Dataset Information

Reduction of Interference Ions Generated in the Enrichment Process of Biotinylated Peptides using Tamavidin 2-REV

ABSTRACT: After tryptic digestion of proteins of RAW264.7 cells expressing TurboID-STING, biotinylated peptides were captured with Tamavidin 2-REV magnetic beads and eluted competitively with excess biotin. When the peptide samples were subjected to LC-MS/MS, high intensity peaks of interference and/or contaminant ions were observed at the MS1 level. To reduce interference ions, we compared the following conditions: surfactants to solubilize proteins (RapiGest vs. PTS); trypsin inactivation (Pefabloc SC vs. heat); beads pre-wash (none vs. 10% ACN); and biotin clean-up (none vs. GL-Tip SDB). To confirm that some contaminant ions were derived from the biotin solution, the biotin solution or biotin-free solution was desalted using GL-Tip SDB and directly analyzed by LC-MS/MS. We assessed reproducibility of each condition by three technical replicates.

ORGANISM(S): Cellular Organisms Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD035218 | JPOST Repository | Thu Aug 25 00:00:00 BST 2022

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	146_Tama_test_1_Original_RapiGest_FusionIT.raw	Raw
	148_Tama_test_4_PTS_buffer_FusionIT.raw	Raw
	150_Tama_test_7_PTS_buffer_Heat_FusionIT.raw	Raw
	152_Tama_test_10_PTS_buffer_BeadsWash_FusionIT.raw	Raw
	154_Tama_test_13_PTS_buffer_SDB_biotin_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

Dataset Information

Reduction of Interference Ions Generated in the Enrichment Process of Biotinylated Peptides using Tamavidin 2-REV

Dataset's files

Publications

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

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