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 cellular protein expression and cysteine oxidation would provide a valuable tool for the field of cysteine-targeted biology. Here, we show a workflow based on protein quantification using metabolic labeling and determination of cysteine oxidation using reporter ion quantification. We applied this approach to determine protein and redox changes in cells after 5-min, 60-min and 32-h exposure to H 2 O 2 , respectively. Based on the functional analysis of our data, we confirmed a biological relevance of this approach and its applicability for parallel mapping of cellular proteomes and redoxomes under diverse conditions. In addition, we revealed a specific pattern of redox changes in peroxiredoxins in a short time-interval cell exposure to H 2 O 2 . Overall, our present study offers an innovative, versatile experimental approach to the multifaceted assessment of cellular proteome and its redox status, with broad implications for biomedical research towards a better understanding of organismal physiology and diverse disease conditions.

Peroxiredoxin 6 protects irradiated cells from oxidative stress and shapes their senescence-associated cytokine landscape

“…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).…”

“…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.…”

“…In this study, we employed our recently published SILAC-iodoTMT methodology to detect and quantitatively estimate the level of protein sulfhydryl oxidation and proteome-level changes [ 48 ] in the IRIS in vitro model of hTERT-RPE-1 cells suffering from elevated ROS formation. We identified PRDX6 as an important player in cellular redox homeostasis in IRIS.…”

Comprehensive proteomic investigation of infectious and inflammatory changes in late preterm prelabour rupture of membranes

“…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.…”

The evolving role of ribosomes in the regulation of protein synthesis

“…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).…”