Peptides vecteurs et protéomique intracellulaire (détection de phosphorylations par spectrométrie de masse MALDI-TOF)

PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

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

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

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)

MALDI-TOFMS identification of "odorant binding proteins" (OBPs) electroblotted onto poly(vinylidence difluoride) membranes

International audienc

Dosage et pistage de peptides Troyens dans les cellules

Les peptides Troyens ou vecteurs sont capables de passer les membranes biologiques et de véhiculer des principes actifs dans le cytoplasme ou le noyau des cellules dans lesquelles ils sont entrés. Les méthodes indirectes utilisées jusqu'à présent pour détecter ces peptides dans les cellules n'ont pas permis d'établir de manière univoque le(s) mécanisme(s) de leur internalisation. La méthode de quantification, basée sur la spectrométrie de masse MALDI-TOF, que nous avons mise au point pour quantifier ces peptides Troyens dans les cellules est développée dans cette revue

Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells.

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Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

11 pagesInternational audienceThe formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods)