PXD035220

PXD035220 is an original dataset announced via ProteomeXchange.

Title	Comparison of Biotinylated Peptides Enriched by the Original and Optimized Workflows using Tamavidin 2-REV.
Description	To evaluate the optimized workflow, biotinylated peptides enriched by the original and optimized workflows from 300 µg proteins of RAW264.7 cells expressing TurboID-STING were compared by LC-MS/MS analysis. We assessed reproducibility of each workflow using six technical replicates. Original workflow: A 15-µL slurry of MagCapture HP Tamavidin 2-REV magnetic beads per sample was washed three times with TBS2 (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). The digested peptides containing 0.1% RapiGest SF were diluted 5-fold with TBS2 and incubated with the Tamavidin 2-REV beads in the presence of 1 mg/mL Pefabloc SC for 3 h at 4 °C. After washing five times with TBS2, biotinylated peptides were eluted with 100 μL of 1 mM biotin in TBS2 for 15 min at 95 °C twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% acetonitrile (ACN). Optimized workflow: A 15-µL slurry of the Tamavidin 2-REV beads per sample was washed twice with 10% ACN and then three times with TBS2. The digested peptides in diluted PTS buffer were heated at 95 °C for 10 min to inactivate trypsin, diluted 2-fold with TBS2, and incubated with the ACN-prewashed Tamavidin 2-REV beads for 3 h at 4 °C. During the incubation period, 1 mM biotin solution in TBS3 (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl) was passed through GL-Tip SDB. After washing five times with TBS2, the beads were incubated with 200 μL of TBS3 for 15 min at 37 °C as a mock elution. After removing TBS3, biotinylated peptides were eluted for 15 min at 37 °C twice with 100 µL of the biotin solution that had been passed through GL-Tip SDB. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.
HostingRepository	jPOST
AnnounceDate	2022-08-25
AnnouncementXML	Submission_2022-09-18_03:26:10.947.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Kohei Nishino
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; N6-biotinyl-L-lysine; L-methionine sulfoxide; alpha-amino acetylated residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2022-07-11 07:41:58	ID requested
1	2022-08-24 08:00:13	announced
⏵ 2	2022-09-18 03:26:11	announced	2022-09-18: Updated FTP location.

Nishino K, Yoshikawa H, Motani K, Kosako H, Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics. J Proteome Res, 21(9):2094-2103(2022) [pubmed]

submitter keyword: Biotinylated Peptides, Tamavidin 2-REV, BioID

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

jPOST dataset URI

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