2D fibrillar osteoid niche mimicry through inclusion of visco-elastic and topographical cues in gelatin-based networks.

Given the clinical need for osteoregenerative materials incorporating controlled biomimetic and biophysical cues, a novel highly-substituted norbornene-modified gelatin was developed enabling thiol-ene crosslinking exploiting thiolated gelatin as cell-interactive crosslinker. Comparing the number of physical crosslinks, the degree of hydrolytic degradation upon modification, the network density and the chemical crosslinking type, the osteogenic effect of visco-elastic and topographical properties was evaluated. This novel network outperformed conventional gelatin-based networks in terms of osteogenesis induction, as evidenced in 2D dental pulp stem cell seeding assays, resulting from the presentation of both a local (substrate elasticity, 25-40 kPa) and a bulk (compressive modulus, 25-45 kPa) osteogenic substrate modulus in combination with adequate fibrillar cell adhesion spacing to optimally transfer traction forces from the fibrillar ECM (as evidenced by mesh size determination with the rubber elasticity theory) and resulting in a 1.7-fold increase in calcium production (compared to the gold standard gelatin methacryloyl (GelMA)).

Robust High-Throughput Proteomics Identification and Deamidation Quantitation of Extinct Species up to Pleistocene with Ultrahigh-Resolution MALDI-FTICR Mass Spectrometry.

Peptide mass fingerprinting (PMF) using MALDI-TOF mass spectrometry allows the identification of bone species based on their type I collagen sequence. In the archaeological or paleontological field, PMF is known as zooarchaeology mass spectrometry (ZooMS) and is widely implemented to find markers for most species, including the extinct ones. In addition to the identification of bone species, ZooMS enables dating estimation by measuring the deamidation value of specific peptides. Herein, we report several enhancements to the classical ZooMS technique, which reduces to 10-fold the required bone sample amount (down to the milligram scale) and achieves robust deamidation value calculation in a high-throughput manner. These improvements rely on a 96-well plate samples preparation, a careful optimization of collagen extraction and digestion to avoid spurious post-translational modification production, and PMF at high resolution using matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance (MALDI-FTICR) analysis. This method was applied to the identification of a hundred bones of herbivores from the Middle Paleolithic site of Caours (Somme, France) well dated from the Eemian Last Interglacial climatic optimum. The method gave reliable species identification to bones already identified by their osteomorphology, as well as to more challenging samples consisting of small or burned bone fragments. Deamidation values of bones originating from the same geological layers have a low standard deviation. The method can be applied to archaeological bone remains and offers a robust capacity to identify traditionally unidentifiable bone fragments, thus increasing the number of identified specimens and providing invaluable information in specific contexts.

The NLRP6 protein is very faintly expressed in several normal and cancerous epithelial cells and may be confused with an unrelated protein.

Nod-Like Receptor Pyrin domain-containing protein 6 (NLRP6), a member of the Nucleotide-oligomerization domain-Like Receptor (NLR) family of proteins, assembles together with the ASC protein to form an inflammasome upon stimulation by bacterial lipoteichoic acid and double-stranded DNA. Besides its expression in myeloid cells, NLRP6 is also expressed in intestinal epithelial cells where it may contribute to the maintenance of gut homeostasis and negatively controls colorectal tumorigenesis. Here, we report that NLRP6 is very faintly expressed in several colon cancer cell lines, detected only in cytoplasmic small dots were it colocalizes with ASC. Consequently, it is very hardly detected by standard western-blotting techniques by several presently available commercial antibodies which, in contrast, highly cross-react with a protein of 90kDa that we demonstrate to be unrelated to NLRP6. We report here these results to caution the community not to confuse the 90kDa protein with the endogenous human NLRP6.

Wild Wheat Rhizosphere-Associated Plant Growth-Promoting Bacteria Exudates: Effect on Root Development in Modern Wheat and Composition.

Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur.

Reliable Approach for Pure Yeast Cell Wall Protein Isolation from Saccharomyces cerevisiae Yeast Cells.

Saccharomyces cerevisiae yeast is a fungus presenting a peripheral organelle called the cell wall. The cell wall protects the yeast cell from stress and provides means for communication with the surrounding environment. It has a complex molecular structure, composed of an internal part of cross-linked polysaccharides and an external part of mannoproteins. These latter are very interesting owing to their functional properties, dependent on their molecular features with massive mannosylations. Therefore, the molecular characterization of mannoproteins is a must relying on the optimal isolation and preparation of the cell wall fraction. Multiple methods are well reported for yeast cell wall isolation. The most applied one consists of yeast cell lysis by mechanical disruption. However, applying this classical approach to S288C yeast cells showed considerable contamination with noncell wall proteins, mainly comprising mitochondrial proteins. Herein, we tried to further purify the yeast cell wall preparation by two means: ultracentrifugation and Triton X-100 addition. While the first strategy showed limited outcomes in mitochondrial protein removal, the second strategy showed optimal results when Triton X-100 was added at 5%, allowing the identification of more mannoproteins and significantly enriching their amounts. This promising method could be reliably implemented on the lab scale for identification of mannoproteins and molecular characterization and industrial processes for "pure" cell wall isolation.

Simple gene signature to assess murine fibroblast polarization.

We provide an original multi-stage approach identifying a gene signature to assess murine fibroblast polarization. Prototypic polarizations (inflammatory/fibrotic) were induced by seeded mouse embryonic fibroblasts (MEFs) with TNFα or TGFß1, respectively. The transcriptomic and proteomic profiles were obtained by RNA microarray and LC-MS/MS. Gene Ontology and pathways analysis were performed among the differentially expressed genes (DEGs) and proteins (DEPs). Balb/c mice underwent daily intradermal injections of HOCl (or PBS) as an experimental murine model of inflammation-mediated fibrosis in a time-dependent manner. As results, 1456 and 2215 DEGs, and 289 and 233 DEPs were respectively found in MEFs in response to TNFα or TGFß1, respectively. Among the most significant pathways, we combined 26 representative genes to encompass the proinflammatory and profibrotic polarizations of fibroblasts. Based on principal component analysis, this signature deciphered baseline state, proinflammatory polarization, and profibrotic polarization as accurately as RNA microarray and LC-MS/MS did. Then, we assessed the gene signature on dermal fibroblasts isolated from the experimental murine model. We observed a proinflammatory polarization at day 7, and a mixture of a proinflammatory and profibrotic polarizations at day 42 in line with histological findings. Our approach provides a small-size and convenient gene signature to assess murine fibroblast polarization.

Effects of Immunoglobulins G From Systemic Sclerosis Patients in Normal Dermal Fibroblasts: A Multi-Omics Study.

Autoantibodies (Aabs) are frequent in systemic sclerosis (SSc). Although recognized as potent biomarkers, their pathogenic role is debated. This study explored the effect of purified immunoglobulin G (IgG) from SSc patients on protein and mRNA expression of dermal fibroblasts (FBs) using an innovative multi-omics approach. Dermal FBs were cultured in the presence of sera or purified IgG from patients with diffuse cutaneous SSc (dcSSc), limited cutaneous SSc or healthy controls (HCs). The FB proteome and transcriptome were explored using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and microarray assays, respectively. Proteomic analysis identified 3,310 proteins. SSc sera and purified IgG induced singular protein profile patterns. These FB proteome changes depended on the Aab serotype, with a singular effect observed with purified IgG from anti-topoisomerase-I autoantibody (ATA) positive patients compared to HC or other SSc serotypes. IgG from ATA positive SSc patients induced enrichment in proteins involved in focal adhesion, cadherin binding, cytosolic part, or lytic vacuole. Multi-omics analysis was performed in two ways: first by restricting the analysis of the transcriptomic data to differentially expressed proteins; and secondly, by performing a global statistical analysis integrating proteomics and transcriptomics. Transcriptomic analysis distinguished 764 differentially expressed genes and revealed that IgG from dcSSc can induce extracellular matrix (ECM) remodeling changes in gene expression profiles in FB. Global statistical analysis integrating proteomics and transcriptomics confirmed that IgG from SSc can induce ECM remodeling and activate FB profiles. This effect depended on the serotype of the patient, suggesting that SSc Aab might play a pathogenic role in some SSc subsets.

Proteomics as a tool to gain next level insights into photo-crosslinkable biopolymer modifications.

The distribution of photo-crosslinkable moieties onto a protein backbone can affect a biomaterial's crosslinking behavior, and therefore also its mechanical and biological properties. A profound insight in this respect is essential for biomaterials exploited in tissue engineering and regenerative medicine. In the present work, photo-crosslinkable moieties have been introduced on the primary amine groups of: (i) a recombinant collagen peptide (RCPhC1) with a known amino acid (AA) sequence, and (ii) bovine skin collagen (COL BS) with an unknown AA sequence. The degree of substitution (DS) was quantified with two conventional techniques: an ortho-phthalic dialdehyde (OPA) assay and 1H NMR spectroscopy. However, neither of both provides information on the exact type and location of the modified AAs. Therefore, for the first time, proteomic analysis was evaluated herein as a tool to identify functionalized AAs as well as the exact position of photo-crosslinkable moieties along the AA sequence, thereby enabling an in-depth, unprecedented characterization of functionalized photo-crosslinkable biopolymers. Moreover, our strategy enabled to visualize the spatial distribution of the modifications within the overall structure of the protein. Proteomics has proven to provide unprecedented insight in the distribution of photo-crosslinkable moieties along the protein backbone, undoubtedly contributing to superior functional biomaterial design to serve regenerative medicine.

Passages in culture and stimulation conditions influence protein expression of primary fibroblasts.

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-ß1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-ß1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-ß1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-ß1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using "omics" approaches.

PDMS Curing Inhibition on 3D-Printed Molds: Why? Also, How to Avoid It?

Three-dimensional (3D)-printing techniques such as stereolithography (SLA) are currently gaining momentum for the production of miniaturized analytical devices and molds for soft lithography. However, most commercially available SLA resins inhibit polydimethylsiloxane (PDMS) curing, impeding reliable replication of the 3D-printed structures in this elastomeric material. Here, we report a systematic study, using 16 commercial resins, to identify a fast and straightforward treatment of 3D-printed structures and to support accurate PDMS replication using UV and/or thermal post-curing. In-depth analysis using Raman spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry revealed that phosphine oxide-based photo-initiators, leaching out of the 3D-printed structures, are poisoning the Pt-based PDMS catalyst. Yet, upon UV and/or thermal treatments, photo-initiators were both eliminated and recombined into high molecular weight species that were sequestered in the molds.

Palladium-Catalyzed Cross-Coupling of Gem-Bromofluoroalkenes with Alkylboronic Acids for the Synthesis of Alkylated Monofluoroalkenes.

Monofluoroalkenes are versatile fluorinated synthons in organic synthesis, medicinal chemistry and materials science. In light of the importance of alkyl-substituted monofluoroalkenes efficient synthesis of these moieties still represents a synthetic challenge. Herein, we described a mild and efficient methodology to obtain monofluoroalkenes through a stereospecific palladium-catalyzed alkylation of gem-bromofluoroalkenes with primary and strained secondary alkylboronic acids under mild conditions. This novel strategy gives access to a wide range of functionalized tri- and tetrasubstituted monofluoroalkenes in high yield, with good functional group tolerance, independently from the gem-bromofluoroalkenes geometry.

Metal-Free ATRP Catalyzed by Visible Light in Continuous Flow.

ATRP of methyl methacrylate catalyzed by Eosin Y, an inexpensive and an environmental benign dye, was performed in a continuous flow reactor made of FEP tubing and irradiated by visible light green LEDs. The reaction under flow conditions was significantly more rapid and controlled compared to that in batch giving 90% of polymerization after only 3 h of irradiation. The formed polymers in flow have M n measured by GPC and DOSY NMR in accordance with the theoretical values and show low dispersities (Ð < 1.5). The livingness of the polymers has been confirmed by LED on and LED off experiments and by the synthesis of block copolymers. The protocol described herein serves as a "proof of concept" of using Eosin Y as a photocatalyst for controlled polymerization and of using 1D and 2D NMR for polymer characterization. The protocol could be replicated in the future for other reversible-deactivation radical polymerizations.

Regio- and Stereo-Specific Chemical Depolymerization of High Molecular Weight Polybutadiene and Polyisoprene for Their Analysis by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: Comparison with Pyrolysis-Comprehensive Two-Dimensional Gas Chromatography/Mass Spectrometry, Atmospheric Solid Analysis Probe, Direct Inlet Probe-Atmospheric Pressure Chemical Ionization Mass Spectrometry, and Ion Mobility Spectrometry-Mass Spectrometry.

Polybutadiene (PB) and polyisoprene (PI), the two most common polydienes (PD), are involved in a large number of materials and used in a wide variety of applications. The characterization of these polymers by mass spectrometry (MS) continues to be very challenging due to their high insolubility and the difficulty to ionize them. In this work, a cross-metathesis reaction was used to generate end-functionalized acetoxy ionizable oligomers for the structural deciphering of different commercial PB and PI samples. A cross-metathesis reaction was carried out between polymers and the Z-1,4-diacetoxy-2-butene as a chain transfer agent in dichloromethane using a Hoveyda-Grubbs second-generation catalyst. Well-defined acetoxy telechelic structures were obtained and analyzed by Fourier transform ion cyclotron resonance (FTICR) high-resolution MS. However, after depolymerization, low molar mass polyolefins contained some units with different configurations, suggesting an olefin isomerization reaction due to the decomposition of the catalyst. The addition of an electron-deficient reagent such as 2,6-dichloro-1,4-benzoquinone suppressed this isomerization in the case of both Z- and E-PB and PI. Ion mobility spectrometry-mass spectrometry (IMS-MS) and energy-resolved tandem mass spectrometry (ERMS) analyses confirmed a successful isomerization suppression. For comparing the results obtained by depolymerization with classical methods for polymer analysis, pyrolysis-comprehensive two-dimensional gas chromatography/mass spectrometry (Py-GC × GC-MS), atmospheric solid analysis probe (ASAP), and direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) analyses were performed on the same polymers. This strategy can be applied on a variety of synthetic and natural not yet characterized polymers.

Heteroleptic Ruthenium(II) Complexes with Bathophenanthroline and Bathophenanthroline Disulfonate Disodium Salt as Fluorescent Dyes for In-Gel Protein Staining.

The in-gel detection of proteins for various proteomic experiments is commonly done with the fluorescent RuII tris(bathophenanthroline disulfonate) complex (Ru(BPS)3), which is more cost-effective compared to commercial Ru-based formulations but requires tedious procedures for its preparation and strongly acidic staining conditions. Herein, we report the synthesis and characterization of heteroleptic RuII complexes Ru(BPS)2(BP) and Ru(BPS)(BP)2 containing bathophenanthroline (BP) and bathophenanthroline disulfonate disodium salt (BPS) in comparison with Ru(BPS)3. It was shown by fluorescent and UV-vis measurements that novel RuII complexes were excitable in both UV and visible light, close to emission bands of classical lasers, which is important for successful in-gel protein detection. Novel fluorescent dyes demonstrated improved protein detection in comparison with commercially available SYPRO Ruby staining solution. In addition, unlike commonly used staining protocols, staining with Ru(BPS)(BP)2 can be performed at nearly neutral pH, thereby reducing artificial post-translational modifications (PTMs).

Intra-Sample Heterogeneity of Potato Starch Reveals Fluctuation of Starch-Binding Proteins According to Granule Morphology.

Starch granule morphology is highly variable depending on the botanical origin. Moreover, all investigated plant species display intra-tissular variability of granule size. In potato tubers, the size distribution of starch granules follows a unimodal pattern with diameters ranging from 5 to 100 µm. Several evidences indicate that granule morphology in plants is related to the complex starch metabolic pathway. However, the intra-sample variability of starch-binding metabolic proteins remains unknown. Here, we report on the molecular characterization of size-fractionated potato starch granules with average diameters of 14.2 ± 3.7 µm, 24.5 ± 6.5 µm, 47.7 ± 12.8 µm, and 61.8 ± 17.4 µm. In addition to changes in the phosphate contents as well as small differences in the amylopectin structure, we found that the starch-binding protein stoichiometry varies significantly according to granule size. Label-free quantitative proteomics of each granule fraction revealed that individual proteins can be grouped according to four distinct abundance patterns. This study corroborates that the starch proteome may influence starch granule growth and architecture and opens up new perspectives in understanding the dynamics of starch biosynthesis.

Photo-crosslinkable recombinant collagen mimics for tissue engineering applications.

Gelatin is frequently used in various biomedical applications. However, gelatin is generally extracted from an animal source, which can result in issues with reproducibility as well as pathogen transmittance. Therefore, we have investigated the potential of a recombinant peptide based on collagen I (RCPhC1) for tissue engineering applications and more specifically for adipose tissue regeneration. In the current paper, RCPhC1 was functionalized with photo-crosslinkable methacrylamide moieties to enable subsequent UV-induced crosslinking in the presence of a photo-initiator. The resulting biomaterial (RCPhC1-MA) was characterized by evaluating the crosslinking behaviour, the mechanical properties, the gel fraction, the swelling properties and the biocompatibility. The obtained results were compared with the data obtained for methacrylamide-modified gelatin (Gel-MA). The results indicated that the properties of RCPhC1-MA networks are comparable to those of animal-derived Gel-MA. RCPhC1-MA is thus an attractive synthetic alternative for animal-derived Gel-MA and is envisioned to be applicable for a wide range of tissue engineering purposes.

Neural Networks Are Promising Tools for the Prediction of the Viscosity of Unsaturated Polyester Resins.

Unsaturated polyester resins are widely used for the preparation of composite materials and fulfill the majority of practical requirements for industrial and domestic applications at low cost. These resins consist of a highly viscous polyester oligomer and a reactive diluent, which allows its process ability and its crosslinking. The viscosity of the initial polyester and the reactive diluent mixture is critical for practical applications. So far, these viscosities were determined by trial and error which implies a time-consuming succession of manipulations, to achieve the targeted viscosities. In this work, we developed a strategy for predicting the viscosities of unsaturated polyesters formulation based on neural networks. In a first step 15 unsaturated polyesters have been synthesized through high-temperature polycondensation using usual monomers. Experimental Hansen solubility parameters (HSP) were determined from solubility experiment with HSPiP software and glass transition temperatures (T g ) were measured by Differential Scanning Calorimetry (DSC). Quantitative Structure-Property Relationship (QSPR) coupled to multiple linear regressions have been used to get a prediction of Hansen solubility parameters δd, δ p , and δ h from structural composition. A second QSPR regression has been done on glass transition temperature (prediction vs. experimental coefficient of determination R 2 = 0.93) of these unsaturated polyesters. These unsaturated polyesters were next diluted in several solvents with different natures (ethers, esters, alcohol, aromatics for example) at different concentrations. Viscosities at room temperature of these polyesters in solution were finally measured in order to create a database of 220 entries with 7 descriptors (polyester molecular weight, T g , dispersity index Ð, polyester-solvent HSP RED, molar volume of the solvent, δ h of the solvent, concentration of polyester in solvent). The QSPR method for predicting the viscosity from these 6 descriptors proved to be ineffective (R 2 = 0.56) as viscosities exhibit non-linear phenomena. A Neural Network with an optimized number of 12 hidden neurons has been trained with 179 entries to predict the viscosity. A correlation between experimental and predicted viscosities based on 41 testing instances gave a correlation coefficient R 2 of 0.88 and a predicted vs. measured slope of 0.98. Thanks to Neural Networks, new developments with eco-friendly reactive diluents can be accelerated.

Two-dimensional mass spectrometry: new perspectives for tandem mass spectrometry.

Fourier transform ion cyclotron resonance mass analysers (FT-ICR MS) can offer the highest resolutions and mass accuracies in mass spectrometry. Mass spectra acquired in an FT-ICR MS can yield accurate elemental compositions of all compounds in a complex sample. Fragmentation caused by ion-neutral, ion-electron, or ion-photon interactions leads to more detailed structural information on compounds. The most often used method to correlate compounds and their fragment ions is to isolate the precursor ions from the sample before fragmentation. Two-dimensional mass spectrometry (2D MS) offers a method to correlate precursor and fragment ions without requiring precursor isolation. 2D MS therefore enables easy access to the fragmentation patterns of all compounds from complex samples. In this article, the principles of FT-ICR MS are reviewed and the 2D MS experiment is explained. Data processing for 2D MS is detailed, and the interpretation of 2D mass spectra is described.

Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBα complex.

The nuclear receptor REV-ERBα integrates the circadian clock with hepatic glucose and lipid metabolism by nucleating transcriptional comodulators at genomic regulatory regions. An interactomic approach identified O-GlcNAc transferase (OGT) as a REV-ERBα-interacting protein. By shielding cytoplasmic OGT from proteasomal degradation and favoring OGT activity in the nucleus, REV-ERBα cyclically increased O-GlcNAcylation of multiple cytoplasmic and nuclear proteins as a function of its rhythmically regulated expression, while REV-ERBα ligands mostly affected cytoplasmic OGT activity. We illustrate this finding by showing that REV-ERBα controls OGT-dependent activities of the cytoplasmic protein kinase AKT, an essential relay in insulin signaling, and of ten-of-eleven translocation (TET) enzymes in the nucleus. AKT phosphorylation was inversely correlated to REV-ERBα expression. REV-ERBα enhanced TET activity and DNA hydroxymethylated cytosine (5hmC) levels in the vicinity of REV-ERBα genomic binding sites. As an example, we show that the REV-ERBα/OGT complex modulates SREBP-1c gene expression throughout the fasting/feeding periods by first repressing AKT phosphorylation and by epigenomically priming the Srebf1 promoter for a further rapid response to insulin. Conclusion: REV-ERBα regulates cytoplasmic and nuclear OGT-controlled processes that integrate at the hepatic SREBF1 locus to control basal and insulin-induced expression of the temporally and nutritionally regulated lipogenic SREBP-1c transcript.

Apart From Rhoptries, Identification of Toxoplasma gondii's O-GlcNAcylated Proteins Reinforces the Universality of the O-GlcNAcome.

O-linked ß-N-acetylglucosaminylation or O-GlcNAcylation is a widespread post-translational modification that belongs to the large and heterogeneous group of glycosylations. The functions managed by O-GlcNAcylation are diverse and include regulation of transcription, replication, protein's fate, trafficking, and signaling. More and more evidences tend to show that deregulations in the homeostasis of O-GlcNAcylation are involved in the etiology of metabolic diseases, cancers and neuropathologies. O-GlcNAc transferase or OGT is the enzyme that transfers the N-acetylglucosamine residue onto target proteins confined within the cytosolic and nuclear compartments. A form of OGT was predicted for Toxoplasma and recently we were the first to show evidence of O-GlcNAcylation in the apicomplexans Toxoplasma gondii and Plasmodium falciparum. Numerous studies have explored the O-GlcNAcome in a wide variety of biological models but very few focus on protists. In the present work, we used enrichment on sWGA-beads and immunopurification to identify putative O-GlcNAcylated proteins in Toxoplasma gondii. Many of the proteins found to be O-GlcNAcylated were originally described in higher eukaryotes and participate in cell shape organization, response to stress, protein synthesis and metabolism. In a more original way, our proteomic analyses, confirmed by sWGA-enrichment and click-chemistry, revealed that rhoptries, proteins necessary for invasion, are glycosylated. Together, these data show that regardless of proteins strictly specific to organisms, O-GlcNAcylated proteins are rather similar among living beings.