TY - JOUR
T1 - Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry
AU - Kang, Jeong Hun
AU - Asai, Daisuke
AU - Toita, Riki
AU - Kawano, Takahito
AU - Murata, Masaharu
N1 - Funding Information:
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.
Publisher Copyright:
© 2015 Springer-Verlag Wien.
PY - 2015/11/1
Y1 - 2015/11/1
N2 - 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.
AB - 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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U2 - 10.1007/s00726-015-2025-y
DO - 10.1007/s00726-015-2025-y
M3 - Article
C2 - 26077716
AN - SCOPUS:84945464968
SN - 0939-4451
VL - 47
SP - 2377
EP - 2383
JO - Amino Acids
JF - Amino Acids
IS - 11
ER -