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VENNTURE--a novel Venn diagram investigational tool for multiple pharmacological dataset analysis.

Martin B, Chadwick W, Yi T, Park SS, Lu D, Ni B, Gadkaree S, Farhang K, Becker KG, Maudsley S - PLoS ONE (2012)

Bottom Line: An improved appreciation of the connectivity between multiple, highly-complex datasets is crucial for the next generation of data analysis of genomic and proteomic data streams.Applied to complex pharmacological datasets, VENNTURE's improved features and ease of analysis are much improved over currently available Venn diagram programs.This study highlights the potential for such a program in fields such as pharmacology, genomics, and bioinformatics.

View Article: PubMed Central - PubMed

Affiliation: Metabolism Unit, Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.

ABSTRACT
As pharmacological data sets become increasingly large and complex, new visual analysis and filtering programs are needed to aid their appreciation. One of the most commonly used methods for visualizing biological data is the Venn diagram. Currently used Venn analysis software often presents multiple problems to biological scientists, in that only a limited number of simultaneous data sets can be analyzed. An improved appreciation of the connectivity between multiple, highly-complex datasets is crucial for the next generation of data analysis of genomic and proteomic data streams. We describe the development of VENNTURE, a program that facilitates visualization of up to six datasets in a user-friendly manner. This program includes versatile output features, where grouped data points can be easily exported into a spreadsheet. To demonstrate its unique experimental utility we applied VENNTURE to a highly complex parallel paradigm, i.e. comparison of multiple G protein-coupled receptor drug dose phosphoproteomic data, in multiple cellular physiological contexts. VENNTURE was able to reliably and simply dissect six complex data sets into easily identifiable groups for straightforward analysis and data output. Applied to complex pharmacological datasets, VENNTURE's improved features and ease of analysis are much improved over currently available Venn diagram programs. VENNTURE enabled the delineation of highly complex patterns of dose-dependent G protein-coupled receptor activity and its dependence on physiological cellular contexts. This study highlights the potential for such a program in fields such as pharmacology, genomics, and bioinformatics.

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MeCh-induced protein phosphorylation in diverse cellular contexts.(A) Proportional diagrams illustrating the number of specifically enriched and identified phosphoproteins, basally occurring, and induced by MeCh stimulation (10 nM to 100 µM) of control or CMP-treated cells. (B) Selective protein immunoprecipitation (IP) and determination of MeCh-induced G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1) serine and threonine phosphorylation, with antisera immunoblot (IB), in MeCh-stimulated control-state SH-SY5Y cells. The associated histogram (representing mean ± SEM data from three independent immunoprecipitations) indicates the extent of immunoprecipitated protein serine and threonine phosphorylation. Panels (C) and (D) indicate similar data from control-state cells for the MeCh-induced phosphorylation of G protein regulated inducer of neurite outgrowth 1 (GPRIN1) and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1). (E) Selective protein immunoprecipitation and determination of MeCh-induced microtubule-associated protein 2 (MAP2) serine and threonine phosphorylation, with antisera immunoblot, in MeCh-stimulated CMP-state SH-SY5Y cells. Panels (F) and (G) indicate similar data from CMP-state cells for the MeCh-induced phosphorylation of reticulon-4 (RTN4) and GRB2-associated binding protein 2 (GAB2). Statistical analysis was performed on three independent experiments using GraphPad Prism version 5.02 with a Student’s t-test (p<0.05, *; p<0.01, **; p<0.001, ***).
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pone-0036911-g002: MeCh-induced protein phosphorylation in diverse cellular contexts.(A) Proportional diagrams illustrating the number of specifically enriched and identified phosphoproteins, basally occurring, and induced by MeCh stimulation (10 nM to 100 µM) of control or CMP-treated cells. (B) Selective protein immunoprecipitation (IP) and determination of MeCh-induced G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1) serine and threonine phosphorylation, with antisera immunoblot (IB), in MeCh-stimulated control-state SH-SY5Y cells. The associated histogram (representing mean ± SEM data from three independent immunoprecipitations) indicates the extent of immunoprecipitated protein serine and threonine phosphorylation. Panels (C) and (D) indicate similar data from control-state cells for the MeCh-induced phosphorylation of G protein regulated inducer of neurite outgrowth 1 (GPRIN1) and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1). (E) Selective protein immunoprecipitation and determination of MeCh-induced microtubule-associated protein 2 (MAP2) serine and threonine phosphorylation, with antisera immunoblot, in MeCh-stimulated CMP-state SH-SY5Y cells. Panels (F) and (G) indicate similar data from CMP-state cells for the MeCh-induced phosphorylation of reticulon-4 (RTN4) and GRB2-associated binding protein 2 (GAB2). Statistical analysis was performed on three independent experiments using GraphPad Prism version 5.02 with a Student’s t-test (p<0.05, *; p<0.01, **; p<0.001, ***).

Mentions: Using a titanium dioxide-mediated phosphoprotein extraction process [22], we were able to identify in SH-SY5Y cells the presence of multiple phosphoproteins in the non-stimulated control and peroxide-treated (CMP) state, as well as after the application of 10 nM, 100 nM, 1 µM, 10 µM and 100 µM acetyl-β-methylcholine (MeCh) in both cellular conditions (Protein identifications listed in Supplementary Tables as follows: control SH-SY5Y cells, non-stimulated-Table S1, 10 nM, 100 nM, 1 µM, 10 µM, 100µM MeCh-Tables S2, S3, S4, S5, S6 respectively; CMP SH-SY5Y cells, non-stimulated-Table S7, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM MeCh-Tables S8, S9, S10, S11, 12 respectively). Therefore, in our paradigm of ligand dose-responses in diverse cellular contexts, we were able to generate six different phosphoprotein datasets for each cellular context, i.e. control or CMP-treated. The total number of phosphoproteins specifically enriched and identified (≥2 peptides required for identification of each specific protein) for each of the MeCh doses applied, as well as the proteins identified in the non-stimulated conditions for each cell context (control or CMP) are depicted in Figure 2A. We validated the presence of several protein serine or threonine phosphorylations, stimulated by MeCh at multiple doses (10 nM, 100 nM, 1 µM), in both cell contexts (control, Figure 2B–D; CMP, Figure 2E–G) using selective immunoprecipitation of the target protein. The isolated target protein was then immunoblotted with specific anti-phosphoserine and anti-phosphothreonine antisera. We did not assess the anti-phosphotyrosine content of the selected proteins due to the considerably lower extent of this form of protein phosphorylation typically observed with TiO2-mediated enrichment techniques [22]. Using immunoblotting, we verified our proteomic identification of the phosphorylation of G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1: 10 nM MeCh), G protein-regulated inducer of neurite outgrowth 1 (GPRIN1: 100 nM MeCh) and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1: 1 µM MeCh) in control SH-SY5Y cells. We also verified the MeCh-induced increase in phosphorylation, identified with our TiO2-enrichment, of microtubule-associated protein 2 (MAP2: 10 nM MeCh), reticulon-4 (RTN4: 100 nM MeCh) and Grb2-associated binding protein 2 (GAB2: 1 µM MeCh) in the CMP-treated SH-SY5Y cells.


VENNTURE--a novel Venn diagram investigational tool for multiple pharmacological dataset analysis.

Martin B, Chadwick W, Yi T, Park SS, Lu D, Ni B, Gadkaree S, Farhang K, Becker KG, Maudsley S - PLoS ONE (2012)

MeCh-induced protein phosphorylation in diverse cellular contexts.(A) Proportional diagrams illustrating the number of specifically enriched and identified phosphoproteins, basally occurring, and induced by MeCh stimulation (10 nM to 100 µM) of control or CMP-treated cells. (B) Selective protein immunoprecipitation (IP) and determination of MeCh-induced G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1) serine and threonine phosphorylation, with antisera immunoblot (IB), in MeCh-stimulated control-state SH-SY5Y cells. The associated histogram (representing mean ± SEM data from three independent immunoprecipitations) indicates the extent of immunoprecipitated protein serine and threonine phosphorylation. Panels (C) and (D) indicate similar data from control-state cells for the MeCh-induced phosphorylation of G protein regulated inducer of neurite outgrowth 1 (GPRIN1) and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1). (E) Selective protein immunoprecipitation and determination of MeCh-induced microtubule-associated protein 2 (MAP2) serine and threonine phosphorylation, with antisera immunoblot, in MeCh-stimulated CMP-state SH-SY5Y cells. Panels (F) and (G) indicate similar data from CMP-state cells for the MeCh-induced phosphorylation of reticulon-4 (RTN4) and GRB2-associated binding protein 2 (GAB2). Statistical analysis was performed on three independent experiments using GraphPad Prism version 5.02 with a Student’s t-test (p<0.05, *; p<0.01, **; p<0.001, ***).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351456&req=5

pone-0036911-g002: MeCh-induced protein phosphorylation in diverse cellular contexts.(A) Proportional diagrams illustrating the number of specifically enriched and identified phosphoproteins, basally occurring, and induced by MeCh stimulation (10 nM to 100 µM) of control or CMP-treated cells. (B) Selective protein immunoprecipitation (IP) and determination of MeCh-induced G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1) serine and threonine phosphorylation, with antisera immunoblot (IB), in MeCh-stimulated control-state SH-SY5Y cells. The associated histogram (representing mean ± SEM data from three independent immunoprecipitations) indicates the extent of immunoprecipitated protein serine and threonine phosphorylation. Panels (C) and (D) indicate similar data from control-state cells for the MeCh-induced phosphorylation of G protein regulated inducer of neurite outgrowth 1 (GPRIN1) and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1). (E) Selective protein immunoprecipitation and determination of MeCh-induced microtubule-associated protein 2 (MAP2) serine and threonine phosphorylation, with antisera immunoblot, in MeCh-stimulated CMP-state SH-SY5Y cells. Panels (F) and (G) indicate similar data from CMP-state cells for the MeCh-induced phosphorylation of reticulon-4 (RTN4) and GRB2-associated binding protein 2 (GAB2). Statistical analysis was performed on three independent experiments using GraphPad Prism version 5.02 with a Student’s t-test (p<0.05, *; p<0.01, **; p<0.001, ***).
Mentions: Using a titanium dioxide-mediated phosphoprotein extraction process [22], we were able to identify in SH-SY5Y cells the presence of multiple phosphoproteins in the non-stimulated control and peroxide-treated (CMP) state, as well as after the application of 10 nM, 100 nM, 1 µM, 10 µM and 100 µM acetyl-β-methylcholine (MeCh) in both cellular conditions (Protein identifications listed in Supplementary Tables as follows: control SH-SY5Y cells, non-stimulated-Table S1, 10 nM, 100 nM, 1 µM, 10 µM, 100µM MeCh-Tables S2, S3, S4, S5, S6 respectively; CMP SH-SY5Y cells, non-stimulated-Table S7, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM MeCh-Tables S8, S9, S10, S11, 12 respectively). Therefore, in our paradigm of ligand dose-responses in diverse cellular contexts, we were able to generate six different phosphoprotein datasets for each cellular context, i.e. control or CMP-treated. The total number of phosphoproteins specifically enriched and identified (≥2 peptides required for identification of each specific protein) for each of the MeCh doses applied, as well as the proteins identified in the non-stimulated conditions for each cell context (control or CMP) are depicted in Figure 2A. We validated the presence of several protein serine or threonine phosphorylations, stimulated by MeCh at multiple doses (10 nM, 100 nM, 1 µM), in both cell contexts (control, Figure 2B–D; CMP, Figure 2E–G) using selective immunoprecipitation of the target protein. The isolated target protein was then immunoblotted with specific anti-phosphoserine and anti-phosphothreonine antisera. We did not assess the anti-phosphotyrosine content of the selected proteins due to the considerably lower extent of this form of protein phosphorylation typically observed with TiO2-mediated enrichment techniques [22]. Using immunoblotting, we verified our proteomic identification of the phosphorylation of G protein-coupled receptor kinase interacting ArfGAP 1 (GIT1: 10 nM MeCh), G protein-regulated inducer of neurite outgrowth 1 (GPRIN1: 100 nM MeCh) and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1: 1 µM MeCh) in control SH-SY5Y cells. We also verified the MeCh-induced increase in phosphorylation, identified with our TiO2-enrichment, of microtubule-associated protein 2 (MAP2: 10 nM MeCh), reticulon-4 (RTN4: 100 nM MeCh) and Grb2-associated binding protein 2 (GAB2: 1 µM MeCh) in the CMP-treated SH-SY5Y cells.

Bottom Line: An improved appreciation of the connectivity between multiple, highly-complex datasets is crucial for the next generation of data analysis of genomic and proteomic data streams.Applied to complex pharmacological datasets, VENNTURE's improved features and ease of analysis are much improved over currently available Venn diagram programs.This study highlights the potential for such a program in fields such as pharmacology, genomics, and bioinformatics.

View Article: PubMed Central - PubMed

Affiliation: Metabolism Unit, Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.

ABSTRACT
As pharmacological data sets become increasingly large and complex, new visual analysis and filtering programs are needed to aid their appreciation. One of the most commonly used methods for visualizing biological data is the Venn diagram. Currently used Venn analysis software often presents multiple problems to biological scientists, in that only a limited number of simultaneous data sets can be analyzed. An improved appreciation of the connectivity between multiple, highly-complex datasets is crucial for the next generation of data analysis of genomic and proteomic data streams. We describe the development of VENNTURE, a program that facilitates visualization of up to six datasets in a user-friendly manner. This program includes versatile output features, where grouped data points can be easily exported into a spreadsheet. To demonstrate its unique experimental utility we applied VENNTURE to a highly complex parallel paradigm, i.e. comparison of multiple G protein-coupled receptor drug dose phosphoproteomic data, in multiple cellular physiological contexts. VENNTURE was able to reliably and simply dissect six complex data sets into easily identifiable groups for straightforward analysis and data output. Applied to complex pharmacological datasets, VENNTURE's improved features and ease of analysis are much improved over currently available Venn diagram programs. VENNTURE enabled the delineation of highly complex patterns of dose-dependent G protein-coupled receptor activity and its dependence on physiological cellular contexts. This study highlights the potential for such a program in fields such as pharmacology, genomics, and bioinformatics.

Show MeSH
Related in: MedlinePlus