Dataset Information

Optimization of Elution Conditions for Biotinylated Peptides from Tamavidin 2-REV

ABSTRACT: We attempted to optimize the conditions for competitive elution of biotinylated peptides from the Tamavidin 2-REV beads. First, to optimize the temperature at which biotinylated peptides were eluted from the beads, biotinylated peptides were eluted with the biotin solution at 4, 37, 56, and 95 ºC. Second, we investigated whether the contamination of non-biotinylated peptides in the eluates could be reduced by adding a step of ‘mock’ elution with biotin-free buffer immediately before elution with the biotin solution. After the beads were incubated with a biotin-free buffer for 15 min at 37 ºC, the buffer was removed, and biotinylated peptides were eluted from the beads with the biotin solution at 37 ºC. Third, we compared biotin solutions containing various concentrations of NaCl (150, 250, 500, 1000, 2000 and 4000 mM). We assessed reproducibility of each condition by three technical replicates.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD035219 | JPOST Repository | Thu Aug 25 00:00:00 BST 2022

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	10_Tama_test_7_ElutionNaCl_500mM_FusionIT.raw	Raw
	12_Tama_test_10_ElutionNaCl_1000mM_FusionIT.raw	Raw
	14_Tama_test_13_ElutionNaCl_2000mM_FusionIT.raw	Raw
	16_Tama_test_16_ElutionNaCl_4000mM_FusionIT.raw	Raw
	195_Tama_test_1_ElutionTemperature4_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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