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

Jeong Hun Kang, Daisuke Asai, Riki Toita, Takahito Kawano, Masaharu Murata

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2377-2383
Number of pages7
JournalAmino Acids
Volume47
Issue number11
DOIs
Publication statusPublished - Nov 1 2015

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry
  • Organic Chemistry

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