Photocross-Linked Peptide-Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes

International audienceProtein–protein interactions are among the keys to organizing cellular processes in space and time. One of the only direct ways to identify such interactions in their cellular environment is to covalently bond the interacting partners to fix the interaction. Photocross-linking in living cells is thus a very promising technique. The feasibility of in cellulo photocross-linking reactions has been shown and mass spectrometry is a tool of choice to analyze photocross-linked proteins. However, the interpretation of the MS and MS/MS spectra of photocross-linked peptides remains one of the most important bottlenecks of the method and still limits its potential for large-scale applications (interactomics). Fundamental studies are still necessary to understand and characterize the fragmentation behavior of photocross-linked peptides. Here, we report the successful identification of the interaction sites in a well-characterized model of in vitro interaction between a protein and a peptide. We describe in detail the fragmentation pattern of these photocross-linked species in order to identify trends that could be generalized. In particular, we compare CID and ETD fragmentation modes (and HCD in a lesser extent), demonstrating the complementarity of both methods and the advantage of ETD for the analysis of photocross-linked species. The information should help further development of dedicated software to properly score MS/MS spectra of photocross-linked species

An integrated cross-linking-MS approach to investigate cell penetrating peptides interacting partners

Cell penetrating peptides (CPPs) are attracting attention because of their ability to deliver biologically active molecules into cells. On their way they can interact with membrane and intracellular proteins. To fully understand and improve CPP efficiency as drug delivery tools, their partners need to be identified. To investigate CPP-protein complexes, chemical cross-linking coupled to mass spectrometry is a relevant method. With this aim, we developed an original approach based on two parallel strategies, an intact complex analysis and a bottom-up one, to have a global characterization of the cross-linked complexes composition as well as a detailed mapping of the interaction zones. Biological significance: The robust and efficient cross-linking-MS workflow presented here can easily be adapted to any CPP-protein interacting system and could thus contribute to a better understanding of CPPs activity as cell-specific drug delivery tools. We validated the relevancy of this cross-linking-MS approach with two biologically active CPPs, (R/W)9 and (R/W)16, and two interacting protein partners, actin and albumin, previously reported using isothermal titration calorimetry (ITC) and NMR. Cross-linking-MS results obtained on these previous studies allowed us to go further by providing a detailed mapping of the interaction zones. The identified interaction zones between actin and CPPs (R/W)9 and (R/W)16 are biologically meaningful. Two cross-linked zones [46–57] and [202–210] of actin are indeed involved in the modulation of its dynamics. Moreover, [46–57] domain has also been described as one interaction domain for thymosin β4 whose actin binding can be displaced by competition with (R/W)16 (NMR experiments)

Proteomic comparison of the EWS-FLI1 expressing cells EF with NIH-3T3 and actin remodeling effect of (R/W)9 cell-penetrating peptide

EWS-FLI1 expression in NIH-3T3 fibroblasts has a profound impact on the phenotype, resulting in the cytoskeleton and adhesive capacity disorganization (EF cells). Besides this, (R/W)9, a cell-penetrating peptide (CPP), has an intrinsic actin remodeling activity in EF cells. To evaluate the impact of the oncogenic protein EWS-FLI1 on proteins expression levels, a quantitative comparison of tumoral EF and non-tumoral 3T3 proteomes was performed. Then to see if we could link the EWS-FLI1 oncogenic transformation to the phenotype reversion induced by (R/W)9, (R/W)9 influence on EF cells proteome was assessed. To our knowledge no such “CPPomic” study has been performed before. Biological significance: Up to now very few global quantitative proteomic studies have been published to help understand the oncogenic transformation induced by EWS-FLI1 fusion protein and leading to Ewing sarcoma development and dissemination. The comparison we did in this study between a model tumoral cell line EF and its non-tumoral counterpart (3T3) allowed us to highlight several features either common to most tumor types or specific to Ewing sarcoma. Particularly, lack of actin cytoskeleton organization could very likely be explained by the down-regulation of many important actin binding proteins. These results are in accordance with the hypothesis of a passive/stochastic mode of dissemination conferring Ewing sarcoma tumoral cell a high metastatic potential

Barium and molybdenum records in bivalve shells: Geochemical proxies for phytoplankton dynamics in coastal environments?

International audienceBarium:calcium and molybdenum:calcium ratios were investigated in shells of the tropical scallop Comptopallium radula. Three juvenile specimens were harvested alive in the southwest lagoon of New Caledonia after a 1-year hydrological survey. Calcite samples representing a few hours of biomineralization were laser-ablated along the maximal growth axis and analyzed for Ba and Mo content using an inductively coupled plasma mass spectrometer. Absolute dates of shell precipitation assigned on the basis of periodic formation of shell growth patterns led to the accurate reconstruction of ontogenetic variations of elemental ratios with subweekly resolution. Inter-individual variability of Ba:Ca and Mo:Ca time series was low, indicating an environmental control on the incorporation of these elements within shells. Both profiles were characterized by a background level punctuated by sharp peaks. The ingestion of diatoms enriched in Ba (adsorbed on iron oxyhydroxides associated with the frustules) is the most likely cause of the formation of Ba:Ca peaks. Some contribution of diatom-associated barite is also possible. In every instance, Ba:Ca would possibly be a proxy for the timing and magnitude of diatom blooms. Among all the theories that could be advanced to explain the occurrence of Mo:Ca peaks, the most plausible appears to be the ingestion of phytoplankton cells grown upon NO3- and therefore containing high levels of Mo required for the activity of nitrate reductase. If this is so, then Mo:Ca could be a new proxy for nitrate uptake by phytoplankton in coastal ecosystems, helping to reconstruct the balance between new and regenerated production in paleoenvironments

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb

International audienceMultispecific antibodies, which target multipleantigens at once, are emerging as promising therapeutic entities tooffer more effective treatment than conventional monoclonalantibodies (mAbs). However, these highly complex mAb formatspose significant analytical challenges. We report here on thecharacterization of a trispecific antibody (tsAb), which presentstwo isomeric forms clearly separated and identified with sizeexclusion chromatography coupled to native mass spectrometry(SEC-nMS). Previous studies showed that these isomers mightoriginate from a proline cis/trans isomerization in one Fab subunit ofthe tsAb. We combined several innovative ion mobility (IM)-basedapproaches to confirm the isomeric nature of the two species and togain new insights into the conformational landscape of both isomers.Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence ofdifferent conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfoldingbehaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our earlygeneration IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of twoconformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument.Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challengingissues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MSmethodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depthconformational characterization of these product

Le conseiller principal d'éducation : un acteur éducatif méconnu ?

Le métier de conseiller principal d’éducation (CPE), spécificité du système scolaire français, est un professionnel dont les missions riches et variées restent au final peu connues, même si paradoxalement, le personnage est souvent très bien identifié des élèves et des familles. Ce numéro thématique s’attache ainsi à mieux faire découvrir les contours et les enjeux du métier, en mettant tout d’abord l’accent sur sa dimension historique dont l’héritage est encore très présent dans les représentations collectives, puis en s’intéressant à l’activité quotidienne du CPE aujourd’hui au sein de l’établissement secondaire et aux différents aspects de sa formation professionnelle

Le conseiller principal d'éducation : un acteur éducatif méconnu ?

Le métier de conseiller principal d’éducation (CPE), spécificité du système scolaire français, est un professionnel dont les missions riches et variées restent au final peu connues, même si paradoxalement, le personnage est souvent très bien identifié des élèves et des familles. Ce numéro thématique s’attache ainsi à mieux faire découvrir les contours et les enjeux du métier, en mettant tout d’abord l’accent sur sa dimension historique dont l’héritage est encore très présent dans les représentations collectives, puis en s’intéressant à l’activité quotidienne du CPE aujourd’hui au sein de l’établissement secondaire et aux différents aspects de sa formation professionnelle

Multi-OMIC profiling of survival and metabolic signaling networks in cells subjected to photodynamic therapy

Photodynamic therapy (PDT) is an established palliative treatment for perihilar cholangiocarcinoma that is clinically promising. However, tumors tend to regrow after PDT, which may result from the PDT-induced activation of survival pathways in sublethally afflicted tumor cells. In this study, tumor-comprising cells (i.e., vascular endothelial cells, macrophages, perihilar cholangiocarcinoma cells, and EGFR-overexpressing epidermoid cancer cells) were treated with the photosensitizer zinc phthalocyanine that was encapsulated in cationic liposomes (ZPCLs). The post-PDT survival pathways and metabolism were studied following sublethal (LC50) and supralethal (LC90) PDT. Sublethal PDT induced survival signaling in perihilar cholangiocarcinoma (SK-ChA-1) cells via mainly HIF-1-, NF-кB-, AP-1-, and heat shock factor (HSF)-mediated pathways. In contrast, supralethal PDT damage was associated with a dampened survival response. PDT-subjected SK-ChA-1 cells downregulated proteins associated with EGFR signaling, particularly at LC90. PDT also affected various components of glycolysis and the tricarboxylic acid cycle as well as metabolites involved in redox signaling. In conclusion, sublethal PDT activates multiple pathways in tumor-associated cell types that transcriptionally regulate cell survival, proliferation, energy metabolism, detoxification, inflammation/angiogenesis, and metastasis. Accordingly, tumor cells sublethally afflicted by PDT are a major therapeutic culprit. Our multi-omic analysis further unveiled multiple druggable targets for pharmacological co-interventio