Application of pulsed electric fields for the biocompatible extraction of proteins from the microalga Haematococcus pluvialis.

This study aims to employ a pulsed electric field (PEF) treatment for the biocompatible (non-destructive) extraction of proteins from living cells of the green microalga Haematococcus pluvialis. Using a field strength of 1 kV cm-1, we achieved the extraction of 10.2 µg protein per mL of culture, which corresponded to 46% of the total amount of proteins that could be extracted by complete destructive extraction (i.e. the grinding of biomass with glass beads). We found that the extraction yield was not improved by stronger field strengths and was not dependent on the pulse frequency. A biocompatibility index (BI) was defined as the relative abundance of cells that remained alive after the PEF treatment. This index relied on measurements of several physiological parameters after a PEF treatment. It was found that at 1 kV cm-1 that cultures recovered after 72 h. Therefore, these PEF conditions constituted a good compromise between protein extraction efficiency and culture survival. To characterize the PEF treatment further at a molecular level, mass spectrometry-based proteomics analyses of PEF-prepared extracts was used. This led to the identification of 52 electro-extracted proteins. Of these, only 16 proteins were identified when proteins were extracted with PEF at 0.5 cm-1. They belong to core metabolism, stress response and cell movement. Unassigned proteins were also extracted. Their physiological implications and possible utilization in food as alimentary complements are discussed.

The ex planta signal activity of a Medicago ribosomal uL2 protein suggests a moonlighting role in controlling secondary rhizobial infection.

We recently described a regulatory loop, which we termed autoregulation of infection (AOI), by which Sinorhizobium meliloti, a Medicago endosymbiont, downregulates the root susceptibility to secondary infection events via ethylene. AOI is initially triggered by so-far unidentified Medicago nodule signals named signal 1 and signal 1' whose transduction in bacteroids requires the S. meliloti outer-membrane-associated NsrA receptor protein and the cognate inner-membrane-associated adenylate cyclases, CyaK and CyaD1/D2, respectively. Here, we report on advances in signal 1 identification. Signal 1 activity is widespread as we robustly detected it in Medicago nodule extracts as well as in yeast and bacteria cell extracts. Biochemical analyses indicated a peptidic nature for signal 1 and, together with proteomic analyses, a universally conserved Medicago ribosomal protein of the uL2 family was identified as a candidate signal 1. Specifically, MtRPuL2A (MtrunA17Chr7g0247311) displays a strong signal activity that requires S. meliloti NsrA and CyaK, as endogenous signal 1. We have shown that MtRPuL2A is active in signaling only in a non-ribosomal form. A Medicago truncatula mutant in the major symbiotic transcriptional regulator MtNF-YA1 lacked most signal 1 activity, suggesting that signal 1 is under developmental control. Altogether, our results point to the MtRPuL2A ribosomal protein as the candidate for signal 1. Based on the Mtnf-ya1 mutant, we suggest a link between root infectiveness and nodule development. We discuss our findings in the context of ribosomal protein moonlighting.

The Consequences of a Disruption in Cyto-Nuclear Coadaptation on the Molecular Response to a Nitrate Starvation in Arabidopsis.

Mitochondria and chloroplasts are important actors in the plant nutritional efficiency. So, it could be expected that a disruption of the coadaptation between nuclear and organellar genomes impact plant response to nutrient stresses. We addressed this issue using two Arabidopsis accessions, namely Ct1 and Jea, and their reciprocal cytolines possessing the nuclear genome from one parent and the organellar genomes of the other one. We measured gene expression, and quantified proteins and metabolites under N starvation and non-limiting conditions. We observed a typical response to N starvation at the phenotype and molecular levels. The phenotypical response to N starvation was similar in the cytolines compared to the parents. However, we observed an effect of the disruption of genomic coadaptation at the molecular levels, distinct from the previously described responses to organellar stresses. Strikingly, genes differentially expressed in cytolines compared to parents were mainly repressed in the cytolines. These genes encoded more mitochondrial and nuclear proteins than randomly expected, while N starvation responsive ones were enriched in genes for chloroplast and nuclear proteins. In cytolines, the non-coadapted cytonuclear genomic combination tends to modulate the response to N starvation observed in the parental lines on various biological processes.

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival.

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination.

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen's genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F-) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F- hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage.

Analysis of durum wheat proteome changes under marine and fungal biostimulant treatments using large-scale quantitative proteomics: A useful dataset of durum wheat proteins.

Durum wheat requires high nitrogen inputs to obtain the high protein concentration necessary to satisfy pasta and semolina quality criteria. Optimizing plant nitrogen use efficiency is therefore of major importance for wheat grain quality. Here, we studied the impact on grain yield, protein concentration, and for the first time on protein composition of a marine (DPI4913) and a fungal (AF086) biostimulants applied to plant leaves. A large-scale quantitative proteomics analysis of wheat flour samples led to a dataset of 1471 identified proteins. Quantitative analysis of 1391 proteins revealed 26 and 38 proteins with a significantly varying abundance after DPI4913 and AF086 treatment, respectively, with 14 proteins in common. Major effects affected proteins involved in grain technological properties like grain hardness, in storage functions with the gluten protein gamma-gliadin, in regulation processes with transcription regulator proteins, and in stress responses with biotic and abiotic stress defense proteins. The involvement of biostimulants in the abiotic stress response was further suggested by an increase in water-use efficiency for both DPI4913 (15.4%) and AF086 (9.9%) treatments. Overall, our work performed in controlled conditions showed that DPI4913 and AF086 treatments promoted grain yield while maintaining protein concentration, and positively affected protein composition for grain quality. Data are available via ProteomeXchange with identifier PXD012469.

Metabolome and proteome changes between biofilm and planktonic phenotypes of the marine bacterium Pseudoalteromonas lipolytica TC8.

A number of bacteria adopt various lifestyles such as planktonic free-living or sessile biofilm stages. This enables their survival and development in a wide range of contrasting environments. With the aim of highlighting specific metabolic shifts between these phenotypes and to improve the overall understanding of marine bacterial adhesion, a dual metabolomics/proteomics approach was applied to planktonic and biofilm cultures of the marine bacterium Pseudoalteromonas lipolytica TC8. The liquid chromatography mass spectrometry (LC-MS) based metabolomics study indicated that membrane lipid composition was highly affected by the culture mode: phosphatidylethanolamine (PEs) derivatives were over-produced in sessile cultures while ornithine lipids (OLs) were more specifically synthesized in planktonic samples. In parallel, differences between proteomes revealed that peptidases, oxidases, transcription factors, membrane proteins and the enzymes involved in histidine biosynthesis were over-expressed in biofilms while proteins involved in heme production, nutrient assimilation, cell division and arginine/ornithine biosynthesis were specifically up-regulated in free-living cells.

Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: A novel approach to characterise markers of oocyte maturation.

Intact cell MALDI-TOF mass spectrometry (ICM-MS) was adapted to bovine follicular cells from individual ovarian follicles to obtain the protein/peptide signatures (<17kDa) of single oocytes, cumulus cells (CC) and granulosa cells (GC), which shared a total of 439 peaks. By comparing the ICM-MS profiles of single oocytes and CC before and after in vitro maturation (IVM), 71 different peaks were characterised, and their relative abundance was found to vary depending on the stage of oocyte meiotic maturation. To identify these endogenous biomolecules, top-down workflow using high resolution MS/MS (TD HR-MS) was performed on the protein extracts from oocytes, CC and GC. The TD HR-MS proteomic approach allowed for: (1) identification of 386 peptide/proteoforms encoded by 194 genes; and (2) characterisation of proteolysis products likely resulting from the action of kallikreins and caspases. In total, 136 peaks observed by ICM-MS were annotated by TD HR-MS (ProteomeXchange PXD004892). Among these, 16 markers of maturation were identified, including IGF2 binding protein 3 and hemoglobin B in the oocyte, thymosins beta-4 and beta-10, histone H2B and ubiquitin in CC. The combination of ICM-MS and TD HR-MS proved to be a suitable strategy to identify non-invasive markers of oocyte quality using limited biological samples. BIOLOGICAL SIGNIFICANCE: Intact cell MALDI-TOF mass spectrometry on single oocytes and their surrounding cumulus cells, coupled to an optimised top-down HR-MS proteomic approach on ovarian follicular cells, was used to identify specific markers of oocyte meiotic maturation represented by whole low molecular weight proteins or products of degradation by specific proteases.

Data on endogenous bovine ovarian follicular cells peptides and small proteins obtained through Top-down High Resolution Mass Spectrometry.

The endogenous peptides and small proteins extracted from bovine ovarian follicular cells (oocytes, cumulus and granulosa cells) were identified by Top-down High Resolution Mass Spectrometry (TD-HR-MS/MS) in order to annotate peptido- and proteoforms detected using qualitative and quantitative profiling method based on ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The description and analysis of these Top-down MS data in the context of oocyte quality biomarkers research are available in the original research article of Labas et al. (2017) http://dx.doi.org/10.1016/j.jprot.2017.03.027[1]. Raw data derived from this peptidomic/proteomic analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD004892). Here, we described the inventory of all identified peptido- and proteoforms including their biochemical and structural features, and functional annotation of correspondent proteins. This peptide/protein inventory revealed that TD-HR-MS/MS was appropriate method for both global and targeted proteomic analysis of ovarian tissues, and it can be further employed as a reference for other studies on follicular cells including single oocytes.

Qualitative and quantitative peptidomic and proteomic approaches to phenotyping chicken semen.

Understanding of the avian male gamete biology is essential to improve the conservation of genetic resources and performance in farming. In this study, the chicken semen peptidome/proteome and the molecular phenotype related to sperm quality were investigated. Spermatozoa (SPZ) and corresponding seminal plasma (SP) from 11 males with different fertilizing capacity were analyzed using three quantitative strategies (fluid and intact cells MALDI-MS, SDS-PAGE combined to LC-MS/MS with spectral counting and XIC methods). Individual MALDI profiling in combination with top-down MS allowed to characterize specific profiles per male and to identify 16 biomolecules (e.g.VMO1, AvBD10 and AvBD9 including polymorphism). Qualitative analysis identified 1165 proteins mainly involved in oxidoreduction mechanisms, energy processes, proteolysis and protein localization. Comparative analyses between the most and the least fertile males were performed. The enzymes involved in energy metabolism, respiratory chain or oxido-reduction activity were over-represented in SPZ of the most fertile males. The SP of the most and the least fertile males differed also on many proteins (e.g. ACE, AvBD10 and AvBD9, NEL precursor, acrosin). Thus proteomic is a "phenomic molecular tool" that may help to discriminate avian males on their reproductive capacity. The data have been deposited with ProteomeXchange (identifiers PXD000287 and PXD001254). BIOLOGICAL SIGNIFICANCE: This peptidomic and proteomic study i) characterized for the first time the semen protein composition of the main domestic avian species (Gallus gallus) by analysis of ejaculated spermatozoa and corresponding seminal plasma; ii) established a characteristic molecular phenotype distinguishing semen and males at an individual level; and iii) proposedthe first evidence of biomarkers related to fertility.

Analysis of epididymal sperm maturation by MALDI profiling and top-down mass spectrometry.

The fertilization ability of male gametes is achieved after their transit through the epididymis where important post-gonadal differentiation occurs in different cellular compartments. Most of these maturational modifications occur at the protein level. The epididymal sperm maturation process was investigated using the ICM-MS (Intact Cell MALDI-TOF MS) approach on boar spermatozoa isolated from four different epididymal regions (immature to mature stage). Differential and quantitative MALDI-TOF profiling for whole cells or sub-cellular fractions was combined with targeted top-down MS in order to identify endogenous biomolecules. Using this approach, 172m/z peaks ranging between 2 and 20kDa were found to be modified during maturation of sperm. Using top-down MS, 62m/z were identified corresponding to peptidoforms/proteoforms with post-translational modifications (MS data are available via ProteomeXchange with identifier PXD001303). Many of the endogenous peptides were characterized as N-, C-terminal sequences or internal fragments of proteins presenting specific cleavages, suggesting the presence of sequential protease activities in the spermatozoa. This is the first time that such proteolytic activities could be evidenced for various sperm proteins through quantification of their proteolytic products. ICM-MS/top-down MS thus proved to be a valid approach for peptidome/degradome studies and provided new contributions to understanding of the maturation process of the male gamete involved in the development of male fertility. BIOLOGICAL SIGNIFICANCE: This peptidomic study (i) characterized the peptidome of epididymal spermatozoa from boar (Sus scrofa); (ii) established characteristic molecular phenotypes distinguishing degrees of maturation of spermatozoa during epididymal transit, and (iii) revealed that protease activities were at the origin of numerous peptides from known and unknown proteins involved in sperm maturation and/or fertility processes.

Data for chicken semen proteome and label free quantitative analyses displaying sperm quality biomarkers.

Understanding of biology of the avian male gamete is essential to improve the conservation of genetic resources and performances in farming. In this study, the semen proteome of the main domestic avian species (Gallus gallus) and evaluation of the molecular phenotype related to sperm quality were investigated using GeLC-MS/MS approach and label-free quantitative proteomic based on Spectral Counting (SC) and extracted ion chromatograms (XIC) methods. Here we describe in details the peptide/protein inventory of chicken ejaculated spermatozoa (SPZ) and seminal plasma (SP). We also show differential analyses of chicken semen (SPZ and corresponding SP) from 11 males demonstrating different levels of fertilizing capacity and sperm motility. The interpretation and description of these data can be found in a research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1]. This is a new resource for exploring the molecular mechanisms involved in fertilizing capacity and to reveal new sets of fertility biomarkers.

Data in support of peptidomic analysis of spermatozoa during epididymal maturation.

The final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several epididymal maturation states could be an informative approach to finding sperm modifications related to maturation and fertility. Here we show the data from differential peptidomic/proteomic analyses on spermatozoa isolated from 4 epididymal regions (immature to mature stage) using a profiling approach based on MALDI-TOF mass spectrometry and, combined to top-down MS in order to identify peptidoforms and proteoforms. By this way, 172m/z peaks ranging between 2 and 20 kDa were found to be modified during maturation of sperm. A total of 62m/z were identified corresponding to 32 different molecular species. The interpretation of these data can be found in the research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1].