Diel Cycle Proteomics: Illuminating Molecular Dynamics in Purple Bacteria for Optimized Biotechnological Applications.

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes-whether under continuous light or alternating light and dark cycles-could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms.

Proteomic differences in seminal fluid of social insects whose sperm differ in heat tolerance.

In the coming years, climate change is likely to increase the frequency and intensity of heatwaves. In many organisms, heat stress provokes physiological perturbations and can lead to decreased male fertility. Bumblebees are endo-heterothermic but display interspecific differences in thermotolerance that could have conservation implications. For the species of concern Bombus magnus, exposure to high temperatures can severely reduce sperm quality and, consequently, reproductive success. Such is not the case for B. terrestris, a ubiquitous species. To decipher the mechanisms at play, we characterized the seminal fluid proteomes of the two species. We quantified 1121 proteins, of which 522 were differentially expressed between B. terrestris and B. magnus. Several proteins with protective functions, such as proteases, antioxidant proteins and various heat-shock proteins, were present at higher levels in B. terrestris than in B. magnus under both control and heat-stress conditions. The same was true for proteins involved in cellular homeostasis, immunity, lipid/sugar metabolism and thermotolerance. Furthermore, proteins involved in the capture and elimination of reactive oxygen species also occurred at much high levels in B. terrestris. Overall, these results clearly indicate differences in the seminal proteome of the more thermotolerant B. terrestris versus B. magnus. The differences may contribute to explaining interspecific differences in sperm survival.

Mass spectrometry analysis of saponins.

Saponins are amphiphilic molecules of pharmaceutical interest and most of their biological activities (i.e., cytotoxic, hemolytic, fungicide, etc.) are associated to their membranolytic properties. These molecules are secondary metabolites present in numerous plants and in some marine animals, such as sea cucumbers and starfishes. Structurally, all saponins correspond to the combination of a hydrophilic glycan, consisting of sugar chain(s), linked to a hydrophobic triterpenoidic or steroidic aglycone, named the sapogenin. Saponins present a high structural diversity and their structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, the extreme structural diversity of saponins makes them challenging from a structural analysis point of view since, most of the time, saponin extracts consist in a huge number of congeners presenting only subtle structural differences. In the present review, we wish to offer an overview of the literature related to the development of mass spectrometry for the study of saponins. This review will demonstrate that most of the past and current mass spectrometry methods, including electron, electrospray and matrix-assisted laser desorption/ionization ionizations, gas/liquid chromatography coupled to (tandem) mass spectrometry, collision-induced dissociation including MS3 experiments, multiple reaction monitoring based quantification, ion mobility experiments, and so forth, have been used for saponin investigations with great success on enriched extracts but also directly on tissues using imaging methods.

Cataglyphis desert ants use distinct behavioral and physiological adaptations to cope with extreme thermal conditions.

Some ant species live in hot and arid environments, such as deserts and savannas. Worker polymorphism-variation in worker size and/or morphology within colonies-is adaptive in such ecosystems because it enhances resistance to heat stress and increases the efficiency of resource exploitation. However, species with small, monomorphic workers are also frequently found in these environments. How species with distinct worker size and degrees of polymorphism deal with such stressful environments remains poorly studied. We investigated the behavioral, physiological, and molecular adaptations that may enhance heat and desiccation tolerance in two sympatric species of Cataglyphis desert ants that differ dramatically in worker size and polymorphism: C. viatica is polymorphic, while C. cubica is small and monomorphic. We found that worker size, water content, water loss, and protein regulation play a key role in thermal resistance. (i) Large C. viatica workers better tolerated heat and desiccation stress than did small C. viatica or C. cubica workers. The former had greater water content and lost proportionally less water to evaporation under thermal stress. (ii) Despite their similar size distribution, workers of C. cubica are more heat tolerant than small C. viatica. This higher degree of tolerance likely stemmed from C. cubica workers having greater relative water content. (iii) Under thermal stress, small C. viatica workers metabolized larger quantities of fat and differentially expressed proteins involved in cellular homeostasis. In contrast, C. cubica downregulated the expression of numerous proteins involved in mitochondrial respiration likely reducing ROS accumulation. (iv) Consistent with these results, large C. viatica workers remained active throughout the day; C. cubica workers displayed a bimodal activity pattern, and small C. viatica remained poorly active outside the nest. Our study shows that ecologically similar ant species with different degrees of worker size polymorphism evolved distinct strategies for coping with extreme heat conditions.

Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process.

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.

Discrimination of Regioisomeric and Stereoisomeric Saponins from Aesculus hippocastanum Seeds by Ion Mobility Mass Spectrometry.

Modern mass spectrometry methods provide a huge benefit to saponin structural characterization, especially when combined with collision-induced dissociation experiments to obtain a partial description of the saponin (ion) structure. However, the complete description of the structures of these ubiquitous secondary metabolites remain challenging, especially since isomeric saponins presenting small differences are often present in a single extract. As a typical example, the horse chestnut triterpene glycosides, the so-called escins, comprise isomeric saponins containing subtle differences such as cis-trans ethylenic configuration (stereoisomers) of a side chain or distinct positions of an acetyl group (regioisomers) on the aglycone. In the present paper, the coupling of liquid chromatography and ion mobility mass spectrometry has been used to distinguish regioisomeric and stereoisomeric saponins. Ion mobility arrival time distributions (ATDs) were recorded for the stereoisomeric and regioisomeric saponin ions demonstrating that isomeric saponins can be partially separated using ion mobility on a commercially available traveling wave ion mobility (TWIMS) mass spectrometer. Small differences in the ATD can only be monitored when the isomeric saponins are separated with liquid chromatography prior to the IM-MS analysis. However, gas phase separation between stereoisomeric and regioisomeric saponin ions can be successfully realized, without any LC separation, on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The main outcome of the present paper is that the structural analysis of regioisomeric and stereoisomeric natural compounds that represents a real challenge can take huge advantages of ion mobility experiments but only if increased ion mobility resolution is attainable.

Triterpenoids in Echinoderms: Fundamental Differences in Diversity and Biosynthetic Pathways.

Echinoderms form a remarkable phylum of marine invertebrates that present specific chemical signatures unique in the animal kingdom. It is particularly the case for essential triterpenoids that evolved separately in each of the five echinoderm classes. Indeed, while most animals have Δ5-sterols, sea cucumbers (Holothuroidea) and sea stars (Asteroidea) also possess Δ7 and Δ9(11)-sterols, a characteristic not shared with brittle stars (Ophiuroidea), sea urchins (Echinoidea), and crinoids (Crinoidea). These particular Δ7 and Δ9(11) sterols emerged as a self-protection against membranolytic saponins that only sea cucumbers and sea stars produce as a defense mechanism. The diversity of saponins is large; several hundred molecules have been described in the two classes of these saponins (i.e., triterpenoid or steroid saponins). This review aims to highlight the diversity of triterpenoids in echinoderms by focusing on sterols and triterpenoid glycosides, but more importantly to provide an updated view of the biosynthesis of these molecules in echinoderms.

Ion mobility mass spectrometry of saponin ions.

RATIONALE: Saponins are natural compounds presenting a high structural diversity whose structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, saponins are also increasingly characterized using tandem mass spectrometry (MS/MS) coupled with liquid chromatography, even if collision-induced dissociation (CID) experiments are often quite limited in accurately determining the saponin structure. METHODS: We consider here ion mobility mass spectrometry (IMMS) as an orthogonal tool for the structural characterization of saponin isomers by comparing the experimental collisional cross sections (CCSs) of saponin ions with theoretical CCSs for candidate ion structures. Indeed, state-of-the-art theoretical calculations perfectly complement the experimental results, allowing the ion structures to be deciphered at the molecular level. RESULTS: We demonstrate that ion mobility can contribute to the structural characterization of saponins because different saponin ions present significantly distinct CCSs. Depending on the nature of the cation (in the positive ion mode), the differences in CCSs can also be exacerbated, optimizing the gas-phase separation. When associated with molecular dynamics simulations, the CCS data can be used to describe the interactions between the cations, i.e. H+ , Na+ and K+ , and the saponin molecules at a molecular level. CONCLUSIONS: Our work contributes to resolve the relationship between the primary and secondary structures of saponin ions. However, it is obvious that the structural diversity and complexity of the saponins cannot be definitively unraveled by measuring a single numerical value, here the CCS. Consequently, the structural characterization of unknown saponins will be difficult to achieve based on IMMS alone. Nevertheless, we demonstrated that monodesmosidic and bidesmosidic saponins can be distinguished via IMMS.

Identification and quantification of spinochromes in body compartments of Echinometra mathaei's coloured types.

Sea urchin pigmentation is mainly due to polyhydroxy-1,4-naphthoquinones called spinochromes. If their molecular structures are well known in test and spines of many species, their abundance and distribution in other body compartments remain unstudied. The aim of this study is to analyse the pigment composition in four body compartments (test/spines, digestive system, gonads and coelomic fluid) of four coloured types of the sea urchin Echinometra mathaei. Qualitative and quantitative measurements by mass spectrometry highlight the existence of 13 different pigments; among which are five isomers of known spinochromes as well as three potentially new ones. The composition comparison shows the largest spinochrome diversity in 'test/spines' body compartments. The spinochrome concentrations vary from 48 to 1279 mg kg-1 of dried body compartment. It is the highest in the digestive system, although it is also important in the organic fraction of the 'test/spines' body compartment. This observation may be explained by higher exposures of some body compartments to external environments and by the protective role fulfilled by spinochromes against microorganisms, ultraviolet radiation and reactive oxygen species. The 'black' type-the most common coloured type in coral reefs-has the highest concentration of spinochromes indicating their importance in Echinoids' fitness by acting as a protective agent.

Is non-host pollen suitable for generalist bumblebees?

Current evidence suggests that pollen is both chemically and structurally protected. Despite increasing interest in studying bee-flower networks, the constraints for bee development related to pollen nutritional content, toxicity and digestibility as well as their role in the shaping of bee-flower interactions have been poorly studied. In this study we combined bioassays of the generalist bee Bombus terrestris on pollen of Cirsium, Trifolium, Salix, and Cistus genera with an assessment of nutritional content, toxicity, and digestibility of pollen. Microcolonies showed significant differences in their development, non-host pollen of Cirsium being the most unfavorable. This pollen was characterized by the presence of quite rare δ7-sterols and a low digestibility. Cirsium consumption seemed increase syrup collection, which is probably related to a detoxification mixing behavior. These results strongly suggest that pollen traits may act as drivers of plant selection by bees and partly explain why Asteraceae pollen is rare in bee generalist diet.

Tackling saponin diversity in marine animals by mass spectrometry: data acquisition and integration.

Saponin analysis by mass spectrometry methods is nowadays progressively supplementing other analytical methods such as nuclear magnetic resonance (NMR). Indeed, saponin extracts from plant or marine animals are often constituted by a complex mixture of (slightly) different saponin molecules that requires extensive purification and separation steps to meet the requirement for NMR spectroscopy measurements. Based on its intrinsic features, mass spectrometry represents an inescapable tool to access the structures of saponins within extracts by using LC-MS, MALDI-MS, and tandem mass spectrometry experiments. The combination of different MS methods nowadays allows for a nice description of saponin structures, without extensive purification. However, the structural characterization process is based on low kinetic energy CID which cannot afford a total structure elucidation as far as stereochemistry is concerned. Moreover, the structural difference between saponins in a same extract is often so small that coelution upon LC-MS analysis is unavoidable, rendering the isomeric distinction and characterization by CID challenging or impossible. In the present paper, we introduce ion mobility in combination with liquid chromatography to better tackle the structural complexity of saponin congeners. When analyzing saponin extracts with MS-based methods, handling the data remains problematic for the comprehensive report of the results, but also for their efficient comparison. We here introduce an original schematic representation using sector diagrams that are constructed from mass spectrometry data. We strongly believe that the proposed data integration could be useful for data interpretation since it allows for a direct and fast comparison, both in terms of composition and relative proportion of the saponin contents in different extracts. Graphical Abstract A combination of state-of-the-art mass spectrometry methods, including ion mobility spectroscopy, is developed to afford a complete description of the saponin molecules in natural extracts.

Inter- and intra-organ spatial distributions of sea star saponins by MALDI imaging.

Saponins are secondary metabolites that are abundant and diversified in echinoderms. Mass spectrometry is increasingly used not only to identify saponin congeners within animal extracts but also to decipher the structure/biological activity relationships of these molecules by determining their inter-organ and inter-individual variability. The usual method requires extensive purification procedures to prepare saponin extracts compatible with mass spectrometry analysis. Here, we selected the sea star Asterias rubens as a model animal to prove that direct analysis of saponins can be performed on tissue sections. We also demonstrated that carboxymethyl cellulose can be used as an embedding medium to facilitate the cryosectioning procedure. Matrix-assisted laser desorption/ionization (MALDI) imaging was also revealed to afford interesting data on the distribution of saponin molecules within the tissues. We indeed highlight that saponins are located not only inside the body wall of the animals but also within the mucus layer that probably protects the animal against external aggressions. Graphical Abstract Saponins are the most abundant secondary metabolites in sea stars. They should therefore participate in important biological activities. Here, MALDI imaging is presented as a powerful method to determine the spatial distribution of saponins within the animal tissues. The inhomogeneity of the intra-organ saponin distribution is highlighted, paving the way for future elegant structure/activity relationship investigations.