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Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

Guo L, Xiao Y, Wang Y - Anal. Chem. (2014)

Bottom Line: Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis.Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant.The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

View Article: PubMed Central - PubMed

Affiliation: Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , Riverside, California 92521-0403, United States.

ABSTRACT
Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

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Targeted human kinases mapped in the dendrogram of the SILAC-compatiblekinome library (A) and a general workflow of SILAC-based multiplereaction monitoring (MRM) analysis for global kinome profiling usingthe ATP affinity probe (B).
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fig1: Targeted human kinases mapped in the dendrogram of the SILAC-compatiblekinome library (A) and a general workflow of SILAC-based multiplereaction monitoring (MRM) analysis for global kinome profiling usingthe ATP affinity probe (B).

Mentions: Recently, we constructed a human MRMkinome library on the basis of large-scale shotgun proteomic analysisof kinases enriched by desthiobiotin-based isotope-coded ATP affinityprobes from the whole cell lysates of six different cell lines (K562,IMR-90, HeLa-S3, Jurkat-T, WM-115, and WM-266-4).13 The acquired tandem mass spectra and retention time informationon kinase-derived peptides with desthiobiotin labeling were then processedusing Skyline for the MRM library construction. However, the use ofisotope-coded ATP affinity probes in the previous study required customizedsynthesis, which may limit its general application in analytical laboratories.To accommodate for a more widely used quantitative approach usingSILAC, we reconstructed the human MRM kinome library by setting upvariable modifications with mass shifts introduced by heavy isotopelabeling with lysine (+8 Da) and arginine (+6 Da). The current SILAC-compatibleMRM kinome library consists of 395 peptides, corresponding to 285kinases, which allows for in-depth kinome profiling that covers allthe seven major human kinase families (Figure 1A).


Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

Guo L, Xiao Y, Wang Y - Anal. Chem. (2014)

Targeted human kinases mapped in the dendrogram of the SILAC-compatiblekinome library (A) and a general workflow of SILAC-based multiplereaction monitoring (MRM) analysis for global kinome profiling usingthe ATP affinity probe (B).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222629&req=5

fig1: Targeted human kinases mapped in the dendrogram of the SILAC-compatiblekinome library (A) and a general workflow of SILAC-based multiplereaction monitoring (MRM) analysis for global kinome profiling usingthe ATP affinity probe (B).
Mentions: Recently, we constructed a human MRMkinome library on the basis of large-scale shotgun proteomic analysisof kinases enriched by desthiobiotin-based isotope-coded ATP affinityprobes from the whole cell lysates of six different cell lines (K562,IMR-90, HeLa-S3, Jurkat-T, WM-115, and WM-266-4).13 The acquired tandem mass spectra and retention time informationon kinase-derived peptides with desthiobiotin labeling were then processedusing Skyline for the MRM library construction. However, the use ofisotope-coded ATP affinity probes in the previous study required customizedsynthesis, which may limit its general application in analytical laboratories.To accommodate for a more widely used quantitative approach usingSILAC, we reconstructed the human MRM kinome library by setting upvariable modifications with mass shifts introduced by heavy isotopelabeling with lysine (+8 Da) and arginine (+6 Da). The current SILAC-compatibleMRM kinome library consists of 395 peptides, corresponding to 285kinases, which allows for in-depth kinome profiling that covers allthe seven major human kinase families (Figure 1A).

Bottom Line: Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis.Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant.The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

View Article: PubMed Central - PubMed

Affiliation: Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , Riverside, California 92521-0403, United States.

ABSTRACT
Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Show MeSH
Related in: MedlinePlus