Deciphering the plant phosphoproteome: tools and strategies for a challenging task.

Protein phosphorylation constitutes a major type of post-translational modification that mobilizes a high number of genes, especially in plants, is involved in many crucial cell functions and largely participates to the complexity of the proteomes. For several biological and technical reasons, the characterization of phosphorylation sites requires complex procedures. In this review, the different approaches presently available to select phosphoproteins and phosphopeptides are described. A special emphasis is then given to the numerous strategies that have emerged for the analysis of phosphorylation sites by various techniques of mass spectrometry. Finally, the few attempts proposed for the quantification of phosphorylation events are presented. In another part, the results of the efforts made in the plant area to analyze the phosphoproteome are compared to those in other biological systems. These overviews are put together to delineate, according to the objectives pursued, the different strategies possible and the corresponding challenges.Journal ArticleResearch Support, Non-U.S. Gov'tReviewinfo:eu-repo/semantics/publishe

Proteome specific sample preparation methods for matrix assisted laser desorption/ionization mass spectrometry

info:eu-repo/semantics/publishedCell Biology 2006: A Laboratory Handbook, Elsevier Science, UK, edited by Julio E. Celi

Proteome analysis of barley seeds and malt

info:eu-repo/semantics/publishe

Wheat proteomics in the healthgrain project

The project HEALTHGRAIN concerns "Exploiting Bioactivity of European Cereal Grains for Improved Nutrition and Health Benefits". The proteome analysis within HEALTHGRAIN includes establishing the seed proteome reference map of the wheat cultivar Chinese Spring as well as proteome profiling of cultivars selected on the basis of properties relevant for health, nutrition and agronomical performance. The purpose of the proteome mapping is to provide data for integration with genetic analysis and for correlation of protein profiles with specific grain quality properties. In addition individual identified seed proteins will be characterized with regard to spatio-temporal occurrence and post-translational modifications. An overview of the current status of the project focusing on albumins and globulins and tissue localization will be given in addition to an outline of future plans.0info:eu-repo/semantics/publishe

Rhodnius prolixus Hemolymph Immuno-Physiology: Deciphering the Systemic Immune Response Triggered by Trypanosoma cruzi Establishment in the Vector Using Quantitative Proteomics

Understanding the development of Trypanosoma cruzi within the triatomine vector at the molecular level should provide novel targets for interrupting parasitic life cycle and affect vectorial competence. The aim of the current study is to provide new insights into triatomines immunology through the characterization of the hemolymph proteome of Rhodnius prolixus, a major Chagas disease vector, in order to gain an overview of its immune physiology. Surprisingly, proteomics investigation of the immunomodulation of T. cruzi-infected blood reveals that the parasite triggers an early systemic response in the hemolymph. The analysis of the expression profiles of hemolymph proteins from 6 h to 24 h allowed the identification of a broad range of immune proteins expressed already in the early hours post-blood-feeding regardless of the presence of the parasite, ready to mount a rapid response exemplified by the significant phenol oxidase activation. Nevertheless, we have also observed a remarkable induction of the immune response triggered by an rpPGRP-LC and the overexpression of defensins 6 h post-T. cruzi infection. Moreover, we have identified novel proteins with immune properties such as the putative c1q-like protein and the immunoglobulin I-set domain-containing protein, which have never been described in triatomines and could play a role in T. cruzi recognition. Twelve proteins with unknown function are modulated by the presence of T. cruzi in the hemolymph. Determining the function of these parasite-induced proteins represents an exciting challenge for increasing our knowledge about the diversity of the immune response from the universal one studied in holometabolous insects. This will provide us with clear answers for misunderstood mechanisms in host–parasite interaction, leading to the development of new generation strategies to control vector populations and pathogen transmission.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification and preliminary characterization of a putative C-type lectin receptor-like protein in the T. cruzi tomato lectin endocytic-enriched proteome

Trypanosoma cruzi, the etiological agent of the Chagas’ disease in Latin America undergoes a complex life cycle involving two hosts, a mammalian host and a reduviid insect vector (triatomine). In the insect midgut the parasite multiplies as epimastigote forms, which rely on endocytosis for their energy requirement. We recently showed that posttranslational modification of endocytic N-glycoproteins by tomato lectin (TL) binding-N-glycans is crucial for receptor-mediated endocytosis (RME) in epimastigote forms. In an attempt to characterize the endocytic proteome we used a TL affinity chromatography, which significantly enriched glycoproteins of the trypanosomal endocytic pathway. In addition to various lysosomal hydrolases, we found an endosomal C-type lectin-like protein, which displays some structural and topological characteristics of the mammalian lectin receptor superfamily. This lectin encoding a large transmembrane protein of around 375 kDa contained three putative extracellular N-terminal C-type lectin domains (CTLD) and located inside the flagellar pocket (FP)/cytostome and endosomal compartments of the insect stage of the parasite and on the surface of the plasma membrane of intracellular amastigote parasites. Noteworthy, this endogenous lectin displayed similar sugar-binding specificity to that of TL and therefore could be important in either the N-glycan mediated endocytosis or parasite adhesion to host cells. We postulated that during the evolution of trypanosomatids, genes encoding lectin harboring 3 CTDLs represent an old acquisition present in free-living, monoxenic and heteroxenic trypanosomatids, which would have been secondarily lost in extracellular parasites from the T. brucei clade.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Early post-prandial regulation of protein expression in the midgut of chagas disease vector rhodnius prolixus highlights new potential targets for vector control strategy

Chagas disease is a vector-borne parasitic disease caused by the flagellated protozoan Trypanosoma cruzi and transmitted to humans by a large group of bloodsucking triatomine bugs. Triatomine insects, such as Rhodnius prolixus, ingest a huge amount of blood in a single meal. Their midgut represents an important interface for triatomine–trypanosome interactions. Furthermore, the development of parasites and their vectorial transmission are closely linked to the blood feeding and digestion; thus, an understanding of their physiology is essential for the development of new strategies to control triatomines. In this study, we used label-free quantitative proteomics to identify and analyze the early effect of blood feeding on protein expression in the midgut of Rhodnius prolixus. We both identified and quantified 124 proteins in the anterior midgut (AM) and 40 in the posterior midgut (PM), which vary significantly 6 h after feeding. The detailed analysis of these proteins revealed their predominant involvement in the primary function of hematophagy, including proteases, proteases inhibitors, amino acids metabolism, primary metabolites processing, and protein folding. Interestingly, our proteomics data show a potential role of the AM in protein digestion. Moreover, proteins related to detoxification processes and innate immunity, which are largely accepted to be triggered by blood ingestion, were mildly modulated. Surprisingly, one third of blood-regulated proteins in the AM have unknown function. This work contributes to the improvement of knowledge on the digestive physiology of triatomines in the early hours post-feeding. It provides key information for selecting new putative targets for the development of triatomine control tools and their potential role in the vector competence, which could be applied to other vector species.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Disclosing the Interactome of Leukemogenic NUP98-HOXA9 and SET-NUP214 Fusion Proteins Using a Proteomic Approach.

The interaction of oncogenes with cellular proteins is a major determinant of cellular transformation. The NUP98-HOXA9 and SET-NUP214 chimeras result from recurrent chromosomal translocations in acute leukemia. Functionally, the two fusion proteins inhibit nuclear export and interact with epigenetic regulators. The full interactome of NUP98-HOXA9 and SET-NUP214 is currently unknown. We used proximity-dependent biotin identification (BioID) to study the landscape of the NUP98-HOXA9 and SET-NUP214 environments. Our results suggest that both fusion proteins interact with major regulators of RNA processing, with translation-associated proteins, and that both chimeras perturb the transcriptional program of the tumor suppressor p53. Other cellular processes appear to be distinctively affected by the particular fusion protein. NUP98-HOXA9 likely perturbs Wnt, MAPK, and estrogen receptor (ER) signaling pathways, as well as the cytoskeleton, the latter likely due to its interaction with the nuclear export receptor CRM1. Conversely, mitochondrial proteins and metabolic regulators are significantly overrepresented in the SET-NUP214 proximal interactome. Our study provides new clues on the mechanistic actions of nucleoporin fusion proteins and might be of particular relevance in the search for new druggable targets for the treatment of nucleoporin-related leukemia.info:eu-repo/semantics/publishe

Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry.

A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe