Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones.

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.

Species identification of ivory and bone museum objects using minimally invasive proteomics.

Ivory is a highly prized material in many cultures since it can be carved into intricate designs and have a highly polished surface. Due to its popularity, the animals from which ivory can be sourced are under threat of extinction. Identification of ivory species is not only important for CITES compliance, it can also provide information about the context in which a work was created. Here, we have developed a minimally invasive workflow to remove minimal amounts of material from precious objects and, using high-resolution mass spectrometry-based proteomics, identified the taxonomy of ivory and bone objects from The Metropolitan Museum of Art collection dating from as early as 4000 B.C. We built a proteomic database of underrepresented species based on exemplars from the American Museum of Natural History, and proposed alternative data analysis workflows for samples containing inconsistently preserved organic material. This application demonstrates extensive ivory species identification using proteomics to unlock sequence uncertainties, e.g., Leu/Ile discrimination.

Tau seeds from Alzheimer's disease brains trigger tau spread in macaques while oligomeric-Aß mediates pathology maturation.

INTRODUCTION: The "prion-like" features of Alzheimer's disease (AD) tauopathy and its relationship with amyloid-ß (Aß) have never been experimentally studied in primates phylogenetically close to humans. METHODS: We injected 17 macaques in the entorhinal cortex with nanograms of seeding-competent tau aggregates purified from AD brains or control extracts from aged-matched healthy brains, with or without intracerebroventricular co-injections of oligomeric-Aß. RESULTS: Pathological tau injection increased cerebrospinal fluid (CSF) p-tau181 concentration after 18 months. Tau pathology spreads from the entorhinal cortex to the hippocampal trisynaptic loop and the cingulate cortex, resuming the experimental progression of Braak stage I to IV. Many AD-related molecular networks were impacted by tau seeds injections regardless of Aß injections in proteomic analyses. However, we found mature neurofibrillary tangles, increased CSF total-tau concentration, and pre- and postsynaptic degeneration only in Aß co-injected macaques. DISCUSSION: Oligomeric-Aß mediates the maturation of tau pathology and its neuronal toxicity in macaques but not its initial spreading. HIGHLIGHTS: This study supports the "prion-like" properties of misfolded tau extracted from AD brains. This study empirically validates the Braak staging in an anthropomorphic brain. This study highlights the role of oligomeric Aß in driving the maturation and toxicity of tau pathology. This work establishes a novel animal model of early sporadic AD that is closer to the human pathology.

Nrf2 Downregulation Contributes to Epithelial-to-Mesenchymal Transition in Helicobacter pylori-Infected Cells.

BACKGROUND: Gastric cancer, the fifth most common cancer worldwide, is mainly linked to Helicobacter pylori infection. H. pylori induces chronic inflammation of the gastric mucosa associated with high oxidative stress. Our study aimed at assessing the implication of Nrf2, a major regulator of cellular redox homeostasis, in H. pylori-induced gastric carcinogenesis. METHODS: Using three different gastric epithelial cell lines, a non-cancerous (HFE-145) and two different subtypes of gastric cancer (AGS and MKN74), we analyzed the modulation of Nrf2 expression over time. After invalidation of Nrf2 by CRISPR-cas9, we assessed its role in H. pylori-induced epithelial-to-mesenchymal transition (EMT). Finally, we evaluated the expression of Nrf2 and ZEB1, a central EMT transcription factor, in human gastric tissues. RESULTS: We first demonstrated that the Nrf2 signaling pathway is differentially regulated depending on the infection stage. Rapidly and transiently activated, Nrf2 was downregulated 24 h post-infection in a VacA-dependent manner. We then demonstrated that Nrf2 invalidation leads to increased EMT, which is even exacerbated after H. pylori infection. Finally, Nrf2 expression tended to decrease in human patients' gastric mucosa infected with H. pylori. CONCLUSIONS: Our work supports the hypothesis that Nrf2 downregulation upon H. pylori infection participates in EMT, one of the most important events in gastric carcinogenesis.

Hydrogen­deuterium exchange mass spectrometry to study interactions and conformational changes of proteins in paints.

Little is known about structural alterations of proteins within the polymeric films of paints. For the first time, hydrogen­deuterium exchange mass spectrometry (HDX-MS) was implemented to explore the conformational alterations of proteins resulting from their interaction with inorganic pigments within the early stages of the paint film formation. Intact protein analysis and bottom-up electrospray-ionisation mass spectrometry strategies combined with progressively increasing deuterium incubation times were used to compare the protein structures of the model protein hen egg-white lysozyme (HEWL) extracted from newly dried non-pigmented films and newly dried films made from a freshly made mixture of HEWL with lead white pigment (2PbCO3 Pb(OH)2). The action of other pigments was also investigated, expanding the HDX study with a global approach to paint models of HEWL mixed with zinc white (ZnO), cinnabar (HgS) and red lead (Pb3O4) pigments. The results show structural modifications of HEWL induced by the interaction with the pigment metal ions during the paint formulation after drying and prior to ageing. Both the charge distribution of HEWL proteoforms, its oxidation rate and its deuterium absorption rate, were influenced by the pigment type, providing the first insights into the correlation of pigment type/metal cation to specific chemistries related to protein stability.

Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in inflammatory bowel diseases.

Human secretory immunoglobulins (SIg) A1 and SIgA2 guide mucosal responses toward tolerance or inflammation, notably through reverse-transcytosis, the apical-to-basal transport of IgA2 immune complexes via M cells of gut Peyer's patches. As such, the maintenance of a diverse gut microbiota requires broad affinity IgA and glycan-glycan interaction. Here, we asked whether IgA1 and IgA2-microbiota interactions might be involved in dysbiosis induction during inflammatory bowel diseases. Using stool HPLC-purified IgA, we show that reverse-transcytosis is abrogated in ulcerative colitis (UC) while it is extended to IgA1 in Crohn's disease (CD). 16S RNA sequencing of IgA-bound microbiota in CD and UC showed distinct IgA1- and IgA2-associated microbiota; the IgA1+ fraction of CD microbiota was notably enriched in beneficial commensals. These features were associated with increased IgA anti-glycan reactivity in CD and an opposite loss of reactivity in UC. Our results highlight previously unknown pathogenic properties of IgA in IBD that could support dysbiosis.

Analysis of the Phospholipid Profile of the Collection Strain PAO1 and Clinical Isolates of Pseudomonas aeruginosa in Relation to Their Attachment Capacity.

Bacteria form multicellular and resistant structures named biofilms. Biofilm formation starts with the attachment phase, and the molecular actors involved in this phase, except adhesins, are poorly characterized. There is growing evidence that phospholipids are more than simple structural bricks. They are involved in bacterial adaptive physiology, but little is known about their role in biofilm formation. Here, we report a mass spectrometry analysis of the phospholipid (PL) profile of several strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients. The aim of our study was to evaluate a possible link between the PL profile of a strain and its attachment phenotype. Our results showed that PL profile is strongly strain-dependent. The PL profile of P. aeruginosa PAO1, a collection strain, was different from those of 10 clinical isolates characterized either by a very low or a very high attachment capacity. We observed also that the clinical strain's PL profiles varied even more importantly between isolates. By comparing groups of strains having similar attachment capacities, we identified one PL, PE 18:1-18:1, as a potential molecular actor involved in attachment, the first step in biofilm formation. This PL represents a possible target in the fight against biofilms.

Characterization of Isomers of Lipid A from Pseudomonas aeruginosa PAO1 by Liquid Chromatography with Tandem Mass Spectrometry with Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.

Lipopolysaccharides (LPS) constitute the outermost layer of Gram-negative bacteria and consequently play an important role in bacterial infections. In order to address public health issues posed by Gram-negative bacteria, it is necessary to elucidate the structure of the molecular actors at the forefront of infections. LPS virulence and toxicity are partially modulated by lipid A, a hydrophobic saccharolipid that anchors LPS to the bacterial outer membrane. Understanding the lipid A structure is inherently intertwined with understanding its role as an endotoxin. Accordingly, several successful strategies incorporating tandem mass spectrometry have been applied toward the structural analysis of lipid A. Herein, a shotgun HCD strategy was applied toward the characterization of the lipid A profile of Pseudomonas aeruginosa PAO1. This analysis was enhanced by the development of an LC-MS/MS approach to eliminate isomeric signals in the MS/MS spectra that confounded characterization. Importantly, combining reverse phase chromatography with HCD and ultraviolet photodissociation analyses of the lipid A profile revealed the presence of previously unreported lipid A acyl chain positional isomers. Altogether, these strategies provide the most in-depth structural and molecular characterization of PAO1 lipid A to date.

Lipid selectivity in detergent extraction from bilayers.

Despite numerous studies on detergent-induced solubilization of membranes and on the underlying mechanisms associated with this process, very little is known regarding the selectivity of detergents for lipids during their extraction from membranes. To get insights about this phenomenon, solubilization of model bilayers prepared from binary lipid mixtures by different detergents was examined. Three commonly used detergents were used: the non-ionic Triton X-100 (TX), the negatively-charged sodium dodecylsulfate (SDS), and the positively-charged n-dodecyltrimethylammonium chloride (DTAC). Two model membranes were used in order to identify if specific intermolecular interactions can lead to lipid selectivity: bilayers made of a binary mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and of a binary mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Therefore, it was possible to describe systems presenting a combination of detergents bearing different charges with bilayers with different polymorphic propensities and charge. In conditions for which partial solubilization was observed, the composition of the extracted lipid phase was quantified with Liquid Chromatography coupled to Mass Spectrometry to elucidate whether a lipid selectivity occurred in the solubilization process. On one hand, it is found that repulsive or attractive electrostatic interactions did not lead to any lipid selectivity. On the other hand, POPE was systematically less extracted than POPC, regardless of the detergent nature. We propose that this lipid selectivity is inherent to the molecular shape of POPE unsuited for micelles curvature properties.

'Palaeoshellomics' reveals the use of freshwater mother-of-pearl in prehistory.

The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including 'palaeoshellomics', the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of 'double-buttons'. This craft is found throughout Europe between 4200-3800 BCE, highlighting the ornament-makers' profound knowledge of the biogeosphere and the existence of cross-cultural traditions.

Proteome Analysis of Potato Starch Reveals the Presence of New Starch Metabolic Proteins as Well as Multiple Protease Inhibitors.

Starch bound proteins mainly include enzymes from the starch biosynthesis pathway. Recently, new functions in starch molecular assembly or active protein targeting were also proposed for starch associated proteins. The potato genome sequence reveals 77 loci encoding starch metabolizing enzymes with the identification of previously unknown putative isoforms. Here we show by bottom-up proteomics that most of the starch biosynthetic enzymes in potato remain associated with starch even after washing with SDS or protease treatment of the granule surface. Moreover, our study confirmed the presence of PTST1 (Protein Targeting to Starch), ESV1 (Early StarVation1) and LESV (Like ESV), that have recently been identified in Arabidopsis. In addition, we report on the presence of a new isoform of starch synthase, SS6, containing both K-X-G-G-L catalytic motifs. Furthermore, multiple protease inhibitors were also identified that are cleared away from starch by SDS and thermolysin treatments. Our results indicate that SS6 may play a yet uncharacterized function in starch biosynthesis and open new perspectives both in understanding storage starch metabolism as well as breeding improved potato lines.

Comparative Proteomic and Transcriptomic Analysis of Follistatin-Induced Skeletal Muscle Hypertrophy.

Skeletal muscle, the most abundant body tissue, plays vital roles in locomotion and metabolism. Myostatin is a negative regulator of skeletal muscle mass. In addition to increasing muscle mass, Myostatin inhibition impacts muscle contractility and energy metabolism. To decipher the mechanisms of action of the Myostatin inhibitors, we used proteomic and transcriptomic approaches to investigate the changes induced in skeletal muscles of transgenic mice overexpressing Follistatin, a physiological Myostatin inhibitor. Our proteomic workflow included a fractionation step to identify weakly expressed proteins and a comparison of fast versus slow muscles. Functional annotation of altered proteins supports the phenotypic changes induced by Myostatin inhibition, including modifications in energy metabolism, fiber type, insulin and calcium signaling, as well as membrane repair and regeneration. Less than 10% of the differentially expressed proteins were found to be also regulated at the mRNA level but the Biological Process annotation, and the KEGG pathways analysis of transcriptomic results shows a great concordance with the proteomic data. Thus this study describes the most extensive omics analysis of muscle overexpressing Follistatin, providing molecular-level insights to explain the observed muscle phenotypic changes.

Plant gum identification in historic artworks.

We describe an integrated and straightforward new analytical protocol that identifies plant gums from various sample sources including cultural heritage. Our approach is based on the identification of saccharidic fingerprints using mass spectrometry following controlled enzymatic hydrolysis. We developed an enzyme cocktail suitable for plant gums of unknown composition. Distinctive MS profiles of gums such as arabic, cherry and locust-bean gums were successfully identified. A wide range of oligosaccharidic combinations of pentose, hexose, deoxyhexose and hexuronic acid were accurately identified in gum arabic whereas cherry and locust bean gums showed respectively PentxHexy and Hexn profiles. Optimized for low sample quantities, the analytical protocol was successfully applied to contemporary and historic samples including 'Colour Box Charles Roberson &Co' dating 1870s and drawings from the American painter Arthur Dove (1880-1946). This is the first time that a gum is accurately identified in a cultural heritage sample using structural information. Furthermore, this methodology is applicable to other domains (food, cosmetic, pharmaceutical, biomedical).

Multianalytical Study of Historical Luminescent Lithopone for the Detection of Impurities and Trace Metal Ions.

We have explored the performance of an integrated multianalytical approach to the analysis of a series of microsamples of historical lithopone (a coprecipitate of ZnS + BaSO4) produced at the beginning of the 20th century, based on the combination of spectrally- and lifetime-resolved photoluminescence (PL) microscopy imaging and electron paramagnetic resonance (EPR) spectroscopy. Multispectral imaging of the PL emission from microsamples revealed the presence of different luminescence centers emitting in the visible spectrum, which we have hypothesized as trace Cu and Mn impurities unintentionally introduced into the ZnS crystal lattice during synthesis, which act as deep traps for electrons. Time-resolved PL imaging analyses highlighted the microsecond decay-kinetic behavior of the emission, confirming the trap state nature of the luminescence centers. EPR confirmed the presence of Cu and Mn, further providing information on the microenvironment of defects in the ZnS crystalline lattice related to specific paramagnetic ions. The multianalytical approach provides important insights into the historical synthesis of lithophone and will be useful for the rapid screening and mapping of impurities in complex semiconductor pigments and other artists' materials.

To settle or to move? The interplay between two classes of cyclic lipopeptides in the biocontrol strain Pseudomonas CMR12a.

Pseudomonas CMR12a is a biocontrol strain that produces phenazine antibiotics and as yet uncharacterized cyclic lipopeptides (CLPs). The CLPs of CMR12a were studied by chemical structure analysis and in silico analysis of the gene clusters encoding the non-ribosomal peptide synthetases responsible for CLP biosynthesis. CMR12a produces two different classes of CLPs: orfamides B, D and E, whereby the latter two represent new derivatives of the orfamide family, and sessilins A-C. The orfamides are made up of a 10 amino acid peptide coupled to a ß-hydroxydodecanoyl or ß-hydroxytetradecanoyl fatty acid moiety, and are related to orfamides produced by biocontrol strain Pseudomonas protegens Pf-5. The sessilins consist of an 18-amino acid peptide linked to a ß-hydroxyoctanoyl fatty acid and differ in one amino acid from tolaasins, toxins produced by the mushroom pathogen Pseudomonas tolaasii. CLP biosynthesis mutants were constructed and tested for biofilm formation and swarming motility. Orfamides appeared indispensable for swarming while sessilin mutants showed reduced biofilm formation, but enhanced swarming motility. The interplay between the two classes of CLPs fine tunes these processes. The presence of sessilins in wild type CMR12a interferes with swarming by hampering the release of orfamides and by co-precipitating orfamides to form a white line in agar.

Proteomics applied to the authentication of fish glue: application to a 17th century artwork sample.

This work provides the first identification of fish glue from a few micrograms of a 17(th) century artwork sample using an adapted proteomics approach. Fish glue has been widely used as a binder in various art objects such as paintings, manuscripts or polychrome objects however its authentication remains particularly challenging. The lack of information on fish species in genomic and proteomic databases represents a major drawback. A supplementary difficulty is provided by the historical sample features, i.e. a few micrograms of a 17(th) century polychrome object with a multilayered structure. SYPRO® Ruby staining was used as a screening technique to probe the presence of proteins in the sample cross-section. Results revealed the presence of several layers containing proteins among which a thin proteinaceous layer located between the silver leaf and the glaze. This thin layer is described as fish glue coating by historical sources but its composition has not been identified yet. The optimized methodology, based on high resolution mass spectrometry and adapted bioinformatic tools, was successfully applied to 50 µg of a polychromy sample and resulted in the identification of several collagen proteins. Extensive interpretation of data generated by tandem mass spectrometry allowed the identification of proteins from different biological origins. In particular, seven peptides specific to fish collagen proteins were identified for the first time proving the presence of fish glue in the sample and corroborating information found in historical texts dealing with the polychromy technique.

The disulfide bond between cysteine 10 and cysteine 34 is required for CCL18 activity.

Asthma is a Th2-mediated disease that involves Th2 cell and eosinophil migration into the bronchial mucosa which is dependent upon the expression of a specific set of chemokines within the lung. Among them, CCL18 seems to play a key role because of its preferential expression in the lung, and its up-regulation by Th2 cytokines. Here, we show that the optimal naïve T cell and basophil chemotaxis, and basophil histamine release induced by rhCCL18 occurred at a 100 time lower concentration with CHO-derived rhCCL18 than with E. coli-derived rhCCL18. FT-ICR mass spectrometry of the intact chemokines showed that the rhCCL18 produced by CHO cells contained the 2 disulfide bonds Cys10-Cys34 and Cys11-Cys50, in clear contrast to the rhCCL18 derived from E. coli where the Cys10-Cys34 bond was absent. We found that reduction of the Cys10-Cys34 of the CHO-derived rhCCL18 resulted in a shift of its activity, reaching the same level as the E. coli-derived rhCCL18. These results demonstrate that the Cys10-Cys34 disulfide bond is involved in the function of CCL18.

Identification of cell wall proteins in the flax (Linum usitatissimum) stem.

Sequential salt (CaCl2 , LiCl) extractions were used to obtain fractions enriched in cell wall proteins (CWPs) from the stem of 60-day-old flax (Linum usitatissimum) plants. High-resolution FT-ICR MS analysis and the use of recently published genomic data allowed the identification of 11 912 peptides corresponding to a total of 1418 different proteins. Subcellular localization using TargetP, Predotar, and WoLF PSORT led to the identification of 152 putative flax CWPs that were classified into nine different functional classes previously established for Arabidopsis thaliana. Examination of different functional classes revealed the presence of a number of proteins known to be involved in, or potentially involved in cell-wall metabolism in plants. The flax stem cell wall proteome was also compared with transcriptomic data previously obtained on comparable samples. This study represents a major contribution to the identification of CWPs in flax and will lead to a better understanding of cell wall biology in this species.