PXD039092

PXD039092 is an original dataset announced via ProteomeXchange.

Title	Immunoprecipitation followed by PRM to detect endogenous interaction between VGLL3 and EWSR1
Description	For immunoprecipitation to detect the endogenous interaction between VGLL3 and EWSR1, cardiac myofibroblasts were fixed with 0.1% PFA and lysed in IP buffer [20 mM HEPES­NaOH (pH 7.5)/1 mM EGTA/1 mM MgCl2/150 mM NaCl /5% glycerol/1% NP­40] containing protease inhibitors (1:100) and phosphatase inhibitors (1:100) at 4 °C for 20 min. The supernatants of cell lysates were incubated for 2 h at 4 °C with SureBeads Protein G (Bio-Rad) coupled with the custom-made rabbit polyclonal anti-VGLL3 antibody, raised against the amino acid sequence of mouse VGLL3 from position 307 to 325. The beads were then washed four times with IP buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested with 200 ng of Trypsin/Lysyl Endopeptidase mixture, and the digested peptides were reduced, alkylated, acidified, desalted, and dissolved in 0.1% trifluoroacetic acid and 3% ACN. To quantify VGLL3 and EWSR1, four peptides of VGLL3 and five peptides of EWSR1 were measured by PRM, an MS/MS-based targeted quantification method using high-resolution MS. Targeted MS/MS scans were acquired by a time-scheduled inclusion list at a resolution of 70,000, an AGC target of 2 × 105, an isolation window of 2.0 m/z, a maximum injection time of 1 s, and a normalised collision energy of 27. Time alignment and relative quantification of transitions were performed using Skyline software.
HostingRepository	jPOST
AnnounceDate	2022-12-24
AnnouncementXML	Submission_2023-02-21_06:50:34.255.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	L-methionine sulfoxide; alpha-amino acetylated residue; S-carboxamidomethyl-L-cysteine
Instrument	instrument

Revision	Datetime	Status	ChangeLog Entry
0	2022-12-23 11:41:22	ID requested
1	2022-12-23 18:05:37	announced
⏵ 2	2023-02-21 06:50:35	announced	2023-02-21: Updated PubMed.

Horii Y, Matsuda S, Toyota C, Morinaga T, Nakaya T, Tsuchiya S, Ohmuraya M, Hironaka T, Yoshiki R, Kasai K, Yamauchi Y, Takizawa N, Nagasaka A, Tanaka A, Kosako H, Nakaya M, VGLL3 is a mechanosensitive protein that promotes cardiac fibrosis through liquid-liquid phase separation. Nat Commun, 14(1):550(2023) [pubmed]

submitter keyword: IP-PRM, VGLL3, EWSR1

Hidetaka Kosako
lab head
Hidetaka Kosako
contact affiliation	Tokushima University
dataset submitter

jPOST dataset URI

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