows for unanimously distinguishing substrates phosphorylated by the target kinase from background phosphorylated proteins by other kinases, facilitating mapping any signaling pathway and our understanding of molecular mechanisms accountable for cancer pathology. The method has the capabilities of defining any signaling pathway and control mechanism that involves phosphorylation or dephosphorylation.
Publications
- Tao, W. A., Wollscheid, B., O Brien, R, Eng, J., Li, X., Bodenmiller, B., Watts, J., Hood, L., Aebersold, R., Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and mass spectrometry, Nature Methods, 2005, 2(8), 591-598.
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