Evaluation of optimized tube-gel methods of sample preparation for large-scale plant proteomics

The so-called tube-gel method is a sample preparation protocol allowing for management of SDS for protein solubilization through in-gel protein trapping. Because of its simplicity, we assumed that once miniaturized, this method could become a standard for large scale experiments. We evaluated the performances of two variants of the miniaturized version of the tube-gel method based on different solubilization buffers (Tris-SDS or urea-SDS). To this end, we compared them to two other digestion methods: (i) liquid digestion after protein solubilization in the absence of SDS (liquid method) and (ii) filter-aided sample preparation (FASP). As large-scale experiments may require long term gel storage, we also examined to which extent gel aging affected the results of the proteomics analysis. We showed that both tube-gel and FASP methods extracted membrane proteins better than the liquid method, while the latter allowed the identification and quantification of a greater number of proteins. All methods were equivalent regarding quantitative stability. However, important differences were observed regarding post-translational modifications. In particular, methionine oxidation was higher with the tube-gel method than with the other methods. Based on these results, and considering time, simplicity, and cost aspects, we conclude that the miniaturized tube-gel method is suitable for sample preparation in the context of large-scale experiments

Physical Contact between Torulaspora delbrueckii and Saccharomyces cerevisiae Alters Cell Growth and Molecular Interactions in Grape Must

The use of multi-starters in oenological conditions (Saccharomyces cerevisiae and non-Saccharomyces species) is becoming increasingly common. For the past ten years, the combination of Torulaspora delbrueckii and S. cerevisiae has been proposed to winemakers to improve the wine aromatic profile compared to pure inoculation with Saccharomyces cerevisiae. In this work, two commercial strains, T. delbrueckii Zymaflore® Alpha and S. cerevisiae Zymaflore® X5 (Laffort compagny, Floirac, France), were investigated in Sauvignon blanc must using a fermentor with a double compartment allowing for physical separation of the two yeast species. The physical separation of the two species resulted in significant differences in the growth, fermentation kinetics (maximum fermentation rate (+13%)), fermentation duration (-14%) and the production of 3SH (+35%) in comparison to mixed cultures with contact. Proteomic analysis confirmed cell–cell contact interactions, as strong differences were observed for both species between mixed cultures with and without physical contact. T. delbrueckii mortality in mixed cultures with physical contact may be explained by an oxidative stress. Indeed two proteins implicated in the oxidative stress response were found in significantly higher amounts: a cytosolic catalase T and a cytoplasmic thioredoxin isoenzyme. For S. cerevisiae, an increase in proteins involved in the respiratory chain and proton transport were found in higher amounts in pure cultures and mixed culture without physical contact. Our results confirmed that the two mixed inoculations increased certain minor esters (ethylpropanoate, ethyl dihydrocinnamate and ethyl isobutanoate) specifically produced by T. delbrueckii, 3.4-fold more compared to in the pure S. cerevisiae culture. In conclusion, these results provide new insights into the underlying mechanisms involved in cell–cell contact and confirm the benefits of using T. delbrueckii species under winemaking conditions

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

Thousand and one ways to quantify and compare protein abundances in label-free bottom-up proteomics

How to process and analyze MS data to quantify and statistically compare protein abundances in bottom-up proteomics has been an open debate for nearly fifteen years. Two main approaches are generally used: the first is based on spectral data generated during the process of identification (e.g. peptide counting, spectral counting), while the second makes use of extracted ion currents to quantify chromatographic pealcs and infer protein abundances based on peptide quantification. These two approaches actually refer to multiple methods which have been developed during the last decade, but were submitted to deep evaluations only recently. In this paper, we compiled these different methods as exhaustively as possible. We also summarized the way they address the different problems raised by bottom-up protein quantification such as normalization, the presence of shared peptides, unequal peptide measurability and missing data. This article is part of a Special Issue entitled: Plant Proteomics- a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. (C) 2016 Elsevier B.V. All rights reserved

Evaluation of Optimized Tube-Gel Methods of Sample Preparation for Large-Scale Plant Proteomics

The so-called tube-gel method is a sample preparation protocol allowing for management of SDS for protein solubilization through in-gel protein trapping. Because of its simplicity, we assumed that once miniaturized, this method could become a standard for large scale experiments. We evaluated the performances of two variants of the miniaturized version of the tube-gel method based on different solubilization buffers (Tris-SDS or urea-SDS). To this end, we compared them to two other digestion methods: (i) liquid digestion after protein solubilization in the absence of SDS (liquid method) and (ii) filter-aided sample preparation (FASP). As large-scale experiments may require long term gel storage, we also examined to which extent gel aging affected the results of the proteomics analysis. We showed that both tube-gel and FASP methods extracted membrane proteins better than the liquid method, while the latter allowed the identification and quantification of a greater number of proteins. All methods were equivalent regarding quantitative stability. However, important differences were observed regarding post-translational modifications. In particular, methionine oxidation was higher with the tube-gel method than with the other methods. Based on these results, and considering time, simplicity, and cost aspects, we conclude that the miniaturized tube-gel method is suitable for sample preparation in the context of large-scale experiments

Proteomic data from leaves of twenty-four sunflower genotypes under water deficit

International audienceThis article describes a proteomic data set produced from sunflower plants subjected to water deficit. Twenty-four sunflower genotypes were selected to represent genetic diversity within cultivated sunflower. They included both inbred lines and their hybrids. Water deficit was applied to plants in pots at the vegetative stage using the high-throughput phenotyping platform Heliaphen. We present here the identification of 3062 proteins and the quantification of 1211 of them in the leaves of the 24 genotypes grown under two watering conditions. These data allow the study of both the effects of genetic variations and watering conditions. They constitute a valuable resource for the community to study adaptation of crops to drought and the molecular basis of heterosis.Cet article décrit un jeu de données protéomiques produites à partir de plantes de tournesol soumises ou non à un déficit hydrique. Vingt-quatre génotypes incluant des lignées pures et leurs hybrides ont été sélectionnés pour représenter la diversité génétique des tournesols cultivés. Les plantes ont été cultivées en pots sur la plateforme Heliaphen de phénotypage à haut débit et le déficit hydrique a été appliqué à un stade végétatif. Nous présentons l’identification de 3062 protéines et la quantification de 1211 d’entre elles dans les feuilles des vingt-quatre génotypes cultivés dans les deux conditions d’arrosage. Ces données permettent d’étudier l’effet des variations génétiques et du déficit hydrique sur le protéome. Elles sont une ressource intéressante pour la communauté, permettant d’étudier l’adaptation des plantes cultivées à la sécheresse et les bases moléculaires de l’hétérosis

Proteomic data from leaves of twenty-four sunflower genotypes under water deficit

This article describes a proteomic data set produced from sunflower plants subjected to water deficit. Twenty-four sunflower genotypes were selected to represent genetic diversity within cultivated sunflower. They included both inbred lines and their hybrids. Water deficit was applied to plants in pots at the vegetative stage using the high-throughput phenotyping platform Heliaphen. We present here the identification of 3062 proteins and the quantification of 1211 of them in the leaves of the 24 genotypes grown under two watering conditions. These data allow the study of both the effects of genetic variations and watering conditions. They constitute a valuable resource for the community to study adaptation of crops to drought and the molecular basis of heterosis