Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.
Phosphoproteome profiling using a fluorescent phosphosensor dye in two-dimensional polyacrylamide gel electrophoresis.
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在二维聚丙烯酰胺凝胶电泳中使用荧光磷酸传感器染料进行磷酸化蛋白质组分析。
DOI：
10.1007/s12010-011-9175-5
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发表时间：
2011
期刊：
影响因子：
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et al.
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作者：
Otani M;Taniguchi T;Sakai A;et al.
我们验证了新型PhosphoQUANTI SolidBlue Complex（PQSC）染料在聚丙烯酰胺和二维凝胶电泳（PAGE和2DE，分别）中用于磷酸化蛋白质的灵敏荧光检测。PQSC可以检测到低至15.6 ng的β-酪蛋白（一种五磷酸化蛋白）和61.3 ng的卵清蛋白（一种二磷酸化蛋白）。荧光强度与蛋白质上磷酸化残基的数量相关。为了证明PQSC对磷蛋白的特异性，在2DE凝胶中分离Swiss 3 T3细胞的酶促去磷酸化裂解物并通过PQSC染色。这些凝胶中的荧光信号在去磷酸化后显著降低。当使用磷蛋白富集柱浓缩Swiss 3 T3细胞裂解物中的磷酸化蛋白质时，大多数磷蛋白显示pI 4-5范围内的荧光信号。最后，我们进行了磷酸化蛋白质组分析，研究在增殖和静止瑞士3 T3细胞的蛋白质磷酸化概况的差异。检测到超过135个可辨别的蛋白质点，从其中选择的15个点进行了鉴定，通过基质辅助激光解吸/电离飞行时间质谱（MALDI TOF-MS）。用于磷蛋白检测的PQSC染色程序简单、可逆，并且与MALDI TOF-MS完全兼容。
We validated the novel PhosphoQUANTI SolidBlue Complex (PQSC) dye for the sensitive fluorescent detection of phosphorylated proteins in polyacrylamide- and two-dimensional gel electrophoresis (PAGE and 2DE, respectively). PQSC can detect as little as 15.6 ng of ß-casein, a pentaphosphorylated protein, and 61.3 ng of ovalbumin, a diphosphorylated protein. Fluorescence intensity correlates with the number of phosphorylated residues on the protein. To demonstrate the specificity of PQSC for phosphoproteins, enzymatically dephosphorylated lysates of Swiss 3T3 cells were separated in 2DE gels and stained by PQSC. The fluorescence signals in these gels were markedly reduced following dephosphorylation. When the phosphorylated proteins in Swiss 3T3 cell lysates were concentrated using a phosphoprotein enrichment column, the majority of phosphoproteins showed fluorescence signals in the pI 4–5 range. Finally, we performed phosphoproteome analysis to study differences in the protein phosphorylation profiles of proliferating and quiescent Swiss 3T3 cells. Over 135 discernible protein spots were detected, from which a selection of 15 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The PQSC staining procedure for phosphoprotein detection is simple, reversible, and fully compatible with MALDI TOF-MS.