mARC1 in MASLD: Modulation of lipid accumulation in human hepatocytes and adipocytes.

BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Transcriptomic and Proteomic Changes Driving Pulmonary Fibrosis Resolution in Young and Old Mice.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFß antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.

Whole lung proteome of an acute epithelial injury mouse model in comparison to spatially resolved proteomes.

Epithelial injury is one of the major drivers of acute pulmonary diseases. Recurring injury followed by aberrant repair is considered as the primary cause of chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Preclinical in vivo models allow studying early disease-driving mechanisms like the recently established adeno-associated virus-diphtheria toxin receptor (AAV-DTR) mouse model of acute epithelial lung injury, which utilises AAV mediated expression of the human DTR. We performed quantitative proteomics of homogenised lung samples from this model and compared the results to spatially resolved proteomics data of epithelial cell regions from the same animals. In whole lung tissue proteins involved in cGAS-STING and interferon pathways, proliferation, DNA replication and the composition of the provisional extracellular matrix were upregulated upon injury. Besides epithelial cell markers SP-A, SP-C and Scgb1a1, proteins involved in cilium assembly, lipid metabolism and redox pathways were among downregulated proteins. Comparison of the bulk to spatially resolved proteomics data revealed a large overlap of protein changes and striking differences. Together our study underpins the broad usability of bulk proteomics and pinpoints to the benefit of sophisticated proteomic analyses of specific tissue regions or single cell types.

Transcriptomic and proteomic profiling of young and old mice in the bleomycin model reveals high similarity.

The most common preclinical, in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2- to 3-mo-old mice. Although this model resembles key aspects of idiopathic pulmonary fibrosis (IPF), there are limitations in its predictability for the human disease. One of the main differences is the juvenile age of animals that are commonly used in experiments, resembling humans of around 20 yr. Because IPF patients are usually older than 60 yr, aging appears to play an important role in the pathogenesis of lung fibrosis. Therefore, we compared young (3 months) and old mice (21 months) 21 days after intratracheal bleomycin instillation. Analyzing lung transcriptomics (mRNAs and miRNAs) and proteomics, we found most pathways to be similarly regulated in young and old mice. However, old mice show imbalanced protein homeostasis as well as an increased inflammatory state in the fibrotic phase compared to young mice. Comparisons with published human transcriptomic data sets (GSE47460, GSE32537, and GSE24206) revealed that the gene signature of old animals correlates significantly better with IPF patients, and it also turned human healthy individuals better into "IPF patients" using an approach based on predictive disease modeling. Both young and old animals show similar molecular hallmarks of IPF in the bleomycin-induced lung fibrosis model, although old mice more closely resemble several features associated with IPF in comparison to young animals.

Mapping the metabolomic and lipidomic changes in the bleomycin model of pulmonary fibrosis in young and aged mice.

Alterations in metabolic pathways were recently recognized as potential underlying drivers of idiopathic pulmonary fibrosis (IPF), translating into novel therapeutic targets. However, knowledge of metabolic and lipid regulation in fibrotic lungs is limited. To comprehensively characterize metabolic perturbations in the bleomycin mouse model of IPF, we analyzed the metabolome and lipidome by mass spectrometry. We identified increased tissue turnover and repair, evident by enhanced breakdown of proteins, nucleic acids and lipids and extracellular matrix turnover. Energy production was upregulated, including glycolysis, the tricarboxylic acid cycle, glutaminolysis, lactate production and fatty acid oxidation. Higher eicosanoid synthesis indicated inflammatory processes. Because the risk of IPF increases with age, we investigated how age influences metabolomic and lipidomic changes in the bleomycin-induced pulmonary fibrosis model. Surprisingly, except for cytidine, we did not detect any significantly differential metabolites or lipids between old and young bleomycin-treated lungs. Together, we identified metabolomic and lipidomic changes in fibrosis that reflect higher energy demand, proliferation, tissue remodeling, collagen deposition and inflammation, which might serve to improve diagnostic and therapeutic options for fibrotic lung diseases in the future.

Biomarker discovery for chronic liver diseases by multi-omics - a preclinical case study.

Nonalcoholic steatohepatitis (NASH) is a major cause of liver fibrosis with increasing prevalence worldwide. Currently there are no approved drugs available. The development of new therapies is difficult as diagnosis and staging requires biopsies. Consequently, predictive plasma biomarkers would be useful for drug development. Here we present a multi-omics approach to characterize the molecular pathophysiology and to identify new plasma biomarkers in a choline-deficient L-amino acid-defined diet rat NASH model. We analyzed liver samples by RNA-Seq and proteomics, revealing disease relevant signatures and a high correlation between mRNA and protein changes. Comparison to human data showed an overlap of inflammatory, metabolic, and developmental pathways. Using proteomics analysis of plasma we identified mainly secreted proteins that correlate with liver RNA and protein levels. We developed a multi-dimensional attribute ranking approach integrating multi-omics data with liver histology and prior knowledge uncovering known human markers, but also novel candidates. Using regression analysis, we show that the top-ranked markers were highly predictive for fibrosis in our model and hence can serve as preclinical plasma biomarkers. Our approach presented here illustrates the power of multi-omics analyses combined with plasma proteomics and is readily applicable to human biomarker discovery.

PROMIS, global analysis of PROtein-metabolite interactions using size separation in Arabidopsis thaliana.

Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein-Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein-small molecule and of protein-protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein-protein (PPIs) and protein-metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr-Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.

Interaction of 2',3'-cAMP with Rbp47b Plays a Role in Stress Granule Formation.

2',3'-cAMP is an intriguing small molecule that is conserved among different kingdoms. 2',3'-cAMP is presumably produced during RNA degradation, with increased cellular levels observed especially under stress conditions. Previously, we observed the presence of 2',3'-cAMP in Arabidopsis (Arabidopsis thaliana) protein complexes isolated from native lysate, suggesting that 2',3'-cAMP has potential protein partners in plants. Here, affinity purification experiments revealed that 2',3'-cAMP associates with the stress granule (SG) proteome. SGs are aggregates composed of protein and mRNA, which enable cells to selectively store mRNA for use in response to stress such as heat whereby translation initiation is impaired. Using size-exclusion chromatography and affinity purification analyses, we identified Rbp47b, the key component of SGs, as a potential interacting partner of 2',3'-cAMP. Furthermore, SG formation was promoted in 2',3'-cAMP-treated Arabidopsis seedlings, and interactions between 2',3'-cAMP and RNA-binding domains of Rbp47b, RRM2 and RRM3, were confirmed in vitro using microscale thermophoresis. Taken together, these results (1) describe novel small-molecule regulation of SG formation, (2) provide evidence for the biological role of 2',3'-cAMP, and (3) demonstrate an original biochemical pipeline for the identification of protein-metabolite interactors.

Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases.

Interactions between metabolites and proteins play an integral role in all cellular functions. Here we describe an affinity purification (AP) approach in combination with LC/MS-based metabolomics and proteomics that allows, to our knowledge for the first time, analysis of protein-metabolite and protein-protein interactions simultaneously in plant systems. More specifically, we examined protein and small-molecule partners of the three (of five) nucleoside diphosphate kinases present in the Arabidopsis genome (NDPK1-NDPK3). The bona fide role of NDPKs is the exchange of terminal phosphate groups between nucleoside diphosphates (NDPs) and triphosphates (NTPs). However, other functions have been reported, which probably depend on both the proteins and small molecules specifically interacting with the NDPK. Using our approach we identified 23, 17, and 8 novel protein partners of NDPK1, NDPK2, and NDPK3, respectively, with nucleotide-dependent proteins such as actin and adenosine kinase 2 being enriched. Particularly interesting, however, was the co-elution of glutathione S-transferases (GSTs) and reduced glutathione (GSH) with the affinity-purified NDPK1 complexes. Following up on this finding, we could demonstrate that NDPK1 undergoes glutathionylation, opening a new paradigm of NDPK regulation in plants. The described results extend our knowledge of NDPKs, the key enzymes regulating NDP/NTP homeostasis.

Combined GC- and UHPLC-HR-MS Based Metabolomics to Analyze Durable Anti-fungal Resistance Processes in Cereals.

Introduction of durable resistance genes in crops is an important strategy to prevent yield loss caused by pathogens. The durable multi-pathogen resistance gene Lr34 originating from wheat is widely used in breeding, and is functionally transferable to barley and rice. The molecular resistance mechanism of Lr34, encoding for an adenosine triphosphate-binding cassette transporter, is not known yet. To understand the molecular function and the defense response of durable disease resistance in cereals, the metabolic response of Lr34 was investigated in, except for the Lr34 gene, genetically identical lines of barley, rice and wheat. A broad range of compounds including primary, secondary and lipophilic metabolites were analyzed by a combination of gas (GC) and liquid chromatography (LC) mass spectrometry (MS) based methods. Data from metabolomics correlated well with transcriptomics data for plant defense responses such as the formation of anti-fungal hordatines or the components of the glyoxylate cycle. Induction of the glyoxylate cycle found in transgenic Lr34 rice grown in the greenhouse was confirmed in field-grown natural Lr34 wheat. Constitutively active plant defense responses were observed in the different cereals.

System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants.

Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems.

Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC-MS/MS.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC-MS/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.

Systems-wide analysis of acclimation responses to long-term heat stress and recovery in the photosynthetic model organism Chlamydomonas reinhardtii.

We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42°C for 24 h and back to 25°C for ≥8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions.

Identification and validation of protein-protein interactions by combining co-immunoprecipitation, antigen competition, and stable isotope labeling.

Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on (15)N stable isotope labeling of cells, (14)N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions.

Rationales and approaches for studying metabolism in eukaryotic microalgae.

The generation of efficient production strains is essential for the use of eukaryotic microalgae for biofuel production. Systems biology approaches including metabolite profiling on promising microalgal strains, will provide a better understanding of their metabolic networks, which is crucial for metabolic engineering efforts. Chlamydomonas reinhardtii represents a suited model system for this purpose. We give an overview to genetically amenable microalgal strains with the potential for biofuel production and provide a critical review of currently used protocols for metabolite profiling on Chlamydomonas. We provide our own experimental data to underpin the validity of the conclusions drawn.

Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii.

We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance.

In vitro characterization of bacterial and chloroplast Hsp70 systems reveals an evolutionary optimization of the co-chaperones for their Hsp70 partner.

The chloroplast Hsp70 (heat-shock protein of 70 kDa) system involved in protein folding in Chlamydomonas reinhardtii consists of HSP70B, the DnaJ homologue CDJ1 and the GrpE-type nucleotide-exchange factor CGE1. The finding that HSP70B needs to be co-expressed with HEP2 (Hsp70 escort protein 2) to become functional allowed the reconstitution of the chloroplast Hsp70 system in vitro and comparison with the homologous Escherichia coli system. Both systems support luciferase refolding and display ATPase and holdase activities. Steady-state activities are low and strongly stimulated by the co-chaperones, whose concentrations need to be balanced to optimally support luciferase refolding. Although the co-chaperones of either system generally stimulate ATPase and folding-assistance activities of the other, luciferase refolding is reduced ~10-fold and <2-fold if either Hsp70 is supplemented with the foreign DnaJ and GrpE protein respectively, suggesting an evolutionary specialization of the co-chaperones for their Hsp70 partner. Distinct features are that HSP70B's steady-state ATPase exhibits ~20-fold higher values for Vmax and Km and that the HSP70B system displays a ~6-fold higher folding assistance on denatured luciferase. Although truncating up to 16 N-terminal amino acids of CGE1 does not affect HSP70B's general ATPase and folding-assistance activities in the physiological temperature range, further deletions hampering dimerization of CGE1 via its N-terminal coiled coil do.

Dissecting the heat stress response in Chlamydomonas by pharmaceutical and RNAi approaches reveals conserved and novel aspects.

To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell's entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.

Chloroplast DnaJ-like proteins 3 and 4 (CDJ3/4) from Chlamydomonas reinhardtii contain redox-active Fe-S clusters and interact with stromal HSP70B.

In the present study we report on the identification and characterization of three novel chloroplast-targeted DnaJ-like proteins CDJ3-5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In sequence databases we could identify homologues of CDJ3-5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed us to distinguish two clades containing CDJ3/4 and CDJ5 that must have diverged early in the ancestor of the 'green lineage' and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both proteins are weakly expressed and appear to be localized to the stroma and to thylakoid membranes respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after a dark-to-light shift. Accordingly, the Arabidopsis orthologue of CDJ5 was also found to be light-inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli and had redox-active Fe-S clusters. In vitro cross-linking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast ATP-bound HSP70B (heat-shock protein 70B), presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were also in a complex with HSP70B in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of approx. 550-2800 kDa, which appeared to contain RNA. We speculate that the CDJ3-5 proteins might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes.

Methods for analysis of protein glutathionylation and their application to photosynthetic organisms.

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.