Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

作者：Kang Jeong Hun^*; Asai Daisuke; Toita Riki; Kawano Takahito; Murata Masaharu

来源：Amino Acids, 2015, 47(11): 2377-2383.

DOI：10.1007/s00726-015-2025-y

• MALDI-TOF-MS
• Negative mode
• Phosphorylation
• Protein kinase
• Counts per minute
• Radioactive assay

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.

• 出版日期2015-11