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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 phosphorylated proteins. Protein mixtureincludes three nonphosphorylated proteins (BSA, catalase, and β-lactoglobulin)and two phosphorylated proteins (ovalbumin and β-casein). (A)VIPing-based detection of 500 ng of the five-protein mixture withand without CIAP treatment. (B) Sypro Ruby-based protein detectionof 500 ng of the five-protein mixture with and without CIAP treatment.(C) VIPing-based phosphorylation detection of different amounts ofthe five-protein mixture separated by SDS-PAGE. (D) Sypro Ruby-basedprotein detection of different amounts of the five-protein mixtureseparated by SDS-PAGE.
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fig2: VIPing-baseddetection of phosphorylated proteins. Protein mixtureincludes three nonphosphorylated proteins (BSA, catalase, and β-lactoglobulin)and two phosphorylated proteins (ovalbumin and β-casein). (A)VIPing-based detection of 500 ng of the five-protein mixture withand without CIAP treatment. (B) Sypro Ruby-based protein detectionof 500 ng of the five-protein mixture with and without CIAP treatment.(C) VIPing-based phosphorylation detection of different amounts ofthe five-protein mixture separated by SDS-PAGE. (D) Sypro Ruby-basedprotein detection of different amounts of the five-protein mixtureseparated by SDS-PAGE.

Mentions: To initially investigate the ability of VIPingto selectively detectphosphorylated proteins in SDS-PAGE, we performed a gel staining ofa five-protein mixture consisting of two standard phosphorylated (β-caseinand ovalbumin) and three nonphosphorylated (BSA, catalase, and β-lactoglobulin)proteins. Five proteins of 500 ng each were mixed and separated bySDS-PAGE. Then the protein gel was incubated with the VIPing reagentand detected by a fluorescence imaging system. As shown in Figure 2A, only the two phosphoproteins (β-caseinand ovalbumin) were stained in the protein gel, indicating good selectivityof VIPing toward phosphoproteins. Also as expected, the signal fromβ-casein appeared much stronger than that from ovalbumin, dueto the relatively larger number of phosphorylated residues presentin β-casein. The signals were no longer detectable after theproteins were treated with calf intestine alkaline phosphatase (CIAP).For comparison, the proteins were also detected by Sypro Ruby (a fluorescentdye for protein gel staining) subsequently on the same gel, as shownin Figure 2B. To further demonstrate the selectivityand sensitivity of VIPing-based phosphoprotein detection, the samefive-protein mixture in different amounts, ranging from 7.85 ng to250 ng were run in SDS-PAGE. The gel staining results (Figure 2C) indicated high specificity and sensitivity, allowingthe detection of as low as 15 ng of β-casein. A comparison withSypro Ruby staining (Figure 2D) indicates thatVIPing has equivalent sensitivity to other fluorescence-based methods.


Specific visualization and identification of phosphoproteome in gels.

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

VIPing-baseddetection of phosphorylated proteins. Protein mixtureincludes three nonphosphorylated proteins (BSA, catalase, and β-lactoglobulin)and two phosphorylated proteins (ovalbumin and β-casein). (A)VIPing-based detection of 500 ng of the five-protein mixture withand without CIAP treatment. (B) Sypro Ruby-based protein detectionof 500 ng of the five-protein mixture with and without CIAP treatment.(C) VIPing-based phosphorylation detection of different amounts ofthe five-protein mixture separated by SDS-PAGE. (D) Sypro Ruby-basedprotein detection of different amounts of the five-protein mixtureseparated by SDS-PAGE.
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Related In: Results  -  Collection

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fig2: VIPing-baseddetection of phosphorylated proteins. Protein mixtureincludes three nonphosphorylated proteins (BSA, catalase, and β-lactoglobulin)and two phosphorylated proteins (ovalbumin and β-casein). (A)VIPing-based detection of 500 ng of the five-protein mixture withand without CIAP treatment. (B) Sypro Ruby-based protein detectionof 500 ng of the five-protein mixture with and without CIAP treatment.(C) VIPing-based phosphorylation detection of different amounts ofthe five-protein mixture separated by SDS-PAGE. (D) Sypro Ruby-basedprotein detection of different amounts of the five-protein mixtureseparated by SDS-PAGE.
Mentions: To initially investigate the ability of VIPingto selectively detectphosphorylated proteins in SDS-PAGE, we performed a gel staining ofa five-protein mixture consisting of two standard phosphorylated (β-caseinand ovalbumin) and three nonphosphorylated (BSA, catalase, and β-lactoglobulin)proteins. Five proteins of 500 ng each were mixed and separated bySDS-PAGE. Then the protein gel was incubated with the VIPing reagentand detected by a fluorescence imaging system. As shown in Figure 2A, only the two phosphoproteins (β-caseinand ovalbumin) were stained in the protein gel, indicating good selectivityof VIPing toward phosphoproteins. Also as expected, the signal fromβ-casein appeared much stronger than that from ovalbumin, dueto the relatively larger number of phosphorylated residues presentin β-casein. The signals were no longer detectable after theproteins were treated with calf intestine alkaline phosphatase (CIAP).For comparison, the proteins were also detected by Sypro Ruby (a fluorescentdye for protein gel staining) subsequently on the same gel, as shownin Figure 2B. To further demonstrate the selectivityand sensitivity of VIPing-based phosphoprotein detection, the samefive-protein mixture in different amounts, ranging from 7.85 ng to250 ng were run in SDS-PAGE. The gel staining results (Figure 2C) indicated high specificity and sensitivity, allowingthe detection of as low as 15 ng of β-casein. A comparison withSypro Ruby staining (Figure 2D) indicates thatVIPing has equivalent sensitivity to other fluorescence-based methods.

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