JMSSJ On-line

JMSSJ On-line, Vol. 51 (2003) No. 1, pp. 324-329

Study of Phosphoprotein and Phosphopeptide by Mass Spectrometry		>> Full Text PDF >> References
Tomoya KINUMI^* ^Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology (AIST) (1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan)*

Protein phosphorylation at serine, threonine and/or tyrosine residue plays a crucial role in cell signaling. Many different classes of protein kinases that are responsible for this reaction have been characterized. A conventional protocol utilizes [³²P-γ] ATP as a radioactive donor and evaluates the radioactivity incorporated into the substrate. I am interested in the structural analysis of phosphopeptides and in an assay system for protein phosphorylation using mass spectrometry which allow a quick and accurate analysis without using radioisotopes. Several methodologies have been developed to determine the phosphorylation sites and kinase activity by using mass spectrometry. In this paper, I present three applications for phosphoprotein and phosphopeptide analysis using mass spectrometry as follows, 1) the determination of in vivo phosphorylation site using a streamed line combination of two-dimensional gel electrophoresis and LC-MS/MS, 2) the structure analysis of long chain phosphopeptide by in-source decay using MALDI-TOF/MS, 3) kinase assay using biotinylated peptide substrate by MALDI-TOF/MS.

Key words: Protein, Peptide, Phosphorylation