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ProteomicsPRM analysis of phosphopeptides of Nab2, Foxk1, HURP, ERK1/2, NPM, and Hspa4 from ΔB-Raf:ER cells.
ΔB-Raf:ER cells were treated with U0126 or 4-HT for 30 min in four biological replicates. Total proteins from cell lysates were digested with Typsin/Lys-C mix, and phosphopeptides were enriched by IMAC using Fe-NTA columns. After data-dependent LC-MS/MS analysis, phosphopeptides of Nab2, Foxk1, HURP...
ORGANISM(S): Mus Musculus (mouse) 
2022-03-03 | PXD028826 | JPOST Repository
ProteomicsPhos-tag SDS-PAGE followed by LC-MS/MS analysis of in vitro phosphorylated Nab2, Foxk1, and HURP
Purified GST-fused Nab2, Foxk1, and HURP were incubated with ATP in the presence or absence of His-tagged active ERK in vitro, separated by Phos-tag SDS-PAGE, and visualized by CBB staining. The most shifted regions of protein bands on the Phos-tag-containing gels were excised, digested with trypsin...
ORGANISM(S): Mus Musculus (mouse) 
2022-03-03 | PXD028837 | JPOST Repository
ProteomicsLabel-free quantitative phosphoproteomics of ΔB-Raf:ER cells treated with U0126 or 4-HT
ΔB-Raf:ER cells were treated with 10 µM U0126 or 1 µM 4-HT for 30 min. The cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication and then centrifuged. The supernatants were recovered, and proteins (...
ORGANISM(S): Mus Musculus (mouse) 
2022-03-03 | PXD028723 | JPOST Repository
ProteomicsSequential Digestion with Different Proteases
We examined three combinations of different proteases for digestion of proteins from TurboID-STING-expressing cells: trypsin alone, Lys-C followed by trypsin, and Glu-C followed by trypsin. First, we tested whether PTS buffer used to solubilize precipitated proteins is compatible with digestion with...
ORGANISM(S): Mus Musculus (mouse) 
2022-08-25 | PXD035242 | JPOST Repository
ProteomicsOptimization of Elution Conditions for Biotinylated Peptides from Tamavidin 2-REV
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. S...
ORGANISM(S): Mus Musculus (mouse) 
2022-08-25 | PXD035219 | JPOST Repository
ProteomicsPurification of Biotinylated Peptides using SDB-SCX StageTip
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 o...
ORGANISM(S): Mus Musculus (mouse) 
2022-08-25 | PXD035229 | JPOST Repository
ProteomicsComparison of Biotinylated Peptides Enriched by the Original and Optimized Workflows using Tamavidin 2-REV.
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 workflo...
ORGANISM(S): Mus Musculus (mouse) 
2022-08-25 | PXD035220 | JPOST Repository
ProteomicsReduction of Interference Ions Generated in the Enrichment Process of Biotinylated Peptides using Tamavidin 2-REV
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 contam...
ORGANISM(S): Cellular Organisms Mus Musculus (mouse) 
2022-08-25 | PXD035218 | JPOST Repository
ProteomicsLFQ of GFP-Trap IP-MS from hTBJ1 cells expressing EGFP-mSTING
Permeabilised hTBJ1 cells expressing EGFP-mSTING were stimulated with or without 1 µM 2’3’-cGAMP at 10°C and cross-linked with 0.1 or 0.5% paraformaldehyde at 4°C for 1 h in three independent experiments. After quenching with 100 mM glycine-NaOH, pH 7.5 at 4°C for 15 min, the cells were lysed in RIP...
ORGANISM(S): Homo Sapiens (human) 
2022-12-09 | PXD029736 | JPOST Repository
ProteomicsLFQ of streptavidin pulldown from hTBJ1 cells expressing APEX2-STING
Permeabilised hTBJ1 cells expressing APEX2-STING were stimulated with or without 1 μM cGAMP at 10°C and incubated for 1 min in the presence of 1 mM H2O2 and 50 µM biotin phenol. The reaction was then quickly stopped by washing three times with ice-cold 500 mM sodium ascorbate, followed by washing tw...
ORGANISM(S): Homo Sapiens (human) 
2022-12-09 | PXD029735 | JPOST Repository
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