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

Research output: Contribution to journalArticle

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 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.

Details

Authors
  • Mikael B. Caspersen
  • Jin-Long Qiu
  • Xumin Zhang
  • Erik Andreasson
  • Henrik Naested
  • John Mundy
  • Birte Svensson
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biological Sciences

Keywords

  • mass spectrometry, phosphorylation site, protein, Arabidopsis, recombinant MKS1, MAP kinase substrate
Original languageEnglish
Pages (from-to)1156-1163
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1774
Issue number9
Publication statusPublished - 2007
Publication categoryResearch
Peer-reviewedYes

Bibliographic note

The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Department of Cell and Organism Biology (Closed 2011.) (011002100)