Detection of Protein Tyrosine Phosphorylation by Open Sandwich Fluoroimmunoassay
Detection of Protein Tyrosine Phosphorylation by Open Sandwich Fluoroimmunoassay
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DOI：
10.1021/bp060104z
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发表时间：
2006
影响因子：
2.9
通讯作者：
Yoshiyuki Sasajima;T. Aburatani;Kenzo Sakamoto;H. Ueda
中科院分区：
文献类型：
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作者：
Yoshiyuki Sasajima;T. Aburatani;Kenzo Sakamoto;H. Ueda
在蛋白质组学时代，高通量筛选蛋白质翻译后修饰状态，特别是蛋白质磷酸化被认为是至关重要的。然而，目前的蛋白质磷酸化检测方法要么依赖于蛋白质分解和质谱学的结合，要么依赖于耗时的免疫分析，这需要不可避免的洗涤过程。作为一种快速检测蛋白质磷酸化事件的方法，我们建议使用开放式夹心免疫分析法来检测抗体可变区(FV)的抗原依赖稳定性。以抗磷酸酪氨酸抗体PY20的重链可变区和轻链可变区(VH/VL)为模型系统对其性能进行了评价。当用噬菌体酶联免疫吸附试验首次估计VH/VL相互作用时，野生型Fv表现出适度的磷酸酪氨酸(PY)依赖的信号增加。然而，在筛选界面残基的突变体后，具有弱VH/VL相互作用的突变体(HQ39R)显示出显著改善(>；200%)的抗原依赖信号。利用该突变体制备了两种融合蛋白(VH-EYFP/VL-eCFP)，其中每个可变区片段都与GFP颜色变异体相连，以监测它们之间的PY诱导的荧光共振能量转移(FRET)。结果表明，在均相溶液中，PY或酪氨酸磷酸化多肽可显著增强FRET的活性，表明该方法适用于体外或原位以及可能的体内快速筛选酪氨酸磷酸化。
In the era of proteomics, high‐throughput screening of posttranslational modification states of proteins, especially protein phosphorylation, is considered of utmost importance. However, current protein phosphorylation detection methods depend on either the combination of proteolysis and mass spectrometry, or time‐consuming immunoassay that requires inevitable washing processes. As a way to rapidly assay protein phosphorylation events, here we propose the use of Open Sandwich immunoassay that detects antigen‐dependent stabilization of antibody variable region (Fv). As a model system, the heavy and light chain variable regions (VH/VL) of anti‐phosphotyrosine antibody PY20 were used to evaluate its performance. When VH/VL interaction was first estimated by phage ELISA, wild‐type Fv showed a modest phosphotyrosine (PY)‐dependent increase in signal. However, after screening of mutants at an interface residue, one with weak VH/VL interaction (HQ39R) showed markedly improved (>200%) antigen‐dependent signals. Using this mutant, two fusion proteins in which each variable region fragment was tethered to a GFP color variant were made (VH‐eYFP/VL‐eCFP) to monitor PY‐induced fluorescence resonance energy transfer (FRET) between them. The results showed significant PY‐ or tyrosine phosphorylated peptide‐induced enhancement in FRET in homogeneous solutions, indicating applicability of the method for rapid screening of tyrosine phosphorylation in vitro or in situ and possibly in vivo.