Dataset Information

Purification of Biotinylated Peptides using SDB-SCX StageTip

ABSTRACT: We tried purification of biotinylated peptides using SDB-SCX StageTip to reduce interference ions. We compared this method with the original and optimized workflows. We assessed reproducibility of each enrichment process by three technical replicates. Original workflow with SDB-SCX: 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 subjected to GL-Tip SDB-SCX. After desalting with the first-stage SDB, biotinylated peptides were eluted from the second-stage SCX with 500 mM ammonium acetate and 30% ACN, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD035229 | JPOST Repository | Thu Aug 25 00:00:00 BST 2022

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	10_Tama_test_4_Original_SCX_FusionIT.raw	Raw
	12_Tama_test_7_Optimilization_FusionIT.raw	Raw
	16_Tama_test_2_Original_FusionIT.raw	Raw
	18_Tama_test_5_Original_SCX_FusionIT.raw	Raw
	20_Tama_test_8_Optimilization_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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