Evolutionarily conserved ovarian fluid proteins are responsible for extending egg viability in salmonid fish.

In contrast to most fishes, salmonids exhibit the unique ability to hold their eggs for several days after ovulation without significant loss of viability. During this period, eggs are held in the body cavity in a biological fluid, the coelomic fluid (CF) that is responsible for preserving egg viability. To identify CF proteins responsible for preserving egg viability, a proteomic comparison was performed using 3 salmonid species and 3 non-salmonid species to identify salmonid-specific highly abundant proteins. In parallel, rainbow trout CF fractions were purified and used in a biological test to estimate their egg viability preservation potential. The most biologically active CF fractions were then subjected to mass spectrometry analysis. We identified 50 proteins overabundant in salmonids and present in analytical fractions with high egg viability preservation potential. The identity of these proteins illuminates the biological processes participating in egg viability preservation. Among identified proteins of interest, the ovarian-specific expression and abundance in CF at ovulation of N-acetylneuraminic acid synthase a (Nansa) suggest a previously unsuspected role. We show that salmonid CF is a complex biological fluid containing a diversity of proteins related to immunity, calcium binding, lipid metabolism, proteolysis, extracellular matrix and sialic acid metabolic pathway that are collectively responsible for preserving egg viability.

Intertidal limits shape covariation between metabolic plasticity, oxidative stress and telomere dynamics in Pacific oyster (Crassostrea gigas).

In intertidal zones, species such as sessile shellfish exhibit extended phenotypic plasticity to face rapid environmental changes, but whether frequent exposure to intertidal limits of the distribution range impose physiological costs for the animal remains elusive. Here, we explored how phenotypic plasticity varied along foreshore range at multiple organization levels, from molecular to cellular and whole organism acclimatization, in the Pacific oyster (Crassostrea gigas). We exposed 7-month-old individuals for up to 16 months to three foreshore levels covering the vertical range for this species, representing 20, 50 and 80% of the time spent submerged monthly. Individuals at the upper range limit produced energy more efficiently, as seen by steeper metabolic reactive norms and unaltered ATP levels despite reduced mitochondrial density. By spending most of their time emerged, oysters mounted an antioxidant shielding concomitant with lower levels of pro-oxidant proteins and postponed age-related telomere attrition. Instead, individuals exposed at the lower limit range near subtidal conditions showed lower energy efficiencies, greater oxidative stress and shorter telomere length. These results unraveled the extended acclimatization strategies and the physiological costs of living too fast in subtidal conditions for an intertidal species.

Metalloprotease-mediated cleavage of CD95 ligand.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.

The Eutopic Endometrium Proteome in Endometriosis Reveals Candidate Markers and Molecular Mechanisms of Physiopathology.

Endometriosis is a common chronic gynaecological disease causing various symptoms, such as infertility and chronic pain. The gold standard for its diagnosis is still laparoscopy and the biopsy of endometriotic lesions. Here, we aimed to compare the eutopic endometrium from women with or without endometriosis to identify proteins that may be considered as potential biomarker candidates. Eutopic endometrium was collected from patients with endometriosis (n = 4) and women without endometriosis (n = 5) during a laparoscopy surgery during the mid-secretory phase of their menstrual cycle. Total proteins from tissues were extracted and digested before LC-MS-MS analysis. Among the 5301 proteins identified, 543 were differentially expressed and enriched in two specific KEGG pathways: focal adhesion and PI3K/AKT signaling. Integration of our data with a large-scale proteomics dataset allowed us to highlight 11 proteins that share the same trend of dysregulation in eutopic endometrium, regardless of the phase of the menstrual cycle. Our results constitute the first step towards the identification of potential promising endometrial diagnostic biomarkers. They provide new insights into the mechanisms underlying endometriosis and its etiology. Our results await further confirmation on a larger sample cohort.

Spatial segmentation and metabolite annotation involved in sperm maturation in the rat epididymis by MALDI imaging mass spectrometry.

Spermatozoa acquire their fertilizing capacity during a complex maturation process that occurs in the epididymis. This process involves a substantial molecular remodeling at the surface of the gamete. Epididymis is divided into three regions (the caput, corpus, and cauda) or into 19 intraregional segments based on histology. Most studies carried out on epididymal maturation have been performed on sperm samples or tissue extracts. Here, we used MALDI imaging mass spectrometry in the positive and negative ion modes combined with spatial segmentation and automated metabolite annotation to study the precise localization of metabolites directly in the rat epididymis. The spatial segmentation revealed that the rat epididymis could be divided into several molecular clusters different from the 19 intraregional segments. The discriminative m/z values that contributed the most to each molecular cluster were then annotated and corresponded mainly to phosphatidylcholines, sphingolipids, glycerophosphates, triacylglycerols, plasmalogens, phosphatidylethanolamines, and lysophosphatidylcholines. A substantial remodeling of lipid composition during epididymal maturation was observed. It was characterized in particular by an increase in the number of sphingolipids and plasmalogens and a decrease in the proportion of triacylglycerols annotated from caput to cauda. Ion images reveal that molecules belonging to the same family can have very different localizations along the epididymis. For some of them, annotation was confirmed by on-tissue MS/MS experiments. A 3D model of the epididymis head was reconstructed from 61 sections analyzed with a lateral resolution of 50 µm and can be used to obtain information on the localization of a given analyte in the whole volume of the tissue.

Deciphering the Dark Proteome: Use of the Testis and Characterization of Two Dark Proteins.

For the C-HPP consortium, dark proteins include not only uPE1, but also missing proteins (MPs, PE2-4), smORFs, proteins from lncRNAs, and products from uncharacterized transcripts. Here, we investigated the expression of dark proteins in the human testis by combining public mRNA and protein expression data for several tissues and performing LC-MS/MS analysis of testis protein extracts. Most uncharacterized proteins are highly expressed in the testis. Thirty could be identified in our data set, of which two were selected for further analyses: (1) A0AOU1RQG5, a putative cancer/testis antigen specifically expressed in the testis, where it accumulates in the cytoplasm of elongated spermatids; and (2) PNMA6E, which is enriched in the testis, where it is found in the germ cell nuclei during most stages of spermatogenesis. Both proteins are coded on Chromosome X. Finally, we studied the expression of other dark proteins, uPE1 and MPs, in a series of human tissues. Most were highly expressed in the testis at both the mRNA and protein levels. The testis appears to be a relevant organ to study the dark proteome, which may have a function related to spermatogenesis and germ cell differentiation. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD009598.

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

Human Spermatozoa as a Model for Detecting Missing Proteins in the Context of the Chromosome-Centric Human Proteome Project.

The Chromosome-Centric Human Proteome Project (C-HPP) aims at cataloguing the proteins as gene products encoded by the human genome in a chromosome-centric manner. The existence of products of about 82% of the genes has been confirmed at the protein level. However, the number of so-called "missing proteins" remains significant. It was recently suggested that the expression of proteins that have been systematically missed might be restricted to particular organs or cell types, for example, the testis. Testicular function, and spermatogenesis in particular, is conditioned by the successive activation or repression of thousands of genes and proteins including numerous germ cell- and testis-specific products. Both the testis and postmeiotic germ cells are thus promising sites at which to search for missing proteins, and ejaculated spermatozoa are a potential source of proteins whose expression is restricted to the germ cell lineage. A trans-chromosome-based data analysis was performed to catalog missing proteins in total protein extracts from isolated human spermatozoa. We have identified and manually validated peptide matches to 89 missing proteins in human spermatozoa. In addition, we carefully validated three proteins that were scored as uncertain in the latest neXtProt release (09.19.2014). A focus was then given to the 12 missing proteins encoded on chromosomes 2 and 14, some of which may putatively play roles in ciliation and flagellum mechanistics. The expression pattern of C2orf57 and TEX37 was confirmed in the adult testis by immunohistochemistry. On the basis of transcript expression during human spermatogenesis, we further consider the potential for discovering additional missing proteins in the testicular postmeiotic germ cell lineage and in ejaculated spermatozoa. This project was conducted as part of the C-HPP initiatives on chromosomes 14 (France) and 2 (Switzerland). The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium under the data set identifier PXD002367.

Using collective expert judgements to evaluate quality measures of mass spectrometry images.

MOTIVATION: Imaging mass spectrometry (IMS) is a maturating technique of molecular imaging. Confidence in the reproducible quality of IMS data is essential for its integration into routine use. However, the predominant method for assessing quality is visual examination, a time consuming, unstandardized and non-scalable approach. So far, the problem of assessing the quality has only been marginally addressed and existing measures do not account for the spatial information of IMS data. Importantly, no approach exists for unbiased evaluation of potential quality measures. RESULTS: We propose a novel approach for evaluating potential measures by creating a gold-standard set using collective expert judgements upon which we evaluated image-based measures. To produce a gold standard, we engaged 80 IMS experts, each to rate the relative quality between 52 pairs of ion images from MALDI-TOF IMS datasets of rat brain coronal sections. Experts' optional feedback on their expertise, the task and the survey showed that (i) they had diverse backgrounds and sufficient expertise, (ii) the task was properly understood, and (iii) the survey was comprehensible. A moderate inter-rater agreement was achieved with Krippendorff's alpha of 0.5. A gold-standard set of 634 pairs of images with accompanying ratings was constructed and showed a high agreement of 0.85. Eight families of potential measures with a range of parameters and statistical descriptors, giving 143 in total, were evaluated. Both signal-to-noise and spatial chaos-based measures performed highly with a correlation of 0.7 to 0.9 with the gold standard ratings. Moreover, we showed that a composite measure with the linear coefficients (trained on the gold standard with regularized least squares optimization and lasso) showed a strong linear correlation of 0.94 and an accuracy of 0.98 in predicting which image in a pair was of higher quality. AVAILABILITY AND IMPLEMENTATION: The anonymized data collected from the survey and the Matlab source code for data processing can be found at: https://github.com/alexandrovteam/IMS_quality.

Localization and in situ absolute quantification of chlordecone in the mouse liver by MALDI imaging.

Chlordecone is an organochlorine pesticide that was extensively used in the French West Indies to fight weevils in banana plantations from 1973 to 1993. This has led to a persistent pollution of the environment and to the contamination of the local population for several decades with effects demonstrated on human health. Chlordecone accumulates mainly in the liver where it is known to potentiate the action of hepatotoxic agents. However, there is currently no information on its in situ localization in the liver. We have thus evaluated a matrix-assisted laser desorption ionization (MALDI) imaging quantification method based on labeled normalization for the in situ localization and quantification of chlordecone. After validating the linearity and the reproducibility of this method, quantitative MALDI imaging was used to study the accumulation of chlordecone in the mouse liver. Our results revealed that normalized intensities measured by MALDI imaging could be first converted in quantitative units. These quantities appeared to be different from absolute quantities of chlordecone determined by gas chromatography (GC), but they were perfectly correlated (R(2) = 0.995). The equation of the corresponding correlation curve was thus efficiently used to convert quantities measured by MALDI imaging into absolute quantities. Our method combining labeled normalization and calibration with an orthogonal technique allowed the in situ absolute quantification of chlordecone by MALDI imaging. Finally, our results obtained on the pathological mouse liver illustrate the advantages of quantitative MALDI imaging which preserves information on in situ localization without radioactive labeling and with a simple sample preparation.

Specific proteomic response of Unio pictorum mussel to a mixture of glyphosate and microcystin-LR.

Cyanobacterial toxins and pesticides regularly impact freshwaters. Microcystin-LR is one of the most toxic and common cyanobacterial toxins whereas glyphosate is the active ingredient of a widely use herbicide. As filter feeders, freshwater mussels are particularly exposed. Like many native bivalve species, Unio pictorum suffers from a continuous decline in Europe. In order to get a deeper insight of its response to contaminants, U. pictorum was exposed to either 10 µg L(-1) of microcystin-LR or 10 µg L(-1) of glyphosate or a mixture of both. Proteins of the digestive glands were extracted and analyzed by DIGE. Gel analysis revealed 103 spots with statistical variations, and the response seems to be less toward glyphosate than to microcystin-LR. Specific spots have variations only when exposed to the mixture, showing that there is an interaction of both contaminants in the responses triggered. The proteins of 30 spots have been identified. They belong mostly to the cytoskeleton family, but proteins of the oxidative pathway, detoxification, and energetic metabolism were affected either by glyphosate or microcystin-LR or by the mixture. These results demonstrate the importance to study contaminants at low concentrations representative of those found in the field and that multicontaminations can lead to different response pathways.

Two-dimensional fluorescence difference gel electrophoresis analysis of Listeria monocytogenes submitted to a redox shock.

The influence of redox alteration on the growth and proteomic pattern of Listeria monocytogenes was investigated. A redox shock was induced in cultures by addition of 3mM ferricyanide (FeCN) and 6mM dithiothreitol (DTT) to increase or to decrease respectively the redox potential naturally occurring at the beginning of growth. In both conditions, the reducing and oxidizing redox shock had a strong influence, decreasing the maximum growth rate by half compared to a control culture. The proteomic analysis of L. monocytogenes performed by two-dimensional difference gel electrophoresis (2D-DIGE) exhibited twenty-three proteins differentially expressed (P<0.05), among these, many were oxidoreductases, and proteins involved in cellular metabolism (glycolysis, protein synthesis), detoxification (kat) or adhesion (Lmo1634).

Metabolic and proteomic profiling of diapause in the aphid parasitoid Praon volucre.

BACKGROUND: Diapause, a condition of developmental arrest and metabolic depression exhibited by a wide range of animals is accompanied by complex physiological and biochemical changes that generally enhance environmental stress tolerance and synchronize reproduction. Even though some aspects of diapause have been well characterized, very little is known about the full range of molecular and biochemical modifications underlying diapause in non-model organisms. METHODOLOGY/PRINCIPAL FINDINGS: In this study we focused on the parasitic wasp, Praon volucre that exhibits a pupal diapause in response to environmental signals. System-wide metabolic changes occurring during diapause were investigated using GC-MS metabolic fingerprinting. Moreover, proteomic changes were studied in diapausing versus non-diapausing phenotypes using a combination of two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry. We found a reduction of Krebs cycle intermediates which most likely resulted from the metabolic depression. Glycolysis was galvanized, probably to favor polyols biosynthesis. Diapausing parasitoids accumulated high levels of cryoprotective polyols, especially sorbitol. A large set of proteins were modulated during diapause and these were involved in various functions such as remodeling of cytoskeleton and cuticle, stress tolerance, protein turnover, lipid metabolism and various metabolic enzymes. CONCLUSIONS/SIGNIFICANCE: The results presented here provide some first clues about the molecular and biochemical events that characterize the diapause syndrome in aphid parasitoids. These data are useful for probing potential commonality of parasitoids diapause with other taxa and they will help creating a general understanding of diapause underpinnings and a background for future interpretations.

Comparative 2D-DIGE proteomic analysis of ovarian carcinoma cells: toward a reorientation of biosynthesis pathways associated with acquired platinum resistance.

Ovarian cancer is the fifth most frequent cause of cancer death in women. Emergence of chemoresistance in the course of treatments with platinum drugs is in part responsible for therapeutic failures. In order to improve the understanding of the complex mechanisms involved in acquired platinum chemoresistance, we decided to compare the basal protein expression profile of the platinum-sensitive cell line OAW42 and that of its resistant counterpart OAW42-R by a proteomic approach. Reversed-phase HPLC pre-fractionated extracts from both cell lines were subjected to 2D-DIGE coupled to mass spectrometry (MS). Forty eight differentially expressed proteins were identified, 39 being up-regulated and 19 down-regulated in OAW42-R versus OAW42 cells. From the current knowledge on biological activities of most differentially expressed proteins, it can be inferred that the acquisition of resistance was associated with a global reorganization of biochemical pathways favoring the production of precursors for biosynthesis, and with the mobilization of macromolecule quality control mechanisms, preserving RNA and protein integrity under damage-inducing conditions.

A comparison of subcutaneous adipose tissue proteomes in juvenile piglets with a contrasted adiposity underscored similarities with human obesity.

Subcutaneous fat tissues from an indigenous fat-type breed and an intensively-lean selected breed were studied in juvenile pigs. Combining DIGE with bioinformatics and target analyses of key genes, enzymes or terminal routes, this study identifies metabolic and homeostatic processes, response to organic substances, and acute-phase responses as the main pathways whose proteins were regulated in association with adiposity. Breed-related differences in abundance and activities of malic enzyme and glucose-6-phosphate dehydrogenase NADPH-supplying enzymes suggested up-regulation of the lipogenic pathway to dispose for a greater adiposity. Over-abundance in the lipolytic protein carboxylesterase-1 was revealed in fat-type piglets. A panel of pro- and anti-inflammatory proteins such as serpins, had an altered abundance in the fat-type piglets, suggesting adverse consequences of fat accumulation even in early post-weaning stages. Propensity to low-grade inflammation in fat pigs was reinforced by the up-regulation of genes encoding pro-inflammatory cytokines IL6 and TNF-α in these piglets. Differential abundance in annexin-A5 and pericentrin suggested a positive regulation of cell apoptosis in lean piglets. Our results are relevant in the context of data linking the accretion of body lipids to the physiology and pathology of adipose tissue in models other than rodents for a better control of human health and nutrition.

Matrix-assisted laser desorption/ionization imaging mass spectrometry: a promising technique for reproductive research.

Matrix-assisted laser desorption/ionization (MALDI) tissue imaging mass spectrometry is particularly promising among the numerous applications of mass spectrometry. It is used for probing and analyzing the spatial arrangement of a wide range of molecules, including proteins, peptides, lipids, drugs, and metabolites, directly in thin slices of tissue. In the field of proteomics, the technology avoids tedious and time-consuming extraction and fractionation steps classically required for sample analysis. MALDI imaging mass spectrometry is increasingly recognized as a powerful method for clinical proteomics, particularly in cancer research. The technology has particular potential for the discovery of new tissue biomarker candidates, classification of tumors, early diagnosis or prognosis, elucidating pathogenesis pathways, and therapy monitoring. Over recent years, MALDI imaging mass spectrometry has been used for molecular profiling and imaging directly in male and female reproductive tissues. This review will consider some of the recent publications in the field, addressing a range of issues covering embryo development, gene expression product profiling during gametogenesis, and seeking and identifying biomarkers of reproductive cancers. The wealth of advances in mass spectrometry imaging will inevitably attract biologists and clinicians as the advantages and power of this technology become more widely known. This review will also discuss bottlenecks and the many technical issues that remain to be resolved before laboratories in the field can adopt the technology. We foresee that MALDI imaging mass spectrometry will have a major impact in reproductive research by opening new avenues to the understanding of various molecular mechanisms and the diagnosis of reproductive pathologies.