Dataset Information

Comparison of Biotinylated Peptides Enriched by the Original and Optimized Workflows using Tamavidin 2-REV.

ABSTRACT: 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.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD035220 | JPOST Repository | Thu Aug 25 00:00:00 BST 2022

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	138_Tama_test_1_Original_FusionIT.raw	Raw
	140_Tama_test_7_Optimlization_FusionIT.raw	Raw
	142_Tama_test_2_Original_FusionIT.raw	Raw
	144_Tama_test_8_Optimlization_FusionIT.raw	Raw
	146_Tama_test_3_Original_FusionIT.raw	Raw

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Publications

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics.

Nishino Kohei K Yoshikawa Harunori H Motani Kou K Kosako Hidetaka H

Journal of proteome research 20220817 9

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular ...[more]

PMID: 35979633

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