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Specific visualization and identification of phosphoproteome in gels.

Wang L, Pan L, Tao WA - Anal. Chem. (2014)

Bottom Line: The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, ‡Department of Medicinal Chemistry & Molecular Pharmacology, and §Center for Cancer Research, Purdue University , West Lafayette, Indiana 47907, United States.

ABSTRACT
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 toward 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.

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VIPing-baseddetection of phosphorylation in the EGFP–Sykprotein complex coimmunoprecipitated from DT-40 cells with or withoutpervanadate stimulation. (A) Chemiluminescent HRP-anti-EGFP antibodydetection, (B) VIPing-based phosphoprotein gel staining, and (C) chemiluminescentHRP-anti-pTyr antibody detection.
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fig4: VIPing-baseddetection of phosphorylation in the EGFP–Sykprotein complex coimmunoprecipitated from DT-40 cells with or withoutpervanadate stimulation. (A) Chemiluminescent HRP-anti-EGFP antibodydetection, (B) VIPing-based phosphoprotein gel staining, and (C) chemiluminescentHRP-anti-pTyr antibody detection.

Mentions: An anti-EGFP Western Blottingwas carried out as the sample loadingcontrol (Figure 4A). As shown in Figure 4B, the VIPing gel staining result indicates thatthe phosphorylation levels of both Syk and interacting proteins increaseddramatically upon treatment. In parallel, the protein complex wasalso detected by an antiphosphotyrosine antibody, showing similarlyincreasing phosphorylation pattern for tyrosine phosphorylation (Figure 4C). All these data suggested the higher phosphorylationlevels of Syk-EGFP and interacting proteins upon stimulation.


Specific visualization and identification of phosphoproteome in gels.

Wang L, Pan L, Tao WA - Anal. Chem. (2014)

VIPing-baseddetection of phosphorylation in the EGFP–Sykprotein complex coimmunoprecipitated from DT-40 cells with or withoutpervanadate stimulation. (A) Chemiluminescent HRP-anti-EGFP antibodydetection, (B) VIPing-based phosphoprotein gel staining, and (C) chemiluminescentHRP-anti-pTyr antibody detection.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215862&req=5

fig4: VIPing-baseddetection of phosphorylation in the EGFP–Sykprotein complex coimmunoprecipitated from DT-40 cells with or withoutpervanadate stimulation. (A) Chemiluminescent HRP-anti-EGFP antibodydetection, (B) VIPing-based phosphoprotein gel staining, and (C) chemiluminescentHRP-anti-pTyr antibody detection.
Mentions: An anti-EGFP Western Blottingwas carried out as the sample loadingcontrol (Figure 4A). As shown in Figure 4B, the VIPing gel staining result indicates thatthe phosphorylation levels of both Syk and interacting proteins increaseddramatically upon treatment. In parallel, the protein complex wasalso detected by an antiphosphotyrosine antibody, showing similarlyincreasing phosphorylation pattern for tyrosine phosphorylation (Figure 4C). All these data suggested the higher phosphorylationlevels of Syk-EGFP and interacting proteins upon stimulation.

Bottom Line: The core of the strategy is a novel compound multifunctionalized with a titanium ion(IV) for outstanding selectivity toward 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, ‡Department of Medicinal Chemistry & Molecular Pharmacology, and §Center for Cancer Research, Purdue University , West Lafayette, Indiana 47907, United States.

ABSTRACT
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 toward 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.

Show MeSH