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Research article - Peer-reviewed, 2007

Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS 1)

Caspersen, Mikael B.; Qiu, Jin-Long; Zhang, Xumin; Andreasson, Erik; Naested, Henrik; Mundy, John; Svensson, Birte


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 TiO2 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 similar to 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, Set108, Ser120) in the phosphorylated form. (c) 2007 Elsevier B.V. All rights reserved.


Arabidopsis; MAP kinase substrate; recombinant MKS1; protein phosphorylation site; mass spectrometry

Published in

BBA - Proteins and Proteomics
2007, volume: 1774, number: 9, pages: 1156-1163

Authors' information

Caspersen, Mikael B.
Technical University of Denmark (DTU)
Qiu, Jin-Long
University of Copenhagen
Zhang, Xumin
University of Southern Denmark
Lund University
Næsted, Henrik
Technical University of Denmark (DTU)
Mundy, John
University of Copenhagen
Svensson, Birte
University of Copenhagen

UKÄ Subject classification

Biochemistry and Molecular Biology

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