Abstract
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.
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Acknowledgments
This work was supported by a Health Labour Sciences Research Grant (Research on Publicly Essential Drugs and Medical Devices) from the Ministry of Health, Labour and Welfare of Japan, and a grant-in-aid for Scientific Research (A) (KAKENHI Grant Number 24245015) and (B) (KAKENHI Grant Number 23310085) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
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Kang, JH., Asai, D., Toita, R. et al. Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Amino Acids 47, 2377–2383 (2015). https://doi.org/10.1007/s00726-015-2025-y
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DOI: https://doi.org/10.1007/s00726-015-2025-y