title: Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS1).
abstract: 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.
authors: Enzyme and Protein Chemistry, BioCentrum-DTU, Søltofts Plads Building 224, DK-2800 Kgs. Lyngby, Denmark.
authors: Mikael B
authors: MB
authors: Caspersen
authors: Jin-Long
authors: JL
authors: Qiu
authors: Xumin
authors: X
authors: Zhang
authors: Erik
authors: E
authors: Andreasson
authors: Henrik
authors: H
authors: Naested
authors: John
authors: J
authors: Mundy
authors: Birte
authors: B
authors: Svensson
mesh_terms: D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029681:Arabidopsis Proteins; C503586:MKS1 protein, Arabidopsis; D009687:Nuclear Proteins; D010750:Phosphoproteins; C436292:AtMPK4 protein, Arabidopsis; D020928:Mitogen-Activated Protein Kinases