Determination of protein phosphorylation by extracellular signalregulated kinase using capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

被引:14
作者
Nam, HS
Ban, E
Yoo, E
Yoo, YS
机构
[1] Korea Inst Sci & Technol, Bioanal & Biotransformat Res Ctr, Seoul 130650, South Korea
[2] Sungshin Womens Univ, Dept Chem, Sungbuk Gu, Seoul 136742, South Korea
关键词
enzymes; proteins; extracellular signal-regulated kinase;
D O I
10.1016/S0021-9673(02)01148-2
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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, micrombule-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. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:79 / 85
页数:7
相关论文
共 20 条
[1]   Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry [J].
Annan, RS ;
Carr, SA .
ANALYTICAL CHEMISTRY, 1996, 68 (19) :3413-3421
[2]   Determination of minute enzymatic activities by means of capillary electrophoretic techniques [J].
Bao, JJ ;
Fujima, JM ;
Danielson, ND .
JOURNAL OF CHROMATOGRAPHY B, 1997, 699 (1-2) :481-497
[3]   DESORPTION IONIZATION MASS-SPECTROMETRY [J].
BUSCH, KL .
JOURNAL OF MASS SPECTROMETRY, 1995, 30 (02) :233-240
[4]   Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry [J].
Chen, J ;
Qi, Y ;
Zhao, RX ;
Zhou, GW ;
Zhao, ZHJ .
ANALYTICAL BIOCHEMISTRY, 2001, 292 (01) :51-58
[5]   Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis [J].
Choi, S ;
Lee, YS ;
Na, DS ;
Yoo, YS .
JOURNAL OF CHROMATOGRAPHY A, 1999, 853 (1-2) :285-293
[6]   MONITORING PROTEIN-KINASE AND PHOSPHATASE REACTIONS WITH MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY AND CAPILLARY ZONE ELECTROPHORESIS - COMPARISON OF THE DETECTION EFFICIENCY OF PEPTIDE-PHOSPHOPEPTIDE MIXTURES [J].
CRAIG, AG ;
HOEGER, CA ;
MILLER, CL ;
GOEDKEN, T ;
RIVIER, JE ;
FISCHER, WH .
BIOLOGICAL MASS SPECTROMETRY, 1994, 23 (08) :519-528
[7]   Capillary electrophoresis of phosphoamino acids with indirect photometric detection [J].
Crowder, MW ;
Numan, AQ ;
Haddadian, F ;
Weitzel, MA ;
Danielson, ND .
ANALYTICA CHIMICA ACTA, 1999, 384 (02) :127-133
[8]   A CAPILLARY ELECTROPHORESIS-BASED ASSAY FOR PROTEIN-KINASES AND PROTEIN PHOSPHATASES USING PEPTIDE-SUBSTRATES [J].
DAWSON, JF ;
BOLAND, MP ;
HOLMES, CFB .
ANALYTICAL BIOCHEMISTRY, 1994, 220 (02) :340-345
[9]   Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases [J].
Gamble, TN ;
Ramachandran, C ;
Bateman, KP .
ANALYTICAL CHEMISTRY, 1999, 71 (16) :3469-3476
[10]   Investigation of enzyme kinetics using quench-flow techniques with MALDI TOF mass spectrometry [J].
Houston, CT ;
Taylor, WP ;
Widlanski, TS ;
Reilly, JP .
ANALYTICAL CHEMISTRY, 2000, 72 (14) :3311-3319