title: Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.
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.
authors.affiliation: 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. jrjhkang@ncvc.go.jp.
authors.forename: Jeong-Hun
authors.initials: JH
authors.lastname: Kang
authors.affiliation: Department of Microbiology, St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki, 216-8511, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Asai
authors.affiliation: Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Riki
authors.initials: R
authors.lastname: Toita
authors.affiliation: Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Takahito
authors.initials: T
authors.lastname: Kawano
authors.affiliation: Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Murata
mesh_terms: D000818:Animals; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000737:chemistry; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D010761:Phosphorus Radioisotopes / Q000737:chemistry*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2
keywords: Counts per minute; MALDI-TOF-MS; Negative mode; Phosphorylation; Protein kinase; Radioactive assay