Abstract Cellular senescence is a complex stress response defined as an essentially irreversible cell cycle arrest mediated by the inhibition of cell cycle-specific cyclin dependent kinases. The imbalance in redox homeostasis and oxidative stress have been repeatedly observed as one of the hallmarks of the senescent phenotype. However, a large-scale study investigating protein oxidation and redox signaling in senescent cells in vitro has been lacking. Here we applied a proteome-wide analysis using SILAC-iodoTMT workflow to quantitatively estimate the level of protein sulfhydryl oxidation and proteome level changes in ionizing radiation-induced senescence (IRIS) in hTERT-RPE-1 cells. We observed that senescent cells mobilized the antioxidant system to buffer the increased oxidation stress. Among the antioxidant proteins with increased relative abundance in IRIS, a unique 1-Cys peroxiredoxin family member, peroxiredoxin 6 (PRDX6), was identified as an important contributor to protection against oxidative stress. PRDX6 silencing increased ROS production in senescent cells, decreased their resistance to oxidative stress-induced cell death, and impaired their viability. Subsequent SILAC-iodoTMT and secretome analysis after PRDX6 silencing showed the downregulation of PRDX6 in IRIS affected protein secretory pathways, decreased expression of extracellular matrix proteins, and led to unexpected attenuation of senescence-associated secretory phenotype (SASP). The latter was exemplified by decreased secretion of pro-inflammatory cytokine IL-6 which was also confirmed after treatment with an inhibitor of PRDX6 iPLA2 activity, MJ33. In conclusion, by combining different methodological approaches we discovered a novel role of PRDX6 in senescent cell viability and SASP development. Our results suggest PRDX6 could have a potential as a drug target for senolytic or senomodulatory therapy. Keywords: Cellular senescence, Interleukin 6, Peroxiredoxin 6, Redox proteomics, Senescence-associated secretory phenotype, SILAC-iodoTMT Graphical abstract [39]Image 1 [40]Open in a new tab Highlights * • SILAC-iodoTMT is a powerful tool to quantify redox imbalance in IRIS. * • Senescence in hTERT-RPE-1 cells is not accompanied by bulk cysteine oxidation. * • Antioxidant proteins are upregulated in senescent hTERT-RPE-1 cells. * • PRDX6 silencing affects redox homeostasis and viability of senescent cells. * • PRDX6 silencing alters secretome of senescent RPE-1 cells and suppresses IL-6. Abbreviations AA arachidonic acid ACN acetonitrile BrdU 5-bromo-2′-deoxyuridine Ctrl control C[p] peroxidatic cysteine Cys cysteine DAMPs damage-associated molecular patterns DAPI 4′,6-diamidino-2-phenylindole dFBS dialyzed fetal bovine serum DMEM Dulbecco's Modified Eagle Medium EDTA ethylenediaminetetraacetic acid EdU 5-ethynyl-2′-deoxyuridine FA formic acid FBS fetal bovine serum f.c. final concentration FDR false discovery rate GAPDH glyceraldehyde 3-phosphate dehydrogenase GRX1 Glutaredoxin 1 H[2]O[2] hydrogen peroxide HBSS Hank's balanced salt solution iodoTMT iodoacetyl tandem mass tag iPLA2 phospholipase A2 IR ionizing radiation IRIS ionizing radiation-induced senescence IW isolation window LC-MS/MS liquid chromatography-tandem mass spectrometry LPCAT lysophosphatidylcholine acyl transferase NCE normalized collision energy NH[4]FA ammonium formate OIS oncogene-induced senescence PBS phosphate buffered saline PCA principal component analysis Prx peroxiredoxin ROS reactive oxygen species RT room temperature SASP senescence-associated secretory phenotype SDC sodium deoxycholate SILAC stable isotope labeling with amino acids in cell culture SIPS stress-induced premature senescence TCEP tris(2-carboxyethyl) phosphine hydrochloride TEAB triethylammonium bicarbonate TFA trifluoroacetic acid TMRE tetramethylrhodamine ethyl ester perchlorate 1. Introduction Cellular senescence is a complex cellular stress response triggered by molecular damage and characterized by a prolonged and generally irreversible cell cycle arrest. This stable arrest, and more importantly, the ability of senescent cells to influence their microenvironment, contribute to their role in the development of age-related pathologies including cancer [[41]1,[42]2]. Various types of stresses including DNA damage and oxidative stress are responsible for expression of protein inhibitors of cyclin-dependent kinases, the pivotal effectors of cellular senescence. The evidence of mechanistic role of oxidative stress in the development of cellular senescence has been laid by the work of Toussaint et al. [[43]3]. The authors showed that in vitro exposure of cells to oxidants such as hydrogen peroxide (H[2]O[2]) resulted in proliferation arrest associated with replicative senescence-like morphological changes, termed stress-induced premature senescence (SIPS) [[44]3]. There has also been accumulating evidence that oxidative stress is involved in the onset of replicative senescence [[45]4,[46]5], oncogene-induced senescence (OIS) [[47]6,[48]7], and ionizing radiation-induced senescence (IRIS) [[49]8]. Furthermore, application of antioxidants can mitigate senescence development [[50]8,[51]9]. It has been proposed that deregulation of NADPH oxidases [[52][10], [53][11], [54][12], [55][13]] or mitochondrial dysfunction [[56][14], [57][15], [58][16]] are the internal sources of reactive oxygen species (ROS) responsible for the development of cellular senescence. Thus, redox homeostasis imbalance and prolonged oxidative stress have been repeatedly observed as one of the major components of the senescent phenotype. Oxidative stress can lead to damage of all macromolecules including nucleic acids, proteins, lipids and their precursors, and metabolites. Thus, all cells must be equipped with cytoprotective antioxidant enzymes. Among them, the peroxiredoxin (Prx) family of peroxidases [[59]17,[60]18] has been shown to maintain cellular redox homeostasis and viability [[61]19], acting as H[2]O[2]-scavenging enzymes, but also to contribute to redox signaling as redox relays [[62]20] or functioning as floodgates [[63]21]. PRDX6 is a unique 1-Cys peroxiredoxin family member [[64]18,[65]22] crucial for maintenance of phospholipid homeostasis due to its unique ability to bind and reduce phospholipid hydroperoxides. Additionally, it has been described to exert phospholipase A2 (iPLA2) [[66]23,[67]24] and lysophosphatidylcholine acyl transferase (LPCAT) activities [[68]25]. The regulation of the PRDX6 enzymatic functions occurs at multiple levels – by subcellular localization, protein-protein interactions, and post-translational modifications [[69]26]. Perhaps most importantly, the irreversible hyperoxidation of the peroxidatic cysteine (Cp)47 has been described as a regulator of the iPLA2 activity of PRDX6 [[70]27]. A number of studies have demonstrated that PRDX6 expression provides protection from ROS of multiple origins in cancer and normal cells, both in vivo and in vitro, prevents lipid peroxidation damage [[71]28], cell death [[72][29], [73][30], [74][31]], and ensures normal proliferation and redox regulation of metabolic pathways [[75]32,[76]33]. Similarly to other members of the Prx family, PRDX6 has been shown to play multifaceted roles in inflammatory processes [[77]34]. While the antioxidant function of PRDX6 can be mostly considered as anti-inflammatory, its iPLA2 activity and the ability to release arachidonic acid (AA) has been linked to inflammation in experiments utilizing the iPLA2 activity inhibitor, MJ33 [[78]28,[79]35,[80]36]. In addition to the iPLA2 activity, peroxiredoxins, including PRDX6, can be released to extracellular space where they might function as damage-associated molecular patterns (DAMPs [[81]37]) activating the pro-inflammatory pathways via the TLR4 receptor [[82]38]. There is a vast research mapping the physiological functions of PRDX6 in multiple organs or cell types such as lungs, brain, eye, or spermatozoa and also pathophysiological functions implying the role of PRDX6 in tumor viability [[83][29], [84][30], [85][31]], progression, and metastasis [[86][39], [87][40], [88][41], [89][42], [90][43]]. It has been shown that PRDX6 protects normal cells from oxidative damage [[91]44,[92]45] and cellular senescence [[93]46,[94]47]. However, to the best of our knowledge, the role of PRDX6 in the development of the senescent phenotype has not been addressed yet. 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 [[95]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. Subsequently, we applied MS-based proteomic strategy and siRNA-mediated knock-down of PRDX6 to evaluate the contribution of PRDX6 to the senescent phenotype. We described that the lack of PRDX6 activity during the development of IRIS led to suppression of important phenotypic features of senescence as well as senescence-associated secretory phenotype (SASP) leading to altered composition of extracellular matrix, protein secretion, and more specifically, decreased secretion of pro-inflammatory cytokine IL-6. 2. Material and methods 2.1. Reagents and chemicals All reagents including antibodies, siRNAs, and primer sequences used in this study are provided in Supplementary Material and Methods section. 2.2. Cell culture Human telomerase-immortalized retinal pigment epithelial cells hTERT-RPE-1 (ATCC CRL-4000) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) with 4.5 g/L glucose and 4 mM l-glutamine supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). Human embryonal lung fibroblasts MRC-5 cells (ATCC CLL-171) were cultured in DMEM with 1 g/L glucose and 4 mM l-glutamine supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), and non-essential amino acids. 2.3. Cell culture SILAC-iodoTMT hTERT-RPE-1 were cultured as described previously [[96]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). SILAC labeling was done by using l-arginine [^13C[6], ^15N[4]] and l-lysine [^13C[6]] for more than six cell population doublings, and the incorporation efficiency was monitored throughout the cultivation. Cell cultures were maintained at 37 °C under 5% CO[2] atmosphere and 95% humidity. 2.4. SDS-PAGE and immunoblotting SDS-PAGE and immunoblotting were performed according to standard protocols. Samples (20–30 μg) in a reducing sample buffer were separated by SDS-PAGE on a 12% gel and detected by immunoblotting. GRX1 detection was performed using Tricine SDS-PAGE [[97]49]. Nitrocellulose membranes were blocked in 5% milk at room temperature (RT) for 1 h, incubated with a primary antibody (1 : 1000) at 4 °C overnight and after washing three times with phosphate buffered saline (PBS)/Tween-20, incubated with a secondary antibody (1 : 10,000) at RT for 1 h (anti-mouse/anti-rabbit IgG HRP, 1 : 10,000; BioRad, Hercules, CA, USA). Proteins were visualized using chemiluminescence (Amersham TM, GE Healthcare) and analyzed for relative band densities (ImageJ software). The list of antibodies used in this study is provided in the Supplementary Material and Methods section. 2.5. Detection of senescence-associated activity of beta-galactosidase The detection of senescence-associated activity of beta-galactosidase was performed as described previously [[98]50]. Briefly, hTERT-RPE-1 cells were seeded at low density (5000 cells/cm^2) and irradiated after 16 h by a dose of 20 Gy. Eight days after irradiation and two days after seeding of proliferating control cells, the cells were fixed using 0.5% glutaraldehyde (15 min, RT) and incubated with the X-gal staining solution at 37 °C until blue color appeared in the senescent cell culture. 2.6. 5-Ethynyl-2′-deoxyuridine incorporation assay 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay was performed using Click-iT™ EdU Cell Proliferation Kit (#[99]C10337, ThermoFisher Scientific). hTERT-RPE-1 cells were seeded at low density (5000 cells/cm^2) and after 16 h irradiated by a dose of 20 Gy. Eight days after irradiation and two days after seeding of a proliferating control cells (seeded 10,000 cells/cm^2), 10 μM EdU was added, and the cells were cultured for 24 h. The cells were fixed using 4% formaldehyde (15 min, RT) and permeabilized using 0.5% Triton X-100 in PBS (20 min, RT). The click reaction was performed according to the manufacturer's protocol, and the nuclei were counterstained using 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution (2 min, RT). 2.7. Fluorescent microscopy – DNA damage foci hTERT-RPE-1 cells were seeded at low density (5000 cells/cm^2) and after 16 h irradiated by a dose of 20 Gy. Eight days after irradiation and two days after seeding of a proliferating control cells (seeded 10,000 cells/cm^2), the cells were fixed using 4% formaldehyde (10 min, RT) and permeabilized using 0.2% Triton X-100 in PBS (10 min, RT). After blocking of non-specific binding using DMEM/10% FBS (30 min, RT), the coverslips were incubated with a primary antibody (1 : 200, 1 h, RT) diluted in blocking solution, following by incubation with the secondary antibody (1 : 1,000, 16 h, 4 °C) in blocking solution. Nuclei were counterstained using 1 μg/mL DAPI solution (2 min, RT). 2.8. Real-time quantitative reverse transcription PCR hTERT-RPE-1 cells were seeded at a density of 20,000 cells/cm^2 and after 16 h irradiated by a dose of 20 Gy [[100]51]. Eight days after irradiation and two days after seeding of proliferating control cells (seeded 10,000 cells/cm^2), cells were harvested, and RNA was isolated using RNeasy Mini Kit (#74106, Qiagen) according to the manufacturer's protocol. Contaminating DNA was removed by RNase-Free DNase Set (#79254, Qiagen) on RNA easy column according to the manufacturer's protocol. cDNA was synthesized using a MultiScribe™ Reverse Transcriptase kit (#4311235, ThermoFisher Scientific). Quantitative reverse transcription PCR was performed in ABI Prism 7300 (Applied Biosystems) using SYBR™ Select Master Mix (#4472919, ThermoFisher Scientific) with the primers shown in the Supplementary Material and Methods section. The relative quantity of cDNA was estimated by ΔΔCt [[101]52], data were normalized to β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or their average. 2.9. siRNA-mediated gene knock-down Cells were transfected with 10 nM siRNA using Lipofectamine® RNAiMAX Transfection Reagent (#13778150; ThermoFisher Scientific) following the manufacturer's protocol. The siRNA sequences are shown in Supplementary Material and Methods section. Silencer™ Select Negative Control No. 1 siRNA (#4390843, ThermoFisher Scientific) was used as a negative control (siNC). For siRNA validation, the cells were seeded at a density of 15,000/cm^2, transfected 16 h after seeding, and harvested after 48 h or 72 h after transfection for mRNA and protein analysis, respectively. For all senescent cells analyses, the cells were seeded at a density of 20,000/cm^2 and irradiated (20 Gy) 16 h after seeding. The cells were first transfected 2 days after irradiation, the second transfection was performed 5 or 6 days after irradiation (both designs were validated to provide comparable results), and the cells were harvested for experiments 8 or 9 days after irradiation as indicated. 2.10. Detection of intracellular ROS, mitochondrial ROS and membrane potential, and lipid peroxidation Intracellular ROS production was detected using CellROX™ Deep Red Reagent (#[102]C10422; Thermo Fisher Scientific) according to the optimized manufacturer's protocol (30 min incubation with 5 μM dye dissolved in the cell culture medium at 37 °C). Mitochondrial ROS production was detected using MitoSOX™ Red Mitochondrial Superoxide Indicator (#[103]M36008; Thermo Fisher Scientific) according to the optimized manufacturer's protocol (60 min incubation with 5 μM dye dissolved in cell culture medium at 37 °C). Lipid peroxidation was detected using BODIPY™ 581/591C11 (Lipid Peroxidation Sensor; #D3861; Thermo Fisher Scientific) according to optimized manufacturer's protocol (30 min incubation with 2.5 μM dye dissolved in the cell culture medium at 37 °C). Mitochondrial membrane potential was analyzed using Tetramethylrhodamine, Ethyl Ester, Perchlorate assay (TMRE; #T669; Thermo Fisher Scientific) according to the optimized manufacturer's protocol (15 min incubation with 0.5 μM dye dissolved in cell culture medium at 37 °C). In all assays, DMSO-treated cells were used as a control to subtract autofluorescence in the post-analysis. Cells were washed with PBS (37 °C), detached with 0.25% trypsin/EDTA, resuspended in PBS, 1 g/L glucose, 1% FBS and centrifuged (300×g, 3 min, 4 °C). Pellets were resuspended in 100 μL PBS, 1 g/L glucose, 1% FBS and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). Hoechst 33258 or propidium iodide were used to exclude non-viable cells from the analysis. 2.11. Detection of extracellular ROS production Extracellular ROS were detected using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit (#A22188; Thermo Fisher Scientific) according to the manufacturer's protocol and a previously published procedure [[104]53]. Briefly, the cells were detached by 0.25% trypsin/EDTA, counted, and 150,000 proliferating or senescent cells (20 Gy, 8 days after irradiation) were used for analysis. Resorufin production was monitored every minute for 60–90 min to estimate kinetics of H[2]O[2] release from the cells. Light microscopy was used to estimate the diameter of the detached cells, and their volume was calculated under the assumption the detached cells have approximately a spherical shape. 2.12. Detection of apoptosis Apoptosis assay was performed using annexin V/Hoechst 33258 staining. Briefly, cells in the cell culture supernatant were collected, and the adherent cells were detached using 0.25% trypsin/EDTA. Both fractions were pooled, and the cells were washed three times in 0.5 mL of 1 × Annexin binding buffer. Annexin V staining was performed in 100 μL of 1 × Annexin binding buffer with Annexin V Dyomics 647 antibody (1 : 100; Exbio, Vestec, Czech Republic) for 15 min on ice with no light exposure. Hoechst 33258 was added prior to the flow cytometry analysis (BD Biosciences, San Jose, CA, USA). 2.13. Viability assays Cells were seeded in 96-well plates at density of 20,000 cells/cm^2 (6440 cells per well). Proliferating or senescent cells (control, siNC, or siPRDX6 transfected as described above) were treated with H[2]O[2] in triplicate in a concentration range 0.025–3.5 mM for proliferating cells and 0.5–10 mM for senescent cells for 24 h. To determine cell viability by the crystal violet assay [[105]54,[106]55], the cells were washed twice with 150 μL PBS and then stained in 30 μL 0.5% (w/v) crystal violet in 20% methanol for 15 min. Plates were washed three times with double distilled H[2]O and left to dry overnight. Crystal violet was solubilized with 75 μL 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO) in PBS for 15 min. Absorbance of crystal violet was measured at 595 nm using a microplate reader (Multiskan EX, Thermo Electron Corporation, Waltham, MA). Alternatively, the MTT assay was performed with CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (#G4000, Promega) according to the manufacturer's protocol. Absorbance of the treated samples was expressed as a percentage of absorbance of untreated cells. IC50 values were estimated using nonlinear regression curve fitting in GraphPad Prism version 8.0.0. 2.14. Inhibition of PRDX6 iPLA2 activity by MJ33 hTERT-RPE-1 cells were seeded at medium density (15,000 cells/cm^2) and irradiated by a dose of 20 Gy 16 h after seeding. Six days after irradiation, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33; #M3315, Sigma-Aldrich), an iPLA2 activity inhibitor [[107]56] was added in a final concentration of 10 μM, 25 μM, and 50 μM. The cells were incubated with the inhibitor for 72 h, and the inhibitor solution in cell culture medium was replaced every 24 h to ensure continuous treatment. Cell culture supernatant was collected on day 9 after irradiation and processed as described below. 2.15. Detection of cytokines in cell culture media The cell culture supernatant was collected 24 h after changing the culture media for fresh. The medium was subjected to a two-step centrifugation to remove cells and debris contamination (200×g, 5 min, 4 °C; 2000×g, 5 min, 4 °C). Adherent cells were detached using 0.25% trypsin/EDTA, and the cell number was estimated using flow cytometry. The relative concentration of 11 cytokines was estimated using a Human Inflammation 11-Plex Panel kit (#C192211, AimPlex Biosciences, Inc., Pomona, CA, USA) according to manufacturer's protocol. Resulting relative fluorescence intensities were corrected for cell count and expressed as fold change relative to control or siNC. 2.16. Redox-proteomic analysis of senescent cells The experiment was performed in four biological replicates with swapped SILAC groups, i.e., the experiment contained two “light” and two “heavy” samples from each condition (proliferating control cells and senescent cells). hTERT-RPE-1 cells were seeded at a density of 10,000 cells/cm^2 and after 16 h irradiated by a dose of 20 Gy. Eight days after irradiation and two days after seeding of proliferating control cells (seeded 15,000 cells/cm^2), the dishes were washed three times with ice-cold PBS and lysed with ice-cold lysis buffer (3% SDC/200 mM TEAB/1 mM EDTA). After the addition of the lysis buffer, the cells were scraped, transferred into microtubes, and the lysate was frozen in liquid nitrogen. The lysates were defrosted in an ice-cold water bath (4 °C) in BioRuptor and sonicated (low intensity, 30 s ON, 30 s OFF, three cycles). Remaining debris was removed by centrifugation (16,100×g, 10 min, 4 °C). During the whole procedure, the tubes were kept on ice and protected from light exposure, and whenever possible, the sample handling was performed in a cold room (4 °C). 2.17. Redox-proteomic analysis of senescent cells after PRDX6 silencing The experiment was performed in two biological replicates per siRNA with swapped SILAC groups, i.e., the experiment contained two “light” and two “heavy” siNC samples, and one “light” and “heavy” samples from each siPRDX6. hTERT-RPE-1 cells were seeded at a density of 20,000 cells/cm^2 and after 16 h irradiated by a dose of 20 Gy. Two sequential transfections were performed on day 2 and day 5 after irradiation as described above, and the cells were harvested 8 days after irradiation using the protocol described in the previous paragraph. 2.18. Sequential iodoTMT labeling, protein digestion and peptide desalting Sequential iodoTMT labeling, protein digestion and peptide desalting were conducted as previously described [[108]48]. Briefly, concentration of all cell lysate samples was adjusted to 1 μg/μL using the lysis buffer and 10 mM neocuproine (f.c. = 0.1 mM). The sample lysate (Condition 1; proliferating cells) labeled in the light/heavy SILAC channel (50 μg) was mixed with an equal amount of the sample lysate (Condition 2; senescent cells) of the complementary heavy/light SILAC group ([109]Supplementary Fig. 1A). Free –SH groups were labeled by the first iodoTMT label (TMT1) at 37 °C for 2 h. Subsequently, proteins were precipitated using cold acetone (−20 °C) for 60 min. The pellet was re-dissolved in lysis buffer with 0.1 mM neocuproine and incubated with TCEP (f.c. = 5 mM) at 50 °C for 60 min to reduce the reversibly oxidized cysteines. The newly reduced cysteines were then labeled using the second iodoTMT label (TMT2) and the reaction was quenched by l-cysteine hydrochloride (f.c. = 20 mM). After the second acetone precipitation, proteins were digested using rLys-C (FUJIFILM Wako, Osaka, Japan) at 37 °C for 3 h followed by sequencing grade trypsin (Promega) digestion at 37 °C overnight at 1 : 50 ratio (enzyme-to-protein). Digestion was stopped by addition of trifluoroacetic acid (TFA, f.c. = 2%), and precipitated SDC was removed by extraction in water-saturated ethyl acetate. Finally, samples were desalted and evaporated to dryness. IodoTMTsixplex™ Isobaric Label Reagent Set for one simplex experiment (Thermo Scientific, Waltham, MA, USA) was used for peptide iodoTMT labeling. 2.19. Basic fractionation liquid chromatography In the first dimension, 85 μg of each sample was re-dissolved in a mobile phase A (2% ACN, 20 mM NH[4]FA, pH 10) and 80 μg was injected for basic fractionation on UltiMate 3000 RSLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with UV detection. Peptides were separated in a XBridge BEH column (2.1 μm × 150 mm, C18, 2.5 μm, 130 Å, from Waters, Milford, MA, USA) using a linear gradient of 3%–50% mobile phase B (80% ACN, 20 mM NH[4]FA, pH 10) at a flow rate 0.4 mL/min, at 40 °C, for 30 min. Peptide fractions were collected in the range of 0.5–37 min per 45 s. In total, 49 fractions were collected and 3rd – 42nd fractions were matched in the final set of 8 fractions ([110]Supplementary Fig. 1C). Those were evaporated to dryness and stored at −80 °C for further analysis. All buffers and mobile phases for LC separation were prepared in LC-MS grade water purchased from Honeywell (Morris Plains, NJ, USA). 2.20. Nano-liquid chromatography coupled to tandem mass spectrometry analysis Evaporated fractions were re-dissolved in a mobile phase A (0.1% TFA in 2% ACN) and 2 μg were injected onto UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Bremen, Germany) for the liquid chromatography separation. The analytical system consisted of PepMap 100C18, 3 μm, 100 Å, 75 μm × 20 mm trap column and PepMap RSLC C18, 2 μm, 100 Å, 75 μm × 500 mm analytical column (both from Thermo Fisher Scientific). The samples were loaded onto the trap column in 0.1% TFA in 2% ACN at 8 μL/min for 3 min. Tryptic peptides were separated by a segment gradient running from 2% to 9% of a mobile phase B (80% ACN with 0.1% FA) for 57 min, further from 9% to 34.5% of B for 160 min, and finally to 45% of B for 23 min at a flow rate of 200 nL/min. Eluted peptides were electrosprayed into Q-Exactive Plus using a Nanospray Flex ion source (both from Thermo Fisher Scientific, Bremen, Germany). Positive ion full scan MS spectra were acquired in the range of 350–1600 m/z using 3 × 10^6 AGC target in the Orbitrap at 70,000 resolution with a maximum ion injection time of 50 ms. Parameters of the isolation window (IW) and normalized collision energy (NCE) were set 1.6 m/z for IW and 30 for NCE as previously described [[111]48]. MS/MS spectra were acquired at resolution of 35,000, with a 1 × 10^6 AGC target and a maximum injection time of 120 ms. Only 15 of the most intensive precursors with minimal AGC target of 2.4 × 10^4 and a charge state ≥2 were fragmented. The dynamic exclusion window was 17 s. The fixed first mass was set to 100 m/z and the scan range from 200 to 2000 m/z. All buffers and mobile phases for LC separation were prepared in LC-MS grade water purchased from Honeywell (Morris Plains, NJ, USA) and Fisher Scientific (Pardubice, Czechia). All additives added to the LC mobile phases were LC-MS grade. 2.21. MS data processing and statistical analysis Survey MS and MS/MS spectra were processed in the MaxQuant 1.6.1.0 [[112]57]. Enzyme specificity was set to trypsin/P, and a maximum of two missed cleavages were allowed. Protein N-terminal acetylation, methionine oxidation, glutamine/asparagine deamidation, and N-terminal glutamate to pyroglutamate conversion were selected as variable modifications based on pre-analysis by Preview (Protein Metrics, Cupertino, CA, USA). The derived peak list was searched using the built-in Andromeda search engine in MaxQuant against human reference proteome downloaded in 11^th October 2018 including contaminants from UniProtKB database. Workflow used for the determination of redox cysteine changes considered heavy arginine (^13C[6]^15N[4]) and lysine (^13C[6]) as variable modifications. Specified iodoTMT labeling has been set as a quantification method. Remaining Group-specific parameters were kept at default values. The minimum Andromeda score needed for modified peptides was set to 0. The minimum ratio count for label-based quantification was set to two quantified peptide pairs. Only unique or razor peptides were considered for calculating protein ratios. For the proteome centric workflow, heavy arginine (^13C[6]^15N[4]) and lysine (^13C[6]) were set to Standard type in Group-specific parameters as heavy labels, and specific iodoTMT labeling was added as a fixed modification. The rest of parameters was set as described above. For the analysis of differently oxidized cysteine peptides shown in [113]Fig. 2C, iodoTMT labeling, cysteine dioxidation (-SO[2]H), and cysteine trioxidation (-SO[3]H) were set as variable modifications. Fig. 2. [114]Fig. 2 [115]Open in a new tab High-throughput comparative analysis of the cysteine oxidation status of proliferating and senescent hTERT-RPE-1 cells using SILAC-iodoTMT. (A) Principal component analysis of the oxidation proportion values (n = 2595) quantified in all samples; the percentage indicates proportion of variability explained by principal components 1 (PC1) and 2 (PC2). (B) Distributions of the average oxidation proportion values of control and senescent cells depicted as a density plot. (C) Relative comparison of PRDX6 peptides with different modifications (iodoTMT-labeled or post-translationally modified by oxidation) between senescent and proliferating cells using the SILAC channel. Each dot represents a ratio from one biological replicate. Statistical analysis was performed using one-way ANOVA; pairwise comparisons relative to unmodified peptides were performed using Dunnett's test. Asterisks indicate statistical significance; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) The volcano plot shows statistically significant differences between senescent and proliferating cells at the p-value (blue and red circles; two-sample t-test) and FDR level (blue and red triangles; Benjamini-Hochberg correction). (E-F) The cysteine residues of proteins were mapped to cellular compartments (GOCC; E) and biological processes (GOBP; F), and paired samples Wilcoxon test was performed with Benjamini-Hochberg correction. The red asterisk indicates Benjamini-Hochberg FDR < 0.05. The most representative examples were selected for visualization. (G) Selected examples of cysteine residues mapped to the two biological processes shown in (F). (For interpretation of the references to color in this figure legend,