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

Caspersen, Mikael B.; Qiu, Jin-Long; Zhang, Xumin; Andreasson, Erik LU ; Naested, Henrik; Mundy, John and Svensson, Birte (2007) In Biochimica et Biophysica Acta - Proteins and Proteomics 1774(9). p.1156-1163
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
mass spectrometry, phosphorylation site, protein, Arabidopsis, recombinant MKS1, MAP kinase substrate
in
Biochimica et Biophysica Acta - Proteins and Proteomics
volume
1774
issue
9
pages
1156 - 1163
publisher
Elsevier
external identifiers
  • wos:000249840000010
  • scopus:34548363771
ISSN
1570-9639
DOI
10.1016/j.bbapap.2007.07.002
language
English
LU publication?
yes
id
ca3496da-5e51-42a4-a44d-e6c26ff7dac7 (old id 656204)
date added to LUP
2007-12-10 09:00:33
date last changed
2017-01-01 07:18:35
@article{ca3496da-5e51-42a4-a44d-e6c26ff7dac7,
  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.},
  author       = {Caspersen, Mikael B. and Qiu, Jin-Long and Zhang, Xumin and Andreasson, Erik and Naested, Henrik and Mundy, John and Svensson, Birte},
  issn         = {1570-9639},
  keyword      = {mass spectrometry,phosphorylation site,protein,Arabidopsis,recombinant MKS1,MAP kinase substrate},
  language     = {eng},
  number       = {9},
  pages        = {1156--1163},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta - Proteins and Proteomics},
  title        = {Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS 1)},
  url          = {http://dx.doi.org/10.1016/j.bbapap.2007.07.002},
  volume       = {1774},
  year         = {2007},
}