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Reduction of Interference Ions Generated in the Enrichment Process of Biotinylated Peptides

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: (a) surfactants to solubilize proteins (RapiGest vs. PTS); (b) trypsin inactivation (Pefabloc SC vs. heat); (c) beads pre-wash (none vs. 10% ACN); and (d) biotin clean-up (none vs. GL-Tip SDB). To confirm that some contaminant ions were not derived from the digested peptides but from the biotin solution, Tamavidin 2-REV beads that had not been incubated with the digested peptides were eluted with the biotin solution or biotin-free solution, and the eluates were analyzed by LC-MS/MS.

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

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD032059 | JPOST Repository | Sat Mar 04 00:00:00 GMT 2023

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	220225_Tamavidin_OptimizedWF_1_PTSbuffer1-5_FusionIT.pdResult	Other
	220225_Tamavidin_OptimizedWF_1_PTSbuffer1-5_FusionIT_PeptideGroups.txt	Txt
	296_Tama_test_1th_1_FusionIT.mgf	Mgf
	296_Tama_test_1th_1_FusionIT.raw	Raw
	298_Tama_test_1th_2_FusionIT.mgf	Mgf

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1 - 5 of 16

Similar Datasets

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

Project description: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.

2022-08-25 | PXD035218 | JPOST Repository

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

Project description: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). Finally, 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.

2023-03-04 | PXD032060 | JPOST Repository

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

Project description: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.

2022-08-25 | PXD035219 | JPOST Repository

Purification of Biotinylated Peptides using SDB-SCX StageTip

Project description: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.

2022-08-25 | PXD035229 | JPOST Repository

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

Project 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.

2022-08-25 | PXD035220 | JPOST Repository

LFQ of biotinylated peptides from Raw264.7 cells expressing BioID-STING

Project description:The STING-null Raw264.7 cells expressing STING-BirA* were treated with DMSO or DMXAA for 15 min, and then biotin was added to the medium. After 8-h incubation, the cells were lysed and digested with trypsin. Biotinylated peptides were captured with tamavidin 2-REV magnetic beads and eluted with 2 mM biotin. The eluates were analysis by LC-MS/MS. Label-free quantification was performed using Proteome Discoverer 2.2 Software.

2023-04-17 | PXD009873 | JPOST Repository

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2021-11-06 | PXD028754 | JPOST Repository

LIN-41 RIP-Seq

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