Skeletal Muscle Proteome Modifications following Antibiotic-Induced Microbial Disturbances in Cancer Cachexia.

Cancer cachexia is an involuntary loss of body weight, mostly of skeletal muscle. Previous research favors the existence of a microbiota-muscle crosstalk, so the aim of the study was to evaluate the impact of microbiota alterations induced by antibiotics on skeletal muscle proteins expression. Skeletal muscle proteome changes were investigated in control (CT) or C26 cachectic mice (C26) with or without antibiotic treatment (CT-ATB or C26-ATB, n = 8 per group). Muscle protein extracts were divided into a sarcoplasmic and myofibrillar fraction and then underwent label-free liquid chromatography separation, mass spectrometry analysis, Mascot protein identification, and METASCAPE platform data analysis. In C26 mice, the atrogen mafbx expression was 353% higher than CT mice and 42.3% higher than C26-ATB mice. No effect on the muscle protein synthesis was observed. Proteomic analyses revealed a strong effect of antibiotics on skeletal muscle proteome outside of cachexia, with adaptative processes involved in protein folding, growth, energy metabolism, and muscle contraction. In C26-ATB mice, proteome adaptations observed in CT-ATB mice were blunted. Differentially expressed proteins were involved in other processes like glucose metabolism, oxidative stress response, and proteolysis. This study confirms the existence of a microbiota-muscle axis, with a muscle response after antibiotics that varies depending on whether cachexia is present.

Characterization of the Skeletal Muscle Proteome in Undernourished Old Rats.

Aging is associated with a progressive loss of skeletal muscle mass and function termed sarcopenia. Various metabolic alterations that occur with aging also increase the risk of undernutrition, which can worsen age-related sarcopenia. However, the impact of undernutrition on aged skeletal muscle remains largely under-researched. To build a deeper understanding of the cellular and molecular mechanisms underlying age-related sarcopenia, we characterized the undernutrition-induced changes in the skeletal muscle proteome in old rats. For this study, 20-month-old male rats were fed 50% or 100% of their spontaneous intake for 12 weeks, and proteomic analysis was performed on both slow- and fast-twitch muscles. Proteomic profiling of undernourished aged skeletal muscle revealed that undernutrition has profound effects on muscle proteome independently of its effect on muscle mass. Undernutrition-induced changes in muscle proteome appear to be muscle-type-specific: slow-twitch muscle showed a broad pattern of differential expression in proteins important for energy metabolism, whereas fast-twitch muscle mainly showed changes in protein turnover between undernourished and control rats. This first proteomic analysis of undernourished aged skeletal muscle provides new molecular-level insight to explain phenotypic changes in undernourished aged muscle. We anticipate this work as a starting point to define new biomarkers associated with undernutrition-induced muscle loss in the elderly.

A Single Bout of Ultra-Endurance Exercise Reveals Early Signs of Muscle Aging in Master Athletes.

Middle-aged and master endurance athletes exhibit similar physical performance and long-term muscle adaptation to aerobic exercise. Nevertheless, we hypothesized that the short-term plasticity of the skeletal muscle might be distinctly altered for master athletes when they are challenged by a single bout of prolonged moderate-intensity exercise. Six middle-aged (37Y) and five older (50Y) master highly-trained athletes performed a 24-h treadmill run (24TR). Vastus lateralis muscle biopsies were collected before and after the run and assessed for proteomics, fiber morphometry, intramyocellular lipid droplets (LD), mitochondrial oxidative activity, extracellular matrix (ECM), and micro-vascularisation. Before 24TR, muscle fiber type morphometry, intramyocellular LD, oxidative activity, ECM and micro-vascularisation were similar between master and middle-aged runners. For 37Y runners, 24TR was associated with ECM thickening, increased capillary-to-fiber interface, and an 89% depletion of LD in type-I fibers. In contrast, for 50Y runners, 24TR did not alter ECM and capillarization and poorly depleted LDs. Moreover, an impaired succinate dehydrogenase activity and functional class scoring of proteomes suggested reduced oxidative phosphorylation post-24TR exclusively in 50Y muscle. Collectively, our data support that middle-aged and master endurance athletes exhibit distinct transient plasticity in response to a single bout of ultra-endurance exercise, which may constitute early signs of muscle aging for master athletes.

The salivary proteome reflects some traits of dietary habits in diabetic and non-diabetic older adults.

PURPOSE: Objective markers of usual diet are of interest as alternative or validating tools in nutritional epidemiology research. The main purpose of the work was to assess whether saliva protein composition can reflect dietary habits in older adults, and how type 2 diabetes impacted on the saliva-diet correlates. METHODS: 214 participants were selected from 2 European cohorts of community-dwelling older adults (3C-Bordeaux and Seniors-ENRICA-2), using a case-control design nested in each cohort. Cases were individuals with type 2 diabetes. Dietary information was obtained using the Mediterranean Diet Adherence Screener (MEDAS). Saliva was successfully obtained from 211 subjects, and its proteome analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: The relative abundance of 246 saliva proteins was obtained across all participants. The salivary proteome differed depending on the intake level of some food groups (especially vegetables, fruits, sweet snacks and red meat), in a diabetic status- and cohort-specific manner. Gene Set Enrichment Analysis suggested that some biological processes were consistently affected by diet across cohorts, for example enhanced platelet degranulation in high consumers of sweet snacks. Minimal models were then fitted to predict dietary variables by sociodemographic, clinical and salivary proteome variables. For the food group «sweet snacks¼, selected salivary proteins contributed to the predictive model and improved its performance in the Seniors-ENRICA-2 cohort and when both cohorts were combined. CONCLUSION: Saliva proteome composition of elderly individuals can reflect some aspects of dietary patterns.

Muscle Proteomic and Transcriptomic Profiling of Healthy Aging and Metabolic Syndrome in Men.

(1) Background: Aging is associated with a progressive decline in muscle mass and function. Aging is also a primary risk factor for metabolic syndrome, which further alters muscle metabolism. However, the molecular mechanisms involved remain to be clarified. Herein we performed omic profiling to decipher in muscle which dominating processes are associated with healthy aging and metabolic syndrome in old men. (2) Methods: This study included 15 healthy young, 15 healthy old, and 9 old men with metabolic syndrome. Old men were selected from a well-characterized cohort, and each vastus lateralis biopsy was used to combine global transcriptomic and proteomic analyses. (3) Results: Over-representation analysis of differentially expressed genes (ORA) and functional class scoring of pathways (FCS) indicated that healthy aging was mainly associated with upregulations of apoptosis and immune function and downregulations of glycolysis and protein catabolism. ORA and FCS indicated that with metabolic syndrome the dominating biological processes were upregulation of proteolysis and downregulation of oxidative phosphorylation. Proteomic profiling matched 586 muscle proteins between individuals. The proteome of healthy aging revealed modifications consistent with a fast-to-slow transition and downregulation of glycolysis. These transitions were reduced with metabolic syndrome, which was more associated with alterations in NADH/NAD+ shuttle and ß-oxidation. Proteomic profiling further showed that all old muscles overexpressed protein chaperones to preserve proteostasis and myofiber integrity. There was also evidence of aging-related increases in reactive oxygen species but better detoxifications of cytotoxic aldehydes and membrane protection in healthy than in metabolic syndrome muscles. (4) Conclusions: Most candidate proteins and mRNAs identified herein constitute putative muscle biomarkers of healthy aging and metabolic syndrome in old men.

Detection of Frozen-Thawed Duck Fatty Liver by MALDI-TOF Mass Spectrometry: A Chemometrics Study.

The marketing of poultry livers is only authorized as fresh, frozen, or deep-frozen. The higher consumer demand for these products for a short period of time may lead to the marketing of frozen-thawed poultry livers: this constitutes fraud. The aim of this study was to design a method for distinguishing frozen-thawed livers from fresh livers. For this, the spectral fingerprint of liver proteins was acquired using Matrix-Assisted Laser Dissociation Ionization-Time-Of-Flight mass spectrometry. The spectra were analyzed using the chemometrics approach. First, principal component analysis studied the expected variability of commercial conditions before and after freezing-thawing. Then, the discriminant power of spectral fingerprint of liver proteins was assessed using supervised model generation. The combined approach of mass spectrometry and chemometrics successfully described the evolution of protein profile during storage time, before and after freezing-thawing, and successfully discriminated the fresh and frozen-thawed livers. These results are promising in terms of fraud detection, providing an opportunity for implementation of a reference method for agencies to fight fraud.

Prediction of the Secretome and the Surfaceome: A Strategy to Decipher the Crosstalk between Adipose Tissue and Muscle during Fetal Growth.

Crosstalk between adipose and muscular tissues is hypothesized to regulate the number of muscular and adipose cells during fetal growth, with post-natal consequences on lean and fat masses. Such crosstalk largely remains, however, to be described. We hypothesized that a characterization of the proteomes of adipose and muscular tissues from bovine fetuses may enhance the understanding of the crosstalk between these tissues through the prediction of their secretomes and surfaceomes. Proteomic experiments have identified 751 and 514 proteins in fetal adipose tissue and muscle. These are mainly involved in the regulation of cell proliferation or differentiation, but also in pathways such as apoptosis, Wnt signalling, or cytokine-mediated signalling. Of the identified proteins, 51 adipokines, 11 myokines, and 37 adipomyokines were predicted, together with 26 adipose and 13 muscular cell surface proteins. Analysis of protein-protein interactions suggested 13 links between secreted and cell surface proteins that may contribute to the adipose-muscular crosstalk. Of these, an interaction between the adipokine plasminogen and the muscular cell surface alpha-enolase may regulate the fetal myogenesis. The in silico secretome and surfaceome analyzed herein exemplify a powerful strategy to enhance the elucidation of the crosstalk between cell types or tissues.

Mammary Gland Transcriptome and Proteome Modifications by Nutrient Restriction in Early Lactation Holstein Cows Challenged with Intra-Mammary Lipopolysaccharide.

: The objective is to study the effects of nutrient restrictions, which induce a metabolic imbalance on the inflammatory response of the mammary gland in early lactation cows. The aim is to decipher the molecular mechanisms involved, by comparing a control, with a restriction group, a transcriptome and proteome, after an intra-mammary lipopolysaccharide challenge. Multi-parous cows were either allowed ad libitum intake of a lactation diet (n = 8), or a ration containing low nutrient density (n = 8; 48% barley straw and dry matter basis) for four days starting at 24 ± 3 days in milk. Three days after the initiation of their treatments, one healthy rear mammary quarter of 12 lactating cows was challenged with 50 µg of lipopolysaccharide (LPS). Transcriptomic and proteomic analyses were performed on mammary biopsies obtained 24 h after the LPS challenge, using bovine 44K microarrays, and nano-LC-MS/MS, respectively. Restriction-induced deficits in energy, led to a marked negative energy balance (41 versus 97 ± 15% of Net Energy for Lactation (NEL) requirements) and metabolic imbalance. A microarray analyses identified 25 differentially expressed genes in response to restriction, suggesting that restriction had modified mammary metabolism, specifically ß-oxidation process. Proteomic analyses identified 53 differentially expressed proteins, which suggests that the modification of protein synthesis from mRNA splicing to folding. Under-nutrition influenced mammary gland expression of the genes involved in metabolism, thereby increasing ß-oxidation and altering protein synthesis, which may affect the response to inflammation.

Subproteomic signature comparison of in vitro selected fluoroquinolone resistance and ciprofloxacin stress in Salmonella Typhimurium DT104B.

BACKGROUND: Fluoroquinolone resistance in nontyphoidal Salmonella is a situation of serious and international concern, particularly in S. Typhimurium DT104B multiresistant strains. Although known to be multifactorial, fluoroquinolone resistance is still far from a complete understanding. METHODS: Subproteome changes between an experimentally selected fluoroquinolone-resistant strain (Se6-M) and its parent strain (Se6), and also in Se6-M under ciprofloxacin (CIP) stress, were evaluated in order to give new insights into the mechanisms involved. Proteomes were compared at the intracellular and membrane levels by a 2-DE~LC-MS/MS and a shotgun LC-MS/MS approach, respectively. RESULTS: In total, 35 differentially abundant proteins were identified when comparing Se6 with Se6-M (25 more abundant in Se6 and 10 more abundant in Se6-M) and 82 were identified between Se6-M and Se6-M+CIP (51 more abundant in Se6-M and 31 more abundant under ciprofloxacin stress). CONCLUSION: Several proteins with known and possible roles in quinolone resistance were identified which provide important information about mechanism-related differential protein expression, supporting the current knowledge and also leading to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

BACKGROUND: Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1A(T). RESULTS: Transcriptomic analyses of B. xylanisolvens XB1A(T) grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. CONCLUSION: This study shows that xylan degradation by B. xylanisolvens XB1A(T) is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1A(T) has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health.

Label-free quantitative protein profiling of vastus lateralis muscle during human aging.

Sarcopenia corresponds to the loss of muscle mass occurring during aging, and is associated with a loss of muscle functionality. Proteomic links the muscle functional changes with protein expression pattern. To better understand the mechanisms involved in muscle aging, we performed a proteomic analysis of Vastus lateralis muscle in mature and older women. For this, a shotgun proteomic method was applied to identify soluble proteins in muscle, using a combination of high performance liquid chromatography and mass spectrometry. A label-free protein profiling was then conducted to quantify proteins and compare profiles from mature and older women. This analysis showed that 35 of the 366 identified proteins were linked to aging in muscle. Most of the proteins were under-represented in older compared with mature women. We built a functional interaction network linking the proteins differentially expressed between mature and older women. The results revealed that the main differences between mature and older women were defined by proteins involved in energy metabolism and proteins from the myofilament and cytoskeleton. This is the first time that label-free quantitative proteomics has been applied to study of aging mechanisms in human skeletal muscle. This approach highlights new elements for elucidating the alterations observed during aging and may lead to novel sarcopenia biomarkers.

Proteomics of muscle chronological ageing in post-menopausal women.

BACKGROUND: Muscle ageing contributes to both loss of functional autonomy and increased morbidity. Muscle atrophy accelerates after 50 years of age, but the mechanisms involved are complex and likely result from the alteration of a variety of interrelated functions. In order to better understand the molecular mechanisms underlying muscle chronological ageing in human, we have undertaken a top-down differential proteomic approach to identify novel biomarkers after the fifth decade of age. RESULTS: Muscle samples were compared between adult (56 years) and old (78 years) post-menopausal women. In addition to total muscle extracts, low-ionic strength extracts were investigated to remove high abundance myofibrillar proteins and improve the detection of low abundance proteins. Two-dimensional gel electrophoreses with overlapping IPGs were used to improve the separation of muscle proteins. Overall, 1919 protein spots were matched between all individuals, 95 were differentially expressed and identified by mass spectrometry, and they corresponded to 67 different proteins. Our results suggested important modifications in cytosolic, mitochondrial and lipid energy metabolism, which may relate to dysfunctions in old muscle force generation. A fraction of the differentially expressed proteins were linked to the sarcomere and cytoskeleton (myosin light-chains, troponin T, ankyrin repeat domain-containing protein-2, vinculin, four and a half LIM domain protein-3), which may account for alterations in contractile properties. In line with muscle contraction, we also identified proteins related to calcium signal transduction (calsequestrin-1, sarcalumenin, myozenin-1, annexins). Muscle ageing was further characterized by the differential regulation of several proteins implicated in cytoprotection (catalase, peroxiredoxins), ion homeostasis (carbonic anhydrases, selenium-binding protein 1) and detoxification (aldo-keto reductases, aldehyde dehydrogenases). Notably, many of the differentially expressed proteins were central for proteostasis, including heat shock proteins and proteins involved in proteolysis (valosin-containing protein, proteasome subunit beta type-4, mitochondrial elongation factor-Tu). CONCLUSIONS: This study describes the most extensive proteomic analysis of muscle ageing in humans, and identified 34 new potential biomarkers. None of them were previously recognized as differentially expressed in old muscles, and each may represent a novel starting point to elucidate the mechanisms of muscle chronological ageing in humans.

Identification of novel GAPDH-derived antimicrobial peptides secreted by Saccharomyces cerevisiae and involved in wine microbial interactions.

Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Peptidomics analysis of in vitro digested wheat breads: Effect of genotype and environment on protein digestibility and release of celiac disease and wheat allergy related epitopes.

Wheat proteins can trigger immunogenic reactions due to their resistance to digestion and immunostimulatory epitopes. Here, we investigated the peptidomic map of partially digested bread samples and the fingerprint of epitope diversity from 16 wheat genotypes grown in two environmental conditions. Flour protein content and composition were characterized; gastric and jejunal peptides were quantified using LC-MS/MS, and genotypes were classified into high or low bread protein digestibility. Differences in flour protein content and peptide composition distinguish high from low digestibility genotypes in both growing environments. No common peptide signature was found between high- and low-digestible genotypes; however, the celiac or allergen epitopes were noted not to be higher in low-digestible genotypes. Overall, this study established a peptidomic and epitope diversity map of digested wheat bread and provided new insights and correlations between weather conditions, genotypes, digestibility and wheat sensitivities such as celiac disease and wheat allergy.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

Expression profiling of starchy endosperm metabolic proteins at 21 stages of wheat grain development.

Proteomic analysis of albumins and globulins (alg) present in starchy endosperm of wheat (Triticum aestivum cv Récital), at 21 stages of grain development, led to the identification of 487 proteins. Four main developmental phases of these metabolic proteins, with three subphases in phase three and two in phase four, were shown. Hierarchical cluster analysis revealed nine major expression profiles throughout grain development. Classification of identified proteins in 17 different biochemical functions provided a uniform picture of temporal coordination among cellular processes. Proteins involved in cell division, transcription/translation, ATP interconversion, protein synthesis, protein transport, along with amino acid, lipid, carbohydrate and nucleotide metabolisms were highly expressed in early and early mid stages of development. Protein folding, cytoskeleton, and storage proteins peaked during the middle of grain development, while in later stages stress/defense, folic acid metabolism, and protein turn over were the abundant functional categories. Detailed analysis of stress/defense enzymes revealed three different evolutionary profiles. A global map with their predicted subcellular localizations and placement in grain developmental scale was constructed. The present study of complete grain development enriched our knowledge on proteome expression of alg, successively from endosperm cell division and differentiation to programmed cell death.

Cellular and molecular large-scale features of fetal adipose tissue: is bovine perirenal adipose tissue brown?.

Epidemiological and fetal programming studies point to the role of fetal growth in adult adipose tissue (AT) mass in large mammals. Despite the incidence of fetal AT growth for human health and animal production outcomes, there is still a lack of relevant studies. We determined the cellular and large-scale-molecular features of bovine fetal perirenal AT sampled at 110, 180, 210, and 260 days post-conception (dpc) with the aim of identifying key cellular and molecular events in AT growth. The increase in AT weight from 110 to 260 dpc resulted from an increase in adipocyte volume and particularly adipocyte number that were concomitant with temporal changes in the abundance of 142 proteins revealed by proteomics. At 110 and 180 dpc, we identified proteins such as TCP1, FKBP4, or HSPD1 that may regulate adipocyte precursor proliferation by controlling cell-cycle progression and/or apoptosis or delaying PPARγ-induced differentiation. From 180 dpc, the up-regulation of PPARγ-induced proteins, lipogenic and lipolytic enzymes, and adipokine expression may underpin the differentiation and increase in adipocyte volume. Also from 180 dpc, we unexpectedly observed up-regulations in the ß-subunit of ATP synthase, which is normally bypassed in brown AT, as well as in aldehyde dehydrogenases ALDH2 and ALDH9A1, which were predominantly expressed in mouse white AT. These results, together with the observed abundant unilocular adipocytes at 180 and 260 dpc, strongly suggest that fetal bovine perirenal AT has much more in common with white than with brown AT.

Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development.

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.

Digestomic data of proteolysis during whether post rumen digestion after tannin supplementation.

The protein degradation of alfalfa hay after tannin supplementation was monitored during wethers digestion. Three rumen-cannulated wethers were infused a tannin solution, and water for control, through the cannula. The digestion time-points samples were collected in vivo in the rumen and in vitro in the abomasum, and the small intestine compartments. The digestomic dataset was acquired by identifying and quantifying the peptides resulting from the protein degradation, using high-resolution LC-MS/MS mass spectrometry and label-free quantitation. The digestomic dataset is the compilation of proteomic data acquired in the rumen and peptidomic data acquired in the abomasum and in the small intestine. The proteomic analysis identified 20 Medicago proteins in the rumen fluid, based on 169 peptides of which 140 are unique. The peptidomic analysis identified 28 Medicago proteins in the abomasum, based on 575 peptides of which 363 are unique, and 11 Medicago proteins in the small intestine, based on 94 peptides of which 63 are unique. This digestomic dataset of proteolysis during sheep post rumen digestion after tannin supplementation reveals the protein regions protected by tannin supplementation, and could be reused in studies related to the protein use efficiency by ruminants.

Impact of Tannin Supplementation on Proteolysis during Post-Ruminal Digestion in Wethers Using a Dynamic In Vitro System: A Plant (Medicago sativa) Digestomic Approach.

The aim of this study was to characterize the effects of tannins on plant protein during sheep digestion using a digestomic approach combining in vivo (rumen) conditions and an in vitro digestive system (abomasum and small intestine). Ruminal fluid from wethers infused with a tannin solution or water (control) was introduced into the digester, and protein degradation was followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tannin infusion in the rumen led to a clear decrease in protein degradation-related fermentation end-products, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) protein was more abundant than in control wethers. In the simulated abomasum, peptidomic analysis showed more degradation products of RuBisCo in the presence of tannins. The effect of RuBisCo protection by tannins continued to impact Rubisco digestion into early-stage intestinal digestion but was no longer detectable in late-stage intestinal digestion. The peptidomics approach proved a potent tool for identifying and quantifying the type of protein hydrolyzed throughout the gastrointestinal tract.