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

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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.

Conflict of interest

The authors declare that they have no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Authors and Affiliations

  1. Division of Biopharmaceutics and Pharmacokinetics, Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka, 565-8565, Japan

    Jeong-Hun Kang

  2. Department of Microbiology, St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki, 216-8511, Japan

    Daisuke Asai

  3. Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Riki Toita

  4. Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Takahito Kawano & Masaharu Murata

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  1. Jeong-Hun Kang
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Correspondence to Jeong-Hun Kang.

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Handling Editor: K. L. Bennett.

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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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