Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS 1)
(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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https://lup.lub.lu.se/record/656204
- author
- Caspersen, Mikael B. ; Qiu, Jin-Long ; Zhang, Xumin ; Andreasson, Erik LU ; Naested, Henrik ; Mundy, John and Svensson, Birte
- organization
- publishing date
- 2007
- 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
- additional info
- 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)
- id
- ca3496da-5e51-42a4-a44d-e6c26ff7dac7 (old id 656204)
- date added to LUP
- 2016-04-01 16:54:33
- date last changed
- 2022-02-13 01:27:29
@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}},
keywords = {{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}},
doi = {{10.1016/j.bbapap.2007.07.002}},
volume = {{1774}},
year = {{2007}},
}