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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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(A) Structure of VIPingreagent. VIPing consists of a titaniumion for selective binding to phosphate groups, a TAMRA for fluorescence-baseddetection and a biotin group for phosphopeptide purification via solid-phasecapture. (B) Experimental workflow for VIPing strategy. After proteinsare separated by SDS-PAGE, phosphorylated proteins are stained withVIPing reagent and then visualized with a fluorescence imager (Ex/Em= 532 nm/580 nm). The phosphorylated proteins of interest are excisedin the gel and digested with trypsin. The phosphorylated tryptic peptidesbound to the VIPing reagent are isolated via streptavidin beads followedby mass spectrometric analysis to identify the proteins and theirphosphorylation sites.
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fig1: (A) Structure of VIPingreagent. VIPing consists of a titaniumion for selective binding to phosphate groups, a TAMRA for fluorescence-baseddetection and a biotin group for phosphopeptide purification via solid-phasecapture. (B) Experimental workflow for VIPing strategy. After proteinsare separated by SDS-PAGE, phosphorylated proteins are stained withVIPing reagent and then visualized with a fluorescence imager (Ex/Em= 532 nm/580 nm). The phosphorylated proteins of interest are excisedin the gel and digested with trypsin. The phosphorylated tryptic peptidesbound to the VIPing reagent are isolated via streptavidin beads followedby mass spectrometric analysis to identify the proteins and theirphosphorylation sites.

Mentions: We devised a gel-compatiblereagent, VIPing, which incorporates three independent functionalities:a titanium ion for highly selective binding to phosphate group, afluorophore such as TAMRA for fluorescence-based detection, and abiotin group for phosphopeptide purification via solid-phase capture(Figure 1A). The VIPing reagent was preparedthrough solid-phase syntheses. Briefly, two lysine building blocks,Fmoc-Lys(Dde)–OH and Fmoc-Lys(Biotin)–OH were conjugatedwith rink beads consequently to form a compound with one biotin groupand two orthogonal protected amine groups. After deprotection, theamine groups were reacted with TAMRA and i-Pro protectedphosphonate groups separately. The product was cleaved from resinby acid cleaving, followed by removal of i-Pro groupswith TMSBr. In the last step, the compound was functionalized withTiOCl2 to generate the VIPing reagent.


Specific visualization and identification of phosphoproteome in gels.

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

(A) Structure of VIPingreagent. VIPing consists of a titaniumion for selective binding to phosphate groups, a TAMRA for fluorescence-baseddetection and a biotin group for phosphopeptide purification via solid-phasecapture. (B) Experimental workflow for VIPing strategy. After proteinsare separated by SDS-PAGE, phosphorylated proteins are stained withVIPing reagent and then visualized with a fluorescence imager (Ex/Em= 532 nm/580 nm). The phosphorylated proteins of interest are excisedin the gel and digested with trypsin. The phosphorylated tryptic peptidesbound to the VIPing reagent are isolated via streptavidin beads followedby mass spectrometric analysis to identify the proteins and theirphosphorylation sites.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: (A) Structure of VIPingreagent. VIPing consists of a titaniumion for selective binding to phosphate groups, a TAMRA for fluorescence-baseddetection and a biotin group for phosphopeptide purification via solid-phasecapture. (B) Experimental workflow for VIPing strategy. After proteinsare separated by SDS-PAGE, phosphorylated proteins are stained withVIPing reagent and then visualized with a fluorescence imager (Ex/Em= 532 nm/580 nm). The phosphorylated proteins of interest are excisedin the gel and digested with trypsin. The phosphorylated tryptic peptidesbound to the VIPing reagent are isolated via streptavidin beads followedby mass spectrometric analysis to identify the proteins and theirphosphorylation sites.
Mentions: We devised a gel-compatiblereagent, VIPing, which incorporates three independent functionalities:a titanium ion for highly selective binding to phosphate group, afluorophore such as TAMRA for fluorescence-based detection, and abiotin group for phosphopeptide purification via solid-phase capture(Figure 1A). The VIPing reagent was preparedthrough solid-phase syntheses. Briefly, two lysine building blocks,Fmoc-Lys(Dde)–OH and Fmoc-Lys(Biotin)–OH were conjugatedwith rink beads consequently to form a compound with one biotin groupand two orthogonal protected amine groups. After deprotection, theamine groups were reacted with TAMRA and i-Pro protectedphosphonate groups separately. The product was cleaved from resinby acid cleaving, followed by removal of i-Pro groupswith TMSBr. In the last step, the compound was functionalized withTiOCl2 to generate the VIPing reagent.

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