Identification of phosphorylation site of Npas4 by LC-MS/MS

Published: 25 Oct 2019 | Version 2 | DOI: 10.17632/3f5p2p7hbs.2
Contributor(s):

Description of this data

To identify the phosphorylation sites of Npas4, GST-Npas4-390-489 aa, GST-Npas4 490-597 aa or GST-Npas4 598-701 aa was expressed in COS7 cells with or without the coexpression of MAP2K1-CA. Cells were lysed in lysis buffer [20 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (PhosStop, Roche), pH 7.5], and sonicated 3 times for 5 sec. After centrifugation at 16,000×g at 4°C for 10 min, the soluble supernatant was incubated in 30 µl of glutathione-Sepharose 4B beads (GE Healthcare) for 1 hr at 4°C with rotation. The beads were then washed three times with lysis buffer and an additional three times with wash buffer (20 mM Tris/HCl, 1 mM EDTA, and 150 mM NaCl, pH 7.5). The bound proteins were extracted from the beads using urea solution, reduced via incubation in 5 mM dithiothreitol for 30 min, and alkylated using 10 mM iodoacetamide for 1 h in the dark. The proteins were digested with Trypsin/Lys-C (Promega) or Glu-C (Promega)/Asp-N (FUJIFILM Wako). Demineralization was performed using SPE c-tips according to the manufacturer’s instructions. The peptides were analyzed by LC−MS using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc).
Dual-phosphorylated peptide DLVCTPPYTPHQPGGCAFLFSLHEPFQTHLPPPSSSLQE, containing T423 and T427 phosphorylation sites, was identified from digested fragments of GST-Npas4-390-489 aa; LPPSPSSPGNGDCTLLALAQLR, containing S577 and S580 phosphorylation sites, was identified from digested fragments of GST-Npas4 490-597 aa; and GLLTPEASPVKQSFFHYTEKE, containing T611 and S615 phosphorylation sites, was identified from digested fragments of GST-Npas4 598-701 aa. Selected ion monitoring (SIM) analysis revealed that the amount of these dual-phosphorylation was significantly increased by coexpression with MAP2K1-CA. These results suggest that Npas4 is phosphorylated by MAPK at T423, T427, S577, S580, T611 and S615 sites.

Experiment data files

Latest version

  • Version 2

    2019-10-25

    Published: 2019-10-25

    DOI: 10.17632/3f5p2p7hbs.2

    Cite this dataset

    Funahashi, Yasuhiro (2019), “ Identification of phosphorylation site of Npas4 by LC-MS/MS”, Mendeley Data, v2 http://dx.doi.org/10.17632/3f5p2p7hbs.2

Statistics

Views: 43
Downloads: 11

Previous versions

Compare to version

Categories

Tandem Mass Spectrometry

Licence

CC BY 4.0 Learn more

The files associated with this dataset are licensed under a Creative Commons Attribution 4.0 International licence.

What does this mean?
You can share, copy and modify this dataset so long as you give appropriate credit, provide a link to the CC BY license, and indicate if changes were made, but you may not do so in a way that suggests the rights holder has endorsed you or your use of the dataset. Note that further permission may be required for any content within the dataset that is identified as belonging to a third party.

Report