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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-based detection of endogenous phosphorylated proteins from E. coli whole cell extract. Lane 1, 25 μg of E. coli lysate protein; Lane 2, 25 μg of E.coli lysate protein with CIAP dephosphorylation treatment;Lane 3, 25 μg of E. coli lysate protein withCIAP dephosphorylation treatment and with 500 ng of the five-proteinmixture spiked in. (A) VIPing-based detection of endogenous phosphorylatedproteins from E. coli. (B) Sypro Ruby detection of E. coli lysate proteins with or without CIAP treatment.
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fig3: VIPing-based detection of endogenous phosphorylated proteins from E. coli whole cell extract. Lane 1, 25 μg of E. coli lysate protein; Lane 2, 25 μg of E.coli lysate protein with CIAP dephosphorylation treatment;Lane 3, 25 μg of E. coli lysate protein withCIAP dephosphorylation treatment and with 500 ng of the five-proteinmixture spiked in. (A) VIPing-based detection of endogenous phosphorylatedproteins from E. coli. (B) Sypro Ruby detection of E. coli lysate proteins with or without CIAP treatment.

Mentions: Next step, we explored the ability of VIPing to detect endogenousphosphorylated proteins in a whole cell extract of E. coli BL21 strain (Figure 3). The whole cell extractwas also treated with CIAP. The CIAP-treated samples were equallydivided and one was spiked with standard phosphoproteins, β-caseinand ovalbumin. As shown in Figure 3A, VIPingwas able to stain phosphoproteins in E. coli cellextract (Lane 1). The signals were due to protein phosphorylationsince all signals disappeared when cell extract was treated with CIAP(Lane 2). Moreover, phosphoproteins β-casein and ovalbumin wereclearly detected when spiking the five-protein mixture into CIAP-treated E. coli cell extract (Lane 3). Protein gel staining withSypro Ruby indicates equal cell extract loading (Figure 3B).


Specific visualization and identification of phosphoproteome in gels.

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

VIPing-based detection of endogenous phosphorylated proteins from E. coli whole cell extract. Lane 1, 25 μg of E. coli lysate protein; Lane 2, 25 μg of E.coli lysate protein with CIAP dephosphorylation treatment;Lane 3, 25 μg of E. coli lysate protein withCIAP dephosphorylation treatment and with 500 ng of the five-proteinmixture spiked in. (A) VIPing-based detection of endogenous phosphorylatedproteins from E. coli. (B) Sypro Ruby detection of E. coli lysate proteins with or without CIAP treatment.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: VIPing-based detection of endogenous phosphorylated proteins from E. coli whole cell extract. Lane 1, 25 μg of E. coli lysate protein; Lane 2, 25 μg of E.coli lysate protein with CIAP dephosphorylation treatment;Lane 3, 25 μg of E. coli lysate protein withCIAP dephosphorylation treatment and with 500 ng of the five-proteinmixture spiked in. (A) VIPing-based detection of endogenous phosphorylatedproteins from E. coli. (B) Sypro Ruby detection of E. coli lysate proteins with or without CIAP treatment.
Mentions: Next step, we explored the ability of VIPing to detect endogenousphosphorylated proteins in a whole cell extract of E. coli BL21 strain (Figure 3). The whole cell extractwas also treated with CIAP. The CIAP-treated samples were equallydivided and one was spiked with standard phosphoproteins, β-caseinand ovalbumin. As shown in Figure 3A, VIPingwas able to stain phosphoproteins in E. coli cellextract (Lane 1). The signals were due to protein phosphorylationsince all signals disappeared when cell extract was treated with CIAP(Lane 2). Moreover, phosphoproteins β-casein and ovalbumin wereclearly detected when spiking the five-protein mixture into CIAP-treated E. coli cell extract (Lane 3). Protein gel staining withSypro Ruby indicates equal cell extract loading (Figure 3B).

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