Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.
Rapid Commun Mass Spectrom. 2010 Aug 15;24(15):2277-82. doi: 10.1002/rcm.4627.
We have developed a novel strategy to improve the efficiency of identification of multiply phosphorylated peptides isolated by hydroxy acid modified metal oxide chromatography (HAMMOC). This strategy consists of alkali-induced chemical dephosphorylation (beta-elimination reaction) of phosphopeptides isolated by HAMMOC prior to analysis by liquid chromatography/mass spectrometry (LC/MS). This approach identified 1.9-fold more multiply phosphorylated peptides than the conventional approach without beta-elimination from a digested mixture of three standard phosphoproteins. In addition, the accuracy of phosphorylation site determination in synthetic phosphopeptides was significantly improved. Finally, we applied this approach to a cell lysate. By combining this dephosphorylation approach with the conventional approach, we successfully identified 1649 unique phosphopeptides, including 325 multiply phosphorylated phosphopeptides, from 200 microg of cultured Arabidopsis cells. These results indicate that chemical dephosphorylation prior to LC/MS analysis increases the efficiency of identification of multiply phosphorylated peptides, as well as the accuracy of phosphorylation site determination.
我们开发了一种新的策略，以提高通过羟基酸修饰的金属氧化物色谱（HAMMOC）分离的多磷酸化肽的鉴定效率。该策略包括在通过液相色谱/质谱（LC/MS）分析之前，对通过 HAMMOC 分离的磷酸肽进行碱诱导的化学去磷酸化（β-消除反应）。与没有β-消除的传统方法相比，该方法从三种标准磷酸化蛋白的消化混合物中鉴定出多磷酸化肽的数量增加了 1.9 倍。此外，合成磷酸肽中磷酸化位点测定的准确性也得到了显著提高。最后，我们将这种方法应用于细胞裂解物。通过将这种去磷酸化方法与传统方法相结合，我们成功地从 200μg 的培养拟南芥细胞中鉴定出 1649 种独特的磷酸肽，包括 325 种多磷酸化磷酸肽。这些结果表明，在 LC/MS 分析之前进行化学去磷酸化可提高多磷酸化肽的鉴定效率以及磷酸化位点测定的准确性。
