2019
DOI: 10.1016/j.redox.2019.101227
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Quantification of cellular protein and redox imbalance using SILAC-iodoTMT methodology

Abstract: Under normal conditions, the cellular redox status is maintained in a steady state by reduction and oxidation processes. These redox alterations in the cell are mainly sensed by protein thiol residues of cysteines thus regulating protein function. The imbalance in redox homeostasis may therefore regulate protein turnover either directly by redox modulating of transcription factors or indirectly by the degradation of damaged proteins due to oxidation. A new analytical method capable of simultaneously assessing … Show more

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Cited by 26 publications
(30 citation statements)
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“…hTERT-RPE-1 were cultured as described previously [ 48 ]. DMEM was supplemented with 200 mg/L l -proline, 10% dialyzed fetal bovine serum (dFBS), 0.1% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).…”
Section: Methodsmentioning
confidence: 99%
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“…hTERT-RPE-1 were cultured as described previously [ 48 ]. DMEM was supplemented with 200 mg/L l -proline, 10% dialyzed fetal bovine serum (dFBS), 0.1% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL).…”
Section: Methodsmentioning
confidence: 99%
“…Sequential iodoTMT labeling, protein digestion and peptide desalting were conducted as previously described [ 48 ]. Briefly, concentration of all cell lysate samples was adjusted to 1 μg/μL using the lysis buffer and 10 mM neocuproine (f.c.…”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…The multiplexes were redissolved in 0.1% trifluoroacetic acid (TFA), 2% acetonitrile (AcN) to a concentration of 1 µg/µL and injected twice on an UltiMate 3000 RSLCnano System (Thermo Scientific, Bremen, Germany) in three technical replicates. The analytical system consisted of a PepMap100 C18, 3 µm, 100 Å, 75 µm × 20 mm trap column and a PepMap RSLC C18, 2 µm, 100 Å, 75 µm × 500 mm analytical column (both from Thermo Scientific, Bremen, Germany) and was previously used in our proteomic analyses 21 . The samples were loaded onto the trap column at a flow rate 8 µl/min of 0.1% TFA, 2% AcN for 3 min.…”
Section: Methodsmentioning
confidence: 99%
“…An advanced quantitative redox proteomics technique, oxidative isotope-coded affinity tags (OxICAT), revealed several RPs from both ribosome subunits to be ROS sensitive in different organisms (Leichert et al, 2008;Menger et al, 2015;Topf et al, 2018). The SILAC-iodoTMT method, which allows to simultaneously monitor the redox state and protein expression level, also confirms RPs to be one of the most significant groups of proteins affected by the hydrogen peroxide treatment (Vajrychova et al, 2019). Topf et al proposed a concept that RPs can serve as redox sensors upon oxidative stress conditions (Topf et al, 2018).…”
Section: Ribosomal Protein Modificationsmentioning
confidence: 99%