Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry
Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry
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通过放射性测定和基质辅助激光解吸电离飞行时间质谱法监测磷酸化肽
DOI：
10.1007/s00726-015-2025-y
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发表时间：
2015
期刊：
影响因子：
3.5
通讯作者：
M.
中科院分区：
文献类型：
--
作者：
Kang;J.H.;Asai;D.;Toita;R.;Kawano;T.;and Murata;M.
基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）经常用于监测磷酸化肽或蛋白激酶活性。然而，很少有报道将放射性测定与MALDI-TOF-MS分析进行比较。我们通过MALDI-TOF-MS分析了具有不同长度和数量的带负电荷的氨基酸的G蛋白偶联受体激酶2（GRK 2）的23种肽底物的磷酸化比率。然后，我们检查了通过MALDI-TOF-MS测定的磷酸化比率与使用放射性测定法测定的放射性水平（每分钟计数，CPM）之间的相关性。使用MALDI-TOF-MS，磷酸化比率在负模式下大于在正模式下。在负模式下测量的磷酸化比率与CPM强烈相关（r= 0.86）。酸性氨基酸的数量与GRK 2对肽底物的磷酸化有关（磷酸化率和CPM分别为r= 0.53和0.46）。这些结果表明，MALDI-TOF-MS是一种替代放射性测定监测磷酸化肽。
Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is frequently used to monitor phosphorylated peptides or protein kinase activities. However, few reports have compared a radioactivity assay with MALDI-TOF-MS analysis. We analyzed the phosphorylation ratios of 23 peptide substrates for G protein-coupled receptor kinase 2 (GRK2) with different lengths and numbers of negatively charged amino acids by MALDI-TOF-MS. We then examined the correlations between the phosphorylation ratios determined by MALDI-TOF-MS and the radioactivity levels (counts per minute, CPM) determined using a radioactive assay. Using MALDI-TOF-MS, the phosphorylation ratios were greater in the negative mode than in the positive mode. The phosphorylation ratio measured in the negative mode was strongly correlated with the CPM (r= 0.86). The number of acidic amino acids was related to the phosphorylation of peptide substrates by GRK2 (r= 0.53 and 0.46 for the phosphorylation ratio and CPM, respectively). These results suggest that MALDI-TOF-MS is an alternative to radioactive assays for monitoring phosphorylated peptides.