TY - JOUR
T1 - Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS1).
AU - Caspersen, Mikael B
AU - Qiu, Jin-Long
AU - Zhang, Xumin
AU - Andreasson, Erik
AU - Naested, Henrik
AU - Mundy, John
AU - Svensson, Birte
N1 - Keywords: Arabidopsis Proteins; Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphorylation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PY - 2007
Y1 - 2007
N2 - The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophoresis and gel-staining with ProQ Diamond and the protein was digested by either trypsin or chymotrypsin for maximum sequence coverage to facilitate identification of phosphorylated positions. Prior to analysis by mass spectrometry, samples were either desalted, passed over TiO(2) or both for improved phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine residues (Ser72, Ser108, Ser120) in the phosphorylated form.
AB - The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophoresis and gel-staining with ProQ Diamond and the protein was digested by either trypsin or chymotrypsin for maximum sequence coverage to facilitate identification of phosphorylated positions. Prior to analysis by mass spectrometry, samples were either desalted, passed over TiO(2) or both for improved phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine residues (Ser72, Ser108, Ser120) in the phosphorylated form.
U2 - 10.1016/j.bbapap.2007.07.002
DO - 10.1016/j.bbapap.2007.07.002
M3 - Journal article
C2 - 17702678
SN - 0304-4165
VL - 1774
SP - 1156
EP - 1163
JO - BBA General Subjects
JF - BBA General Subjects
IS - 9
ER -