Determination of protein phosphorylation by extracellular signal-regulated kinase using capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Extracellular signal-regulated kinase (ERK) is a key regulatory enzyme mediating cell responses to mitogenic stimulation and is one of the key components in linking growth factor receptor activation to serine/threonine protein phosphorylation processes. Phosphorylation reaction by ERK plays an important role in many signal transduction pathways. ERK phosphorylates numerous substrates such as MBP, microtubule-associated protein 2 (MAP2) and nuclear protein. In particular, MBP is a substrate commonly employed for the detection of ERK activity and contains the consensus primary sequence PRT97P. In this paper, we compared the degree of the phosphorylation reaction of MBP substrate peptides by ERK with the three different MBP substrate peptides, MBP1(KNIVTPRTPPPSQGK), MBP2(VPRTPGGRR) and MBP3(APRTPGGRR) in order to select an efficient substrate peptide for phosphorylation reaction by ERK. The results showed that the MBP3 peptide is the most efficient substrate for phosphorylation reaction by ERK. Using MBP3 peptide, the phosphorylation reaction of MBP by ERK was monitored with both matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis (CE). Our results demonstrate the feasibility of the CE method, the method being a simple and reliable technique in determining and characterizing various kinds of enzyme reaction especially including kinase enzymes.
Nam
Hui-Sun
HS
Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, South Korea.
Ban
Eunmi
E
Yoo
Eunah
E
Yoo
Young Sook
YS
D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011506:Proteins; D020928:Mitogen-Activated Protein Kinases