When suspended in cell culture medium, nano-objects composed of soluble metals such as silver can dissolve resulting in ion formation, altered particle properties (e.g. mass, morphology, etc.), and ...modulated cellular dose. Cultured cells are exposed not just to nanoparticles but to a complex, dynamic mixture of altered nanoparticles, unbound ions, and ion-ligand complexes. Here, three different cell types (RAW 264.7 macrophages and bone marrow derived macrophages from wild-type C57BL/6 J mice and Scavenger Receptor A deficient (SR-A
) mice) were exposed to 20 and 110 nm silver nanoparticles, and RAW 264.7 cells were exposed to freshly mixed silver ions, aged silver ions (ions incubated in cell culture medium), and ions formed from nanoparticle dissolution. The In Vitro Sedimentation, Diffusion, Dissolution, and Dosimetry Model (ISD3) was used to predict dose metrics for each exposure scenario.
Silver nanoparticles, freshly mixed ions, and ions from nanoparticle dissolution were toxic, while aged ions were not toxic. Macrophages from SR-A
mice did not take up 20 nm silver nanoparticles as well as wild-types but demonstrated no differences in silver levels after exposure to 110 nm nanoparticles. Dose response modeling with ISD3 predicted dose metrics suggest that amount of ions in cells and area under the curve (AUC) of ion amount in cells are the most predictive of cell viability after nanoparticle and combined nanoparticle/dissolution-formed-ions exposures, respectively.
Results of this study suggest that the unbound silver cation is the ultimate toxicant, and ions formed extracellularly drive toxicity after exposure to nanoparticles. Applying computational modeling (ISD3) to better understand dose metrics for soluble nanoparticles allows for better interpretation of in vitro hazard assessments.
Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can ...substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.
Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot ...for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.
Reversible modifications of cysteine thiols have a key role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation (SNO) and ...S-glutathionylation (SSG), have been recognized for their significance in various physiological and pathological processes. Here we describe a procedure for the enrichment of peptides containing reversible cysteine modifications. Starting with tissue or cell lysate samples, all of the unmodified free thiols are blocked using N-ethylmaleimide (NEM). This is followed by the selective reduction of those cysteines bearing the reversible modification(s) of interest. The reduction is achieved by using different reducing reagents that react specifically with each type of cysteine modification (e.g., ascorbate for SNO). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach uses a commercially available thiol-affinity resin (thiopropyl Sepharose 6B) to directly capture free thiol-containing proteins through a disulfide exchange reaction, followed by on-resin protein digestion and multiplexed isobaric labeling to facilitate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared with the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of SNO and the level of total reversible cysteine modifications (or total oxidation) is presented to demonstrate the utility of this general strategy. The entire protocol requires ∼3 d for sample processing with an additional day for LC-MS/MS and data analysis.
The thiol redox proteome refers to all proteins whose cysteine thiols are subjected to various redox-dependent posttranslational modifications (PTMs) including
glutathionylation (SSG),
nitrosylation ...(SNO),
sulfenylation (SOH), and
sulfhydration (SSH). These modifications can impact various aspects of protein function such as activity, binding, conformation, localization, and interactions with other molecules. To identify novel redox proteins in signaling and regulation, it is highly desirable to have robust redox proteomics methods that can provide global, site-specific, and stoichiometric quantification of redox PTMs. Mass spectrometry (MS)-based redox proteomics has emerged as the primary platform for broad characterization of thiol PTMs in cells and tissues. Herein, we review recent advances in MS-based redox proteomics approaches for quantitative profiling of redox PTMs at physiological or oxidative stress conditions and highlight some recent applications. Considering the relative maturity of available methods, emphasis will be on two types of modifications:
) total oxidation (i.e., all reversible thiol modifications), the level of which represents the overall redox state, and
)
glutathionylation, a major form of reversible thiol oxidation. We also discuss the significance of stoichiometric measurements of thiol PTMs as well as future perspectives toward a better understanding of cellular redox regulatory networks in cells and tissues.
Proteins with deamidated/citrullinated amino acids play critical roles in the pathogenesis of many human diseases; however, identifying these modifications in complex biological samples has been an ...ongoing challenge. Herein we present a method to accurately identify these modifications from shotgun proteomics data generated by a deep proteome profiling study of human pancreatic islets obtained by laser capture microdissection. All MS/MS spectra were searched twice using MSGF+ database matching, with and without a dynamic +0.9840 Da mass shift modification on amino acids asparagine, glutamine, and arginine (NQR). Consequently, each spectrum generates two peptide-to-spectrum matches (PSMs) with MSGF+ scores, which were used for the Delta Score calculation. It was observed that all PSMs with positive Delta Score values were clustered with mass errors around 0 ppm, while PSMs with negative Delta Score values were distributed nearly equally within the defined mass error range (20 ppm) for database searching. To estimate false discovery rate (FDR) of modified peptides, a “target-mock” strategy was applied in which data sets were searched against a concatenated database containing “real-modified” (+0.9840 Da) and “mock-modified” (+1.0227 Da) peptide masses. The FDR was controlled to ∼2% using a Delta Score filter value greater than zero. Manual inspection of spectra showed that PSMs with positive Delta Score values contained deamidated/citrullinated fragments in their MS/MS spectra. Many citrullinated sites identified in this study were biochemically confirmed as autoimmunogenic epitopes of autoimmune diseases in literature. The results demonstrated that in situ deamidated/citrullinated peptides can be accurately identified from shotgun tissue proteomics data using this dual-search Delta Score strategy. Raw MS data is available at ProteomeXchange (PXD010150).
Sarcopenia and exercise intolerance are major contributors to reduced quality of life in the elderly for which there are few effective treatments. We tested whether enhancing mitochondrial function ...and reducing mitochondrial oxidant production with SS-31 (elamipretide) could restore redox balance and improve skeletal muscle function in aged mice. Young (5 mo) and aged (26 mo) female C57BL/6Nia mice were treated for 8-weeks with 3 mg/kg/day SS-31. Mitochondrial function was assessed in vivo using 31P and optical spectroscopy. SS-31 reversed age-related decline in maximum mitochondrial ATP production (ATPmax) and coupling of oxidative phosphorylation (P/O). Despite the increased in vivo mitochondrial capacity, mitochondrial protein expression was either unchanged or reduced in the treated aged mice and respiration in permeabilized gastrocnemius (GAS) fibers was not different between the aged and aged+SS-31 mice. Treatment with SS-31 also restored redox homeostasis in the aged skeletal muscle. The glutathione redox status was more reduced and thiol redox proteomics indicated a robust reversal of cysteine S-glutathionylation post-translational modifications across the skeletal muscle proteome. The gastrocnemius in the age+SS-31 mice was more fatigue resistant with significantly greater mass compared to aged controls. This contributed to a significant increase in treadmill endurance compared to both pretreatment and untreated control values. These results demonstrate that the shift of redox homeostasis due to mitochondrial oxidant production in aged muscle is a key factor in energetic defects and exercise intolerance. Treatment with SS-31 restores redox homeostasis, improves mitochondrial quality, and increases exercise tolerance without an increase in mitochondrial content. Since elamipretide is currently in clinical trials these results indicate it may have direct translational value for improving exercise tolerance and quality of life in the elderly.
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The widespread use of engineered nanomaterials or nanotechnology makes the characterization of biological responses to nanomaterials an important area of research. The application of omics ...approaches, such as mass spectrometry-based proteomics, has revealed new insights into the cellular responses of exposure to nanomaterials, including how nanomaterials interact and alter cellular pathways. In addition, exposure to engineered nanomaterials often leads to the generation of reactive oxygen species and cellular oxidative stress, which implicates a redox-dependent regulation of cellular responses under such conditions. In this review, we discuss quantitative proteomics-based approaches, with an emphasis on redox proteomics, as a tool for system-level characterization of the biological responses induced by engineered nanomaterials.
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Protein thiols play a crucial role in redox signaling, in the regulation of enzymatic activity and protein function, and in maintaining redox homeostasis in living systems. The unique chemical ...reactivity of the thiol group makes protein cysteines susceptible to reactions with reactive oxygen and nitrogen species that form various reversible and irreversible post-translational modifications (PTMs). The reversible PTMs in particular are major components of redox signaling and are involved in the regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in both healthy and disease states has been increasingly recognized. Herein, we review recent advances in quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including general considerations of sample processing, chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for specific biological applications. Although technical limitations remain, redox proteomics is paving the way to a better understanding of redox signaling and regulation in both healthy and disease states.
Thiol-based post-translational modifications (PTMs) play a key role in redox-dependent regulation and signaling. Functional cysteine (Cys) sites serve as redox switches, regulated through multiple ...types of PTMs. Herein, we aim to characterize the complexity of thiol PTMs at the proteome level through the establishment of a direct detection workflow. The LC-MS/MS based workflow allows for simultaneous quantification of protein abundances and multiple types of thiol PTMs. To demonstrate its utility, the workflow was applied to mouse pancreatic β-cells (β-TC-6) treated with thapsigargin to induce endoplasmic reticulum (ER) stress. This resulted in the quantification of >9000 proteins and multiple types of thiol PTMs, including intra-peptide disulfide (S–S), S-glutathionylation (SSG), S-sulfinylation (SO2H), S-sulfonylation (SO3H), S-persulfidation (SSH), and S-trisulfidation (SSSH). Proteins with significant changes in abundance were observed to be involved in canonical pathways such as autophagy, unfolded protein response, protein ubiquitination pathway, and EIF2 signaling. Moreover, ~500 Cys sites were observed with one or multiple types of PTMs with SSH and S–S as the predominant types of modifications. In many cases, significant changes in the levels of different PTMs were observed on various enzymes and their active sites, while their protein abundance exhibited little change. These results provide evidence of independent translational and post-translational regulation of enzyme activity. The observed complexity of thiol modifications on the same Cys residues illustrates the challenge in the characterization and interpretation of protein thiol modifications and their functional regulation.
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•Simultaneous quantification of protein abundances and multiple types of thiol PTMs.•Multiple types PTMs observed on the same Cys sites for redox-regulated proteins.•Data revealed complexity of thiol PTMs and their regulation.•Distinctive translational and post-translational regulation under ER stress in β-cells.
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